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Controlling Superselectivity of Multivalent Interactions with Cofactors and Competitors
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Journal of the American Chemical Society

Cite this: J. Am. Chem. Soc. 2022, 144, 38, 17346–17350
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https://doi.org/10.1021/jacs.2c06942
Published September 14, 2022

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Abstract

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Moieties that compete with multivalent interactions or act as cofactors are common in living systems, but their effect on multivalent binding remains poorly understood. We derive a theoretical model that shows how the superselectivity of multivalent interactions is modulated by the presence of cofactors or competitors. We find that the role of these participating moieties can be fully captured by a simple rescaling of the affinity constant of the individual ligand–receptor bonds. Theoretical predictions are supported by experimental data of the membrane repair protein annexin A5 binding to anionic lipid membranes in the presence of Ca2+ cofactors and of the extracellular matrix polysaccharide hyaluronan (HA) binding to CD44 cell surface receptors in the presence of HA oligosaccharide competitors. The obtained findings should facilitate understanding of multivalent recognition in biological systems and open new routes for fine-tuning the selectivity of multivalent nanoprobes in medicinal chemistry.

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Subjects

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Multivalent interactions involve the simultaneous formation of multiple supramolecular bonds, such as ligand–receptor binding (1) or host–guest complexation. (2,3) The combinatorial entropy of possible binding configurations gives rise to a supralinear change in the number of bound multivalent probes as a function of receptor concentration. (4,5) This superselective behavior (6) allows specific targeting of surfaces displaying binding sites above a threshold surface concentration, while leaving surfaces with lower coverages virtually unaffected (Figure 1A). The types of multivalent entities that display superselectivity vary widely, including proteins, (7) antibodies, (4,8) polymers, (9,10) viruses, (11−13) liposomes, and nanoparticles. (14,15) Resolving the mechanism of multivalent interactions is crucial both to understand the selectivity of biomolecular interactions and to facilitate the design of highly selective nanoprobes for diagnostics and therapies. (16)

Figure 1

Figure 1. Multivalent interactions in the presence of competitors and cofactors. Superselectivity of multivalent probes to changes in receptor density (A, top) is modulated by the presence of cofactors (B) or competitors (C). Illustrative plot of probe surface density (solid black line) and corresponding selectivity parameters α (dashed red line) vs receptor density, and cofactor or competitor concentrations (A, bottom). Insets show the relevant reaction equilibria.

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Previous studies of superselectivity in synthetic and living systems have clarified the roles of the affinity of individual ligand–receptor bonds, probe valency, receptor surface density, and in-plane mobility in multivalent binding. (2,14,17) In addition to these factors, biological systems commonly involve interacting moieties that modulate multivalent interactions. For example, many specific interactions in biochemistry require a cofactor (e.g., a multivalent ion or a small molecule) to form a bond, and the strength of the interaction can be tuned by varying the concentration of cofactors. (7,18) Likewise, competing interactions such as agonists vs antagonists are common in biology. The effect of cofactors (Figure 1B) or competitors (Figure 1C) on multivalent binding remains largely unexplored, hampering the wider application of superselectivity concepts. Cofactors and competitors modulate the effective number of available receptors, and we hypothesize that a superselective response toward changes in receptor density naturally extends to modulations in cofactor or competitor concentrations (Figure 1A, bottom).
Here, we demonstrate based on simple theoretical arguments that cofactors and monovalent competitors impact superselective binding by effectively rescaling the ligand–receptor affinity. We apply this insight to two important yet distinct examples of biomolecular interactions, namely, the Ca2+-dependent binding of the membrane repair protein annexin A5 (AnxA5) to anionic lipid membranes (7) and the effect of competing oligosaccharides on the recognition of the extracellular matrix polysaccharide hyaluronan (HA) by cell surface receptors. (19)
The theory of multivalent binding (2,5,6) predicts that the strength of the multivalent interaction, or avidity constant Kav, depends supralinearly on the receptor density, ΓR, and the ligand–receptor dissociation constant, Kd (Figure 1A), as
(1)
where a and nL are the size and valency of the multivalent probe, respectively, nR = a2ΓR the number of accessible receptors, NA Avogadro’s number, and veff the effective free volume that each unbound ligand can explore (the ratio nR/veff is also called effective molarity (1,20)). When binding to a surface, this equation can be used as an input to the Langmuir isotherm, which predicts the surface density of adsorbed probes to be
(2)
with the maximum possible density Γmax and the concentration of unbound probes cP. The binding is said to be superselective if the surface density increases faster than linearly with the receptor density, i.e., if the selectivity parameter
(3)
is larger than unity (Figure 1A). Here we extend this theory to fully capture the effect of cofactors and competitors, including selectivity with regard to cofactor concentration ccf (αcf = d log Γp/d log ccf) and competitor concentration cmc (αmc = −d log Γp/d log cmc; the minus sign ensures that αmc > 0, since binding generally decreases with cmc). The full theoretical derivation that considers the distribution of all possible binding states in equilibrium is provided in the Supporting Information, with only the main results being shown here.

Cofactors

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We consider monovalent cofactors at (unbound) concentration ccf that bind to ligands and receptors with the dissociation constants Kd,L–cf and Kd,R–cf, while the ligand–cofactor (or receptor–cofactor) complex binds to the receptors (or ligands) with constant Kd,Lcf–R (or Kd,Rcf–L) (Figure 1B). The effect of cofactors can be fully captured by using a generalized ligand–receptor “affinity”, with an effective dissociation constant
(4)
where Kd,L–cf–R = Kd,L–cfKd,Lcf–R = Kd,R–cfKd,Rcf–L is the tripartite affinity constant.
At low cofactor concentrations, ccf < Kd,L–cf and ccf < Kd,R–cf, we can approximate Kd(cf) ≈ Kd,L–cf–R/ccf, and thus changing the cofactor concentration has the same effect as changing the receptor density nR (eq 1) and yields an equivalent superselective response (αcf ≈ αR; Figure 1A, bottom). At intermediate concentrations, Kd,R–cf > ccf > Kd,L–cf or Kd,L–cf > ccf > Kd,R–cf, either the ligands or receptors are saturated with cofactors, and changing the cofactor concentration has no effect: Kd(cf) ≈ max[Kd,Lcf–R, Kd,Rcf–L]. Lastly, at very high concentrations, ccf > Kd,L–cf and ccf > Kd,R–cf, the oversaturation with cofactors weakens the effective binding: Kd(cf) ≈ ccfKd,L–cf–R/(Kd,R–cfKd,L–cf), and thus changing ccf has the same effect as changing the inverse receptor density nR–1 (Figure 2A). Often, however, only the low-concentration regime is biologically relevant. These features can be employed to control the range of superselective receptor recognition by tuning the cofactor concentration (Figure 2A). Thus, the influence of cofactors does not change the nature of multivalent binding; rather, it simply rescales the affinity constant according to eq 4.

Figure 2

Figure 2. Effect of cofactors. (A) Example of the dependence of the selectivity parameter αR on the receptor surface density and cofactor concentration (eqs 1–4; nL = 8, cPa3NA = 0.001, Kd,R–cf = 100Kd,L–cf). (B) Schematic of AnxA5 (PDB code 1AVR (21)) binding to supported lipid bilayers presenting PS lipids in a background of PC lipids. (C) Experimental dependence of AnxA5 (nonoligomerizing mutant at cP = 0.56 μM) binding on PS density at different Ca2+ concentrations (symbols; error bars represent experimental precision) is well reproduced by the theory (solid lines in matching colors) that explicitly models binding to the two types of lipids and membrane fluidity (see Supporting Information). (D) The sets of data at different Ca2+ concentration collapse onto a master curve when plotted as a function of fPS × [Ca2+]. Slopes with α values are included in (C) and (D) for reference.

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A salient biological example of how cofactors influence multivalent interactions is AnxA5 binding to lipid membranes (Figure 2B). AnxA5 functions as a cell membrane scaffolding and repair protein. (22) It preferentially binds anionic phospholipids and requires Ca2+ as a cofactor for membrane binding. (7) In intact cells, anionic phospholipids reside in the inner (but not the outer) leaflet of the plasma membrane, whereas Ca2+ ions are virtually absent in the cytoplasm but present (in mM concentrations) outside the cell. AnxA5 thus binds to the cell membrane only upon membrane damage leading to an influx of Ca2+ ions into the cell and possibly also to interleaflet lipid content mixing near the damage site.
Experimental data reveal superselective binding of AnxA5 to lipid membranes presenting anionic phosphatidyl serine (PS) in a background of zwitterionic phosphatidyl choline (PC) lipids, and our theoretical model predicts well AnxA5 binding over 4 orders of magnitude of Ca2+ concentrations (Figure 2C). Moreover, within the range of the investigated calcium concentrations, the binding of Ca2+ to both AnxA5 and PS lipids appears to be weak: ccf/Kd,L–cf < 1 and ccf/Kd,R–cf < 1. Thus, eq 4 can be approximated as Kd(cf) = Kd,L–cf–R/ccf, which implies that AnxA5 binding depends only on the product nRccf (eq 1), where nR = fPS(a/l)2, with the protein cross-section a2 = 25 nm2, the lipid cross-section l2 = 0.7 nm2, and the PS lipid fraction fPS. Indeed, when the AnxA5 binding data are plotted as a function of fPSccf, all experimental data collapse onto a single master curve (Figure 2D), thus validating our theory.
Our analysis identifies membrane recognition by AnxA5 as a striking example of superselective binding, demonstrating that binding is strongly superselective with respect to the cofactor Ca2+ as well as the receptor PS lipids, with maximal α values αcf,max ≈ αR,max ≈ 4 (Figure 2D). This enables the protein to effectively respond to slight changes in the concentration of either of these two factors, which is crucial for its function as a membrane repair protein. We note that effective membrane repair additionally requires AnxA5 to organize into trimers and two-dimensional crystals on the membrane. (22) To probe superselective binding of the AnxA5 monomers, we have in Figure 2 probed an AnxA5 mutant that does not oligomerize yet retains the membrane binding properties of the wild-type protein. However, the superselective effects are retained, and even further accentuated, by the self-organization of the wild-type protein on the membrane (see Supporting Information).

Competitors

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Similar to the theoretical treatment of cofactors, monovalent competitors are assumed to bind to surface receptors with the affinity constant Kd,R–mc. As shown in the full derivation of our analytical model, competitors at (unbound) concentration cmc effectively rescale the ligand–receptor affinity Kd to
(5)
The impact of this rescaling on superselective binding is illustrated in Figure 3A and shows that increasing the competitor concentration pushes the range of superselective binding toward higher receptor densities. Equation 5 is well known for monovalent interactions; (29) we here establish that it also applies to multivalent interactions and can be generalized to multiple competitor types (see Supporting Information).

Figure 3

Figure 3. Effect of monovalent competitors. (A) Illustrative example of the dependence of the selectivity parameter αR on the receptor surface density and competitor concentration (eqs 1–3, 5; nL = 8, cPa3NA = 0.001). (B) Schematic of HA binding to CD44 obtained from a crystal structure. (24) (C) Competition of HA polysaccharides (HApoly) with octasaccharides (HA8) binding CD44 monovalently: experimental data from ref (19) (blue symbols), analytical fit (blue line), and the competitor selectivity αmc (red line).

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We tested our simple model on data reported by Lesley et al. (19) on the inhibition of HA polysaccharide binding to CD44 cell surface receptors by HA oligosaccharides (Figure 3B). That HA binding to cells depends sharply on receptor surface density is evident from previous work. (10,23) Such superselective recognition is important for cell–extracellular matrix communication, and changes in HA presentation can dramatically affect recognition, e.g., inflammation entails degradation of large HA polysaccharides (MDa range) into small oligosaccharides. HA octasaccharides (HA8) just about fill the binding groove in a CD44 receptor (24) and thus are effective monovalent competitors.
The simple analytical model (eqs 1 and 2) with the rescaled affinity Kd(mc) (eq 5) reproduces the experimental data well (Figure 3C), illustrating that it captures the salient features of the competition process. In the model, we fixed nL = 500 distinct sites for binding to CD44 receptors (consistent with an HA molecular mass of ∼1 MDa and a decasaccharide “footprint” per receptor), a coil volume of a3 = 4πRg3/3 (with the radius of gyration, Rg ≈ 90 nm, (25) and a concentration of cP ≈ 0.5 nM), and Kd,R–mc ≈ 50 μM (within the broad range of reported values (19,24,26)). As the only fitting parameter, we determined nR/(Kdveff) ≈ 0.03, a value that is consistent with typical CD44 cell surface densities (see Supporting Information); that is, the simple model makes reasonable quantitative predictions.
Importantly, we demonstrate that the binding response can be superselective with respect to the competitor concentration cmc (αmc > 1; Figure 3C). The fact that the experimental dependence is less sharp then predicted theoretically is attributed to the relatively large polydispersity of HA polymers (ranging from 0.5 to 3 MDa) used in the experiments, (19) which is not considered by the analytical model.
The above reanalysis of data from the literature demonstrates the tangible benefits of superselectivity concepts. It is well known that small vs large HA can exert opposing functional effects, (27) but the underpinning mechanisms have long remained elusive. With the theoretical tool presented here, we can rationalize how HA molecules of different sizes bind and compete with each other for receptors. Moreover, we can predict how changes in the presentation of HA (e.g., the effective mean size and size dispersity, which may be modulated by degradation or by cross-linking with soluble HA binding proteins) and its receptors (e.g., their affinity, surface density, and clustering) modulate HA binding and downstream physiological processes.
In conclusion, we have developed a new mechanistic understanding of multivalent recognition with cofactors and competitors. Rather than modifying the multivalent probe itself, the addition of monovalent binders as competitors or cofactors is a simple, and thus attractive, avenue to modulate superselective binding. This effect can be exploited, for example, to tune the threshold receptor density Γ* of a given probe (Figure 3A), to target surfaces with low receptor density, (28) and for superselective discrimination of cofactor concentrations (Figure 2D). Our theory thus helps design superselective probes for targeting and analytical purposes controlled by cofactors and competitors. While the simple multivalent model (eqs 1 and 2) assumes each ligand can bind to many receptors, the scaling expressions (eqs 4 and 5) are general: they expand on similar and well-known expressions for monovalent interactions (29) and also apply to systems with few receptors and many ligands (see Supporting Information).

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.2c06942.

  • Theoretical derivations, details of the AnxA5-to-membrane binding experiments and binding model, and details of the HA-to-CD44 binding analysis (PDF)

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Author Information

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  • Corresponding Authors
    • Tine Curk - Department of Materials Science and Engineering, Northwestern University, Evanston, Illinois 60208, United States;  Orcidhttps://orcid.org/0000-0002-2167-5336;  Email: [email protected]
    • Ralf P. Richter - School of Biomedical Sciences, Faculty of Biological Sciences, School of Physics and Astronomy, Faculty of Engineering and Physical Sciences, Astbury Centre for Structural Molecular Biology, and Bragg Centre for Materials Research, University of Leeds, Leeds, LS2 9JT, United Kingdom;  Orcidhttps://orcid.org/0000-0003-3071-2837;  Email: [email protected]
  • Authors
    • Galina V. Dubacheva - Département de Chimie Moléculaire, Université Grenoble Alpes, CNRS UMR 5250, 38000 Grenoble, France;  Orcidhttps://orcid.org/0000-0003-1417-5381
    • Alain R. Brisson - UMR-CBMN, CNRS-University of Bordeaux-IPB, 33600 Pessac, France
  • Author Contributions

    T.K. and G.V.D. contributed equally.

  • Funding

    Marie Skłodowska-Curie Fellowship “ELNANO” (845032) to T.C., Marie Curie Career Integration Grant “CELLMULTIVINT” (293803) to G.V.D., and European Research Council Starting Grant “JELLY” (306435) and Biotechnology and Biological Sciences Research Council grant BB/T001631/1 to R.P.R.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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We thank Céline Gounou for her expert assistance in producing the AnxA5protein, and Chunyue Wang for help with rendering the CD44/HA structure.

References

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This article references 29 other publications.

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    Binding of hyaluronan to the native lymphatic vessel endothelial receptor LYVE-1 is critically dependent on receptor clustering and hyaluronan organization
    Lawrance, William; Banerji, Suneale; Day, Anthony J.; Bhattacharjee, Shaumick; Jackson, David G.
    Journal of Biological Chemistry (2016), 291 (15), 8014-8030CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The lymphatic endothelial receptor LYVE-1 has been implicated in both uptake of hyaluronan (HA) from tissue matrix and in facilitating transit of leukocytes and tumor cells through lymphatic vessels based largely on in vitro studies with recombinant receptor in transfected fibroblasts. Curiously, however, LYVE-1 in lymphatic endothelium displays little if any binding to HA in vitro, and this has led to the conclusion that the native receptor is functionally silenced, a feature that is difficult to reconcile with its proposed in vivo functions. Nonetheless, as we reported recently, LYVE-1 can function as a receptor for HA-encapsulated Group A streptococci and mediate lymphatic dissemination in mice. Here we resolve these paradoxical findings and show that the capacity of LYVE-1 to bind HA is strictly dependent on avidity, demanding appropriate receptor self-assocn. and/or HA multimerization. In particular, we demonstrate the prerequisite of a crit. LYVE-1 threshold d. and show that HA binding may be elicited in lymphatic endothelium by surface clustering with divalent LYVE-1 mAbs. In addn., we show that crosslinking of biotinylated HA in streptavidin multimers or supramol. complexes with the inflammation-induced protein TSG-6 enables binding even in the absence of LYVE-1 crosslinking. Finally, we show that endogenous HA on the surface of macrophages can engage LYVE-1, facilitating their adhesion and transit across lymphatic endothelium. These results reveal LYVE-1 as a low affinity receptor tuned to discriminate between different HA configurations through avidity and establish a new mechanistic basis for the functions ascribed to LYVE-1 in matrix HA binding and leukocyte trafficking in vivo.
  11. 11
    Vachieri, S. G.; Xiong, X.; Collins, P. J.; Walker, P. A.; Martin, S. R.; Haire, L. F.; Zhang, Y.; McCauley, J. W.; Gamblin, S. J.; Skehel, J. J. Receptor binding by H10 influenza viruses. Nature 2014, 511, 475– 7,  DOI: 10.1038/nature13443
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    11
    Receptor binding by H10 influenza viruses
    Vachieri, Sebastien G.; Xiong, Xiaoli; Collins, Patrick J.; Walker, Philip A.; Martin, Stephen R.; Haire, Lesley F.; Zhang, Ying; McCauley, John W.; Gamblin, Steven J.; Skehel, John J.
    Nature (London, United Kingdom) (2014), 511 (7510), 475-477CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
    H10N8 follows H7N9 and H5N1 as the latest in a line of avian influenza viruses that cause serious disease in humans and have become a threat to public health. Since Dec. 2013, three human cases of H10N8 infection have been reported, two of whom are known to have died. To gather evidence relating to the epidemic potential of H10 we have detd. the structure of the hemagglutinin of a previously isolated avian H10 virus and we present here results relating esp. to its receptor-binding properties, as these are likely to be major determinants of virus transmissibility. Our results show, first, that the H10 virus possesses high avidity for human receptors and second, from the crystal structure of the complex formed by avian H10 hemagglutinin with human receptor, it is clear that the conformation of the bound receptor has characteristics of both the 1918 H1N1 pandemic virus and the human H7 viruses isolated from patients in 2013 (ref. 3). We conclude that avian H10N8 virus has sufficient avidity for human receptors to account for its infection of humans but that its preference for avian receptors should make avian-receptor-rich human airway mucins an effective block to widespread infection. In terms of surveillance, particular attention will be paid to the detection of mutations in the receptor-binding site of the H10 hemagglutinin that decrease its avidity for avian receptor, and could enable it to be more readily transmitted between humans.
  12. 12
    Overeem, N. J.; Hamming, P. H. E.; Grant, O. C.; Di Iorio, D.; Tieke, M.; Bertolino, M. C.; Li, Z.; Vos, G.; de Vries, R. P.; Woods, R. J.; Tito, N. B.; Boons, G. P. H.; van der Vries, E.; Huskens, J. Hierarchical multivalent effects control influenza host specificity. ACS Cent Sci. 2020, 6, 2311– 8,  DOI: 10.1021/acscentsci.0c01175
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    12
    Hierarchical Multivalent Effects Control Influenza Host Specificity
    Overeem, Nico J.; Hamming, P. H. Erik; Grant, Oliver C.; Di Iorio, Daniele; Tieke, Malte; Bertolino, M. Candelaria; Li, Zeshi; Vos, Gael; de Vries, Robert P.; Woods, Robert J.; Tito, Nicholas B.; Boons, Geert-Jan P. H.; van der Vries, Erhard; Huskens, Jurriaan
    ACS Central Science (2020), 6 (12), 2311-2318CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
    Understanding how emerging influenza viruses recognize host cells is crit. in evaluating their zoonotic potential, pathogenicity, and transmissibility between humans. The surface of the influenza virus is covered with hemagglutinin (HA) proteins that can form multiple interactions with sialic acid-terminated glycans on the host cell surface. This multivalent binding affects the selectivity of the virus in ways that cannot be predicted from the individual receptor-ligand interactions alone. Here, the intrinsic structural and energetic differences between the interactions of avian- or human-type receptors with influenza HA translate from individual site affinity and orientation through receptor length and d. on the surface into virus avidity and specificity. The authors introduce a method to measure virus avidity using receptor d. gradients. Influenza viruses attached stably to a surface at receptor densities that correspond to a min. no. of ∼8 HA-glycan interactions, but more interactions were required if the receptors were short and human-type. Thus, the avidity and specificity of influenza viruses for a host cell depend not on the sialic acid linkage alone but on a combination of linkage and the length and d. of receptors on the cell surface. The authors' findings suggest that threshold receptor densities play a key role in virus tropism, which is a predicting factor for both their virulence and zoonotic potential. Virus binding to receptor d. gradients at cell surface mimics shows that the avidity and selectivity of influenza A virus are governed by receptor structure and d.
  13. 13
    Overeem, N. J.; Hamming, P. H. E.; Tieke, M.; van der Vries, E.; Huskens, J. Multivalent affinity profiling: Direct visualization of the superselective binding of influenza viruses. ACS Nano 2021, 15, 8525– 36,  DOI: 10.1021/acsnano.1c00166
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    Multivalent Affinity Profiling: Direct Visualization of the Superselective Binding of Influenza Viruses
    Overeem, Nico J.; Hamming, P. H.; Tieke, Malte; van der Vries, Erhard; Huskens, Jurriaan
    ACS Nano (2021), 15 (5), 8525-8536CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
    The influenza A virus (IAV) interacts with the glycocalyx of host cells through its surface proteins hemagglutinin (HA) and neuraminidase (NA). Quant. biophys. measurements of these interactions may help to understand these interactions at the mol. level with the long-term aim to predict influenza infectivity and answer other biol. questions. We developed a method, called multivalent affinity profiling (MAP), to measure virus binding profiles on receptor d. gradients to det. the threshold receptor d., which is a quant. measure of virus avidity toward a receptor. Here, we show that imaging of IAVs on receptor d. gradients allows the direct visualization and efficient assessment of their superselective binding. We show how the multivalent binding of IAVs can be quant. assessed using MAP if the receptor d. gradients are prepd. around the threshold receptor d. without crowding at the higher densities. The threshold receptor d. increases strongly with increasing flow rate, showing that the superselective binding of IAV is influenced by shear force. This method of visualization and quant. assessment of superselective binding allows not only comparative studies of IAV-receptor interactions, but also more fundamental studies of how superselectivity arises and is influenced by exptl. conditions.
  14. 14
    Scheepers, M. R. W.; van Ijzendoorn, L. J.; Prins, M. W. J. Multivalent weak interactions enhance selectivity of interparticle binding. Proc. Natl. Acad. Sci. U. S. A. 2020, 117, 22690– 7,  DOI: 10.1073/pnas.2003968117
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    14
    Multivalent weak interactions enhance selectivity of interparticle binding
    Scheepers, M. R. W.; van IJzendoorn, L. J.; Prins, M. W. J.
    Proceedings of the National Academy of Sciences of the United States of America (2020), 117 (37), 22690-22697CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
    Targeted drug delivery critically depends on the binding selectivity of cargo-transporting colloidal particles. Extensive theor. work has shown that two factors are necessary to achieve high selectivity for a threshold receptor d.: multivalency and weak interactions. Here, we study a model system of DNA-coated particles with multivalent and weak interactions that mimics ligand-receptor interactions between particles and cells. Using an optomagnetic cluster expt., particle aggregation rates are measured as a function of ligand and receptor densities. The measured aggregation rates show that the binding becomes more selective for shorter DNA ligand-receptor pairs, proving that multivalent weak interactions lead to enhanced selectivity in interparticle binding. Simulations confirm the exptl. findings and show the role of ligand-receptor dissocn. in the selectivity of the weak multivalent binding.
  15. 15
    Linne, C.; Visco, D.; Angioletti-Uberti, S.; Laan, L.; Kraft, D. J. Direct visualization of superselective colloid-surface binding mediated by multivalent interactions. Proc. Natl. Acad. Sci. U. S. A. 2021, 118, e210603611  DOI: 10.1073/pnas.2106036118
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    There is no corresponding record for this reference.
  16. 16
    Schroeder, A.; Heller, D. A.; Winslow, M. M.; Dahlman, J. E.; Pratt, G. W.; Langer, R.; Jacks, T.; Anderson, D. G. Treating metastatic cancer with nanotechnology. Nat. Rev. Cancer 2012, 12, 39– 50,  DOI: 10.1038/nrc3180
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    Treating metastatic cancer with nanotechnology
    Schroeder, Avi; Heller, Daniel A.; Winslow, Monte M.; Dahlman, James E.; Pratt, George W.; Langer, Robert; Jacks, Tyler; Anderson, Daniel G.
    Nature Reviews Cancer (2012), 12 (1), 39-50CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)
    A review. Metastasis accounts for the vast majority of cancer deaths. The unique challenges for treating metastases include their small size, high multiplicity and dispersion to diverse organ environments. Nanoparticles have many potential benefits for diagnosing and treating metastatic cancer, including the ability to transport complex mol. cargoes to the major sites of metastasis, such as the lungs, liver and lymph nodes, as well as targeting to specific cell populations within these organs. This Review highlights the research, opportunities and challenges for integrating engineering sciences with cancer biol. and medicine to develop nanotechnol.-based tools for treating metastatic disease.
  17. 17
    Dubacheva, G. V.; Curk, T.; Frenkel, D.; Richter, R. P. Multivalent recognition at fluid surfaces: The interplay of receptor clustering and superselectivity. J. Am. Chem. Soc. 2019, 141, 2577– 88,  DOI: 10.1021/jacs.8b12553
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    17
    Multivalent recognition at fluid surfaces: The interplay of receptor clustering and superselectivity
    Dubacheva, Galina V.; Curk, Tine; Frenkel, Daan; Richter, Ralf P.
    Journal of the American Chemical Society (2019), 141 (6), 2577-2588CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
    The interaction between a biol. membrane and its environment is a complex process, as it involves multivalent binding between ligand/receptor pairs, which can self-organize in patches. Any description of the specific binding of biomols. to membranes must account for the key characteristics of multivalent binding, namely, its unique ability to discriminate sharply between high and low receptor densities (superselectivity), but also for the effect of the lateral mobility of membrane-bound receptors to cluster upon binding. Here, we present an exptl. model system that allows us to compare systematically the effects of multivalent interactions on fluid and immobile surfaces. A crucial feature of our model system is that it allows us to control the membrane surface chem., the properties of the multivalent binder, and the binding affinity. We found that multivalent probes retained their superselective binding behavior at fluid interfaces. Supported by numerical simulations, we demonstrated that, as a consequence of receptor clustering, superselective binding was enhanced and shifted to lower receptor densities at fluid interfaces. To translate our findings into a simple, predictive tool, we propose an anal. model that enables rapid predictions of how the superselective binding behavior is affected by the lateral receptor mobility as a function of the physicochem. characteristics of the multivalent probe. We believe that our model, which captures the key phys. mechanisms underpinning multivalent binding to biol. membranes, will greatly facilitate the rational design of nanoprobes for the superselective targeting of cells.
  18. 18
    Jeppesen, B.; Smith, C.; Gibson, D. F.; Tait, J. F. Entropic and enthalpic contributions to annexin V-membrane binding: A comprehensive quantitative model. J. Biol. Chem. 2008, 283, 6126– 35,  DOI: 10.1074/jbc.M707637200
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    Entropic and Enthalpic Contributions to Annexin V-Membrane Binding: A Comprehensive Quantitative Model
    Jeppesen, Brian; Smith, Christina; Gibson, Donald F.; Tait, Jonathan F.
    Journal of Biological Chemistry (2008), 283 (10), 6126-6135CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    Annexin V binds to membranes with very high affinity, but the factors responsible remain to be quant. elucidated. Anal. by isothermal microcalorimetry and calcium titrn. under conditions of low membrane occupancy showed that there was a strongly pos. entropy change upon binding. For vesicles contg. 25% phosphatidylserine at 0.15 M ionic strength, the free energy of binding was -53 kcal/mol protein, whereas the enthalpy of binding was -38 kcal/mol. Addn. of 4 M urea decreased the free energy of binding by about 30% without denaturing the protein, suggesting that hydrophobic forces make a significant contribution to binding affinity. This was confirmed by mutagenesis studies that showed that binding affinity was modulated by the hydrophobicity of surface residues that are likely to enter the interfacial region upon protein-membrane binding. The change in free energy was quant. consistent with predictions from the Wimley-White scale of interfacial hydrophobicity. In contrast, binding affinity was not increased by making the protein surface more pos. charged, nor decreased by making it more neg. charged, ruling out general ionic interactions as major contributors to binding affinity. The affinity of annexin V was the same regardless of the head group present on the anionic phospholipids tested (phosphatidylserine, phosphatidylglycerol, phosphatidylmethanol, and cardiolipin), ruling out specific interactions between the protein and non-phosphate moieties of the head group as a significant contributor to binding affinity. Anal. by fluorescence resonance energy transfer showed that multimers did not form on phosphatidylserine membranes at low occupancy, indicating that annexin-annexin interactions did not contribute to binding affinity. In summary, binding of annexin V to membranes is driven by both enthalpic and entropic forces. Dehydration of hydrophobic regions of the protein surface as they enter the interfacial region makes an important contribution to overall binding affinity, supplementing the role of protein-calcium-phosphate chelates.
  19. 19
    Lesley, J.; Hascall, V. C.; Tammi, M.; Hyman, R. Hyaluronan binding by cell surface CD44. J. Biol. Chem. 2000, 275, 26967– 75,  DOI: 10.1016/S0021-9258(19)61467-5
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    19
    Hyaluronan binding by cell surface CD44
    Lesley, Jayne; Hascall, Vincent C.; Tammi, Markku; Hyman, Robert
    Journal of Biological Chemistry (2000), 275 (35), 26967-26975CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    CD44 is the primary cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan. Here the authors detd. the relative avidities of unlabeled hyaluronan prepns. for cell surface CD44 by their ability to block the binding of fluorescein-conjugated hyaluronan to a variety of cells. The authors show that hyaluronan binding at the cell surface is a complex interplay of multivalent binding events affected by the size of the multivalent hyaluronan ligand, the quantity and d. of cell surface CD44, and the activation state of CD44 as detd. by cell-specific factors and/or treatment with CD44-specific monoclonal antibody (mAb). Using low Mr hyaluronan oligomers of defined sizes, the authors obsd. monovalent binding between 6 and 18 sugars. At ∼20 to ∼38 sugars, there was an increase in avidity (∼3x), suggesting that divalent binding was occurring. In the presence of the inducing mAb IRAWB14, monovalent binding avidity was similar to that of noninduced CD44, but beginning at ∼20 residues, there was a dramatic and progressive increase in avidity with increasing oligomer size (∼22 < 26 < 30 < 34 < 38 sugars). Kinetic studies of binding and dissocn. of fluorescein-conjugated hyaluronan indicated that inducing mAb treatment had little effect on the binding kinetics, but dissocn. from the cell surface was greatly delayed by inducing mAb.
  20. 20
    Ercolani, G.; Schiaffino, L. Allosteric, chelate, and interannular cooperativity: A mise au point. Angew. Chem., Int. Ed. Engl. 2011, 50, 1762– 8,  DOI: 10.1002/anie.201004201
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    Allosteric, chelate, and interannular cooperativity: a mise au point
    Ercolani Gianfranco; Schiaffino Luca
    Angewandte Chemie (International ed. in English) (2011), 50 (8), 1762-8 ISSN:.
    There is no expanded citation for this reference.
  21. 21
    Huber, R.; Berendes, R.; Burger, A.; Schneider, M.; Karshikov, A.; Luecke, H.; Romisch, J.; Paques, E. Crystal and molecular structure of human annexin V after refinement. Implications for structure, membrane binding and ion channel formation of the annexin family of proteins. J. Mol. Biol. 1992, 223, 683– 704,  DOI: 10.1016/0022-2836(92)90984-R
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    Crystal and molecular structure of human annexin V after refinement. Implications for structure, membrane binding and ion channel formation of the annexin family of proteins
    Huber, Robert; Berendes, Robert; Burger, Alexander; Schneider, Monika; Karshikov, Andrej; Luecke, Hartmut; Roemisch, Juergen; Paques, Eric
    Journal of Molecular Biology (1992), 223 (3), 683-704CODEN: JMOBAK; ISSN:0022-2836.
    Two crystal forms (P63 and R3) of human annexin V have been crystallog. refined at 2.3 and 2.0 Å resoln. to R-values of 0.184 and 0.174, resp., applying very tight stereochem. restraints with deviations from ideal geometry of 0.01 Å and 2°. The three independent mols. (2 in P63, 1 in R3) are similar, with deviations in Cα positions of 0.6 Å. The polypeptide chain of 320 amino acid residues is folded into a planar cyclic arrangement of four repeats. The repeats have similar structures of five α-helical segments wound into a right-handed compact superhelix. Three calcium ion sites in repeats I, II and IV and two lanthanum ion sites in repeat I have been found in the R3 crystals. They are located at the convex face of the mol. opposite the N terminus. Repeat III has a different conformation at this site and no calcium bound. The calcium sites are similar to the phospholinase A2 calcium-binding site, suggesting analogy also in phospholipid interaction. The center of the mol. is formed by a channel of polar charged residues and also harbors a chain of ordered water mols. conserved in the different crystal forms. Comparison with amino acid sequences of other annexins shows a high degree of similarity between them. Long insertions are found only at the N termini. Most conserved are the residues forming the metal-binding sites and the polar channel. Annexins V and VII form voltage-gated calcium ion channels when bound to membranes in vitro. It is suggested that annexins bind with their convex face to membranes, causing local disorder and permeability of the phospholipid bilayers. Annexins are Janus-faced proteins that face phospholipid and water and mediate calcium transport.
  22. 22
    Bouter, A.; Gounou, C.; Berat, R.; Tan, S.; Gallois, B.; Granier, T.; d’Estaintot, B. L.; Poschl, E.; Brachvogel, B.; Brisson, A. R. Annexin-A5 assembled into two-dimensional arrays promotes cell membrane repair. Nat. Commun. 2011, 2, 270,  DOI: 10.1038/ncomms1270
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    Annexin-A5 assembled into two-dimensional arrays promotes cell membrane repair
    Bouter Anthony; Gounou Celine; Berat Remi; Tan Sisareuth; Gallois Bernard; Granier Thierry; d'Estaintot Beatrice Langlois; Poschl Ernst; Brachvogel Bent; Brisson Alain R
    Nature communications (2011), 2 (), 270 ISSN:.
    Eukaryotic cells possess a universal repair machinery that ensures rapid resealing of plasma membrane disruptions. Before resealing, the torn membrane is submitted to considerable tension, which functions to expand the disruption. Here we show that annexin-A5 (AnxA5), a protein that self-assembles into two-dimensional (2D) arrays on membranes upon Ca(2+) activation, promotes membrane repair. Compared with wild-type mouse perivascular cells, AnxA5-null cells exhibit a severe membrane repair defect. Membrane repair in AnxA5-null cells is rescued by addition of AnxA5, which binds exclusively to disrupted membrane areas. In contrast, an AnxA5 mutant that lacks the ability of forming 2D arrays is unable to promote membrane repair. We propose that AnxA5 participates in a previously unrecognized step of the membrane repair process: triggered by the local influx of Ca(2+), AnxA5 proteins bind to torn membrane edges and form a 2D array, which prevents wound expansion and promotes membrane resealing.
  23. 23
    Perschl, A.; Lesley, J.; English, N.; Trowbridge, I.; Hyman, R. Role of CD44 cytoplasmic domain in hyaluronan binding. Eur. J. Immunol. 1995, 25, 495– 501,  DOI: 10.1002/eji.1830250228
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    23
    Role of CD44 cytoplasmic domain in hyaluronan binding
    Perschl, Astrid; Lesley, Jayne; English, Nicole; Trowbridge, Ian; Hyman, Robert
    European Journal of Immunology (1995), 25 (2), 495-501CODEN: EJIMAF; ISSN:0014-2980. (VCH)
    The hyaluronan (HA) binding activity of mutant CD44 constructs expressed in AKR1 T-lymphoma cells was evaluated by flow cytometry using fluorescein-conjugated HA (Fl-HA). Previous studies showed that wild-type hematopoietic CD44 bound Fl-HA when expressed in AKR1, but that truncated "tailless" CD44, lacking all but six amino acids of the cytoplasmic domain, did not bind. Here, we show that a disulfide-bonded dimer of CD44, formed by substituting the transmembrane region of CD3ζ chain for that of CD44, binds Fl-HA, even when the cytoplasmic domain of the CD44 dimer is absent. We conclude that dimerization of CD44 abrogates the requirement for the cytoplasmic domain, suggesting that the cytoplasmic domain of CD44 may contribute to HA binding by promoting CD44 clustering. These results suggest that changes in the distribution of CD44 on the cell surface, induced by mol. interactions either from within the cell or from outside, may regulate its role as a receptor. Further studies sought to localize the region of the CD44 cytoplasmic domain contributing to HA binding by the construction of a series of cytoplasmic domain truncation mutants and internal deletion mutants. All of the mutant CD44 mols. bound Fl-HA similarly to wild-type CD44. Thus, it was not possible to assign the function mediating HA binding to a specific region of the cytoplasmic domain, suggesting either that multiple regions of the cytoplasmic domain can promote enhancement of HA binding, or that the role of the cytoplasmic domain in mediating this function does not require a specific amino acid sequence.
  24. 24
    Banerji, S.; Wright, A. J.; Noble, M.; Mahoney, D. J.; Campbell, I. D.; Day, A. J.; Jackson, D. G. Structures of the CD44–hyaluronan complex provide insight into a fundamental carbohydrateprotein interaction. Nat. Struct Mol. Biol. 2007, 14, 234– 9,  DOI: 10.1038/nsmb1201
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    Structures of the Cd44-hyaluronan complex provide insight into a fundamental carbohydrate-protein interaction
    Banerji, Suneale; Wright, Alan J.; Noble, Martin; Mahoney, David J.; Campbell, Iain D.; Day, Anthony J.; Jackson, David G.
    Nature Structural & Molecular Biology (2007), 14 (3), 234-239CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
    Regulation of transient interactions between cells and the ubiquitous matrix glycosaminoglycan hyaluronan is crucial to such fundamental processes as embryonic development and leukocyte homing. Cd44, the primary cell surface receptor for hyaluronan, binds ligand via a lectin-like fold termed the Link module, but only after appropriate functional activation. The mol. details of the Cd44-hyaluronan interaction and hence the structural basis for this activation are unknown. Here we present the first crystal structure of Cd44 complexed with hyaluronan. This reveals that the interaction with hyaluronan is dominated by shape and hydrogen-bonding complementarity and identifies two conformational forms of the receptor that differ in orientation of a crucial hyaluronan-binding residue (Arg45, equiv. to Arg41 in human CD44). Measurements by NMR indicate that the conformational transition can be induced by hyaluronan binding, providing further insight into possible mechanisms for regulation of Cd44.
  25. 25
    Takahashi, R.; Al-Assaf, S.; Williams, P. A.; Kubota, K.; Okamoto, A.; Nishinari, K. Asymmetrical-flow field-flow fractionation with on-line multiangle light scattering detection. 1. Application to wormlike chain analysis of weakly stiff polymer chains. Biomacromolecules 2003, 4, 404– 9,  DOI: 10.1021/bm025706v
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    Asymmetrical-Flow Field-Flow Fractionation with On-Line Multiangle Light Scattering Detection. 1. Application to Wormlike Chain Analysis of Weakly Stiff Polymer Chains
    Takahashi, Rheo; Al-Assaf, Saphwan; Williams, Peter A.; Kubota, Kenji; Okamoto, Akio; Nishinari, Katsuyoshi
    Biomacromolecules (2003), 4 (2), 404-409CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
    Four samples of hyaluronan in the sodium form, ranging in wt.-av. mol. wt., Mw, from 6.67 × 105 to 4.23 × 106 were investigated by asym.-flow field-flow fractionation coupled to multiangle light scattering (FlFFF-MALS) in 0.2 M aq. NaCl at 25 °C. Mw and z-av. radii of gyration, RGz, obtained via FlFFF-MALS showed a good agreement with the results obtained by conventional static light scattering. Furthermore, the mol. wt. dependence of the radius of gyration for sodium hyaluronan obtained via FlFFF-MALS was analyzed on the basis of the Kratky-Porod model for unperturbed wormlike chains combined with the Yamakawa theory for radius expansion factor, and a sufficiently good agreement was obsd. between the theor. prediction and exptl. data. These results show the potential usage of FlFFF-MALS regarding size sepn. and mol. characterization even for weakly stiff chains.
  26. 26
    Banerji, S.; Hide, B. R. S.; James, J. R.; Noble, M. E. M.; Jackson, D. G. Distinctive properties of the hyaluronan binding domain in the lymphatic endothelial receptor LYVE-1 and their implications for receptor function. J. Biol. Chem. 2010, 285, 10724– 35,  DOI: 10.1074/jbc.M109.047647
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    26
    Distinctive Properties of the Hyaluronan-binding Domain in the Lymphatic Endothelial Receptor Lyve-1 and Their Implications for Receptor Function
    Banerji, Suneale; Hide, Branwen R. S.; James, John R.; Noble, Martin E. M.; Jackson, David G.
    Journal of Biological Chemistry (2010), 285 (14), 10724-10735CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
    The lymphatic endothelial hyaluronan (HA) receptor Lyve-1 is a member of the Link protein superfamily most similar to the leukocyte HA receptor CD44. However, the structure of Lyve-1 and the nature of its interaction with ligand are obscure. Here we present new evidence that Lyve-1 is functionally distinct from CD44. Using truncation mutagenesis we confirm that Lyve-1 in common with CD44 contains an extended HA-binding unit, comprising elements flanking the N and C termini of the consensus lectin-like Link module, bridged by a third conserved disulfide linkage that is crit. for HA binding. In addn., we identify six essential residues Tyr-87, Ile-97, Arg-99, Asn-103, Lys-105, and Lys-108 that define a compact HA-binding surface on Lyve-1, encompassing the epitope for an adhesion-blocking monoclonal antibody 3A, in an analogous position to the HA-binding surface in CD44. The overtly electrostatic character of HA binding in Lyve-1 and its sensitivity to ionic strength (IC50 of 150 mM NaCl) contrast markedly with CD44 (IC50 > 2 M NaCl) in which HA binding is mediated by hydrogen bonding and hydrophobic interactions. In addn., unlike the extended Link module in CD44, which binds HA efficiently when expressed as a sol. monomer (Kd = 65.7 μM), that of Lyve-1 requires artificial dimerization, although the full ectodomain is active as a monomer (Kd = 35.6 μM). Finally, full-length Lyve-1 did not form stable dimers in binding-competent 293T transfectants when assessed using bioluminescent resonance energy transfer. These results reveal that elements addnl. to the extended Link module are required to stabilize HA binding in Lyve-1 and indicate important structural and functional differences with CD44.
  27. 27
    Cyphert, J. M.; Trempus, C. S.; Garantziotis, S. Size matters: Molecular weight specificity of hyaluronan effects in cell biology. Int. J. Cell Biol. 2015, 2015, 563818,  DOI: 10.1155/2015/563818
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    Size Matters: Molecular Weight Specificity of Hyaluronan Effects in Cell Biology
    Cyphert Jaime M; Trempus Carol S; Garantziotis Stavros
    International journal of cell biology (2015), 2015 (), 563818 ISSN:1687-8876.
    Hyaluronan signaling properties are unique among other biologically active molecules, that they are apparently not influenced by postsynthetic molecular modification, but by hyaluronan fragment size. This review summarizes the current knowledge about the generation of hyaluronan fragments of different size and size-dependent differences in hyaluronan signaling as well as their downstream biological effects.
  28. 28
    Tito, N. B. Multivalent ″attacker and guard″ strategy for targeting surfaces with low receptor density. J. Chem. Phys. 2019, 150, 184907,  DOI: 10.1063/1.5086277
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    Multivalent "attacker and guard" strategy for targeting surfaces with low receptor density
    Tito, Nicholas B.
    Journal of Chemical Physics (2019), 150 (18), 184907/1-184907/16CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
    Multivalent particles, i.e., microscopic constructs having multiple ligands, can be used to target surfaces selectively depending on their receptor d. Typically, there is a sharp onset of multivalent binding as the receptor d. exceeds a given threshold. However, the opposite case, selectively binding to surfaces with a receptor d. below a given threshold, is much harder. Here, the authors present a simple strategy for selectively targeting a surface with a low d. of receptors, within a system also having a surface with a higher d. of the same receptors. The authors' strategy exploits competitive adsorption of two species. The first species, called "guards," are receptor-sized monovalent particles designed to occupy the high-d. surface at equil., while the second multivalent "attacker" species outcompetes the guards for binding onto the low-d. surface. Surprisingly, the recipe for attackers and guards yields more selective binding with stronger ligand-receptor assocn. consts., in contrast to std. multivalency. The authors derive explicit expressions for the attacker and guard mol. design parameters and concns., optimized within bounds of what is exptl. accessible, thereby facilitating implementation of the proposed approach. (c) 2019 American Institute of Physics.
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    Berg, J. M.; Tymoczko, J. L.; Gatto, G. J., Jr.; Stryer, L. Biochemistry; Macmillan Learning, 2019.
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  • Abstract

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    Figure 1

    Figure 1. Multivalent interactions in the presence of competitors and cofactors. Superselectivity of multivalent probes to changes in receptor density (A, top) is modulated by the presence of cofactors (B) or competitors (C). Illustrative plot of probe surface density (solid black line) and corresponding selectivity parameters α (dashed red line) vs receptor density, and cofactor or competitor concentrations (A, bottom). Insets show the relevant reaction equilibria.

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    Figure 2

    Figure 2. Effect of cofactors. (A) Example of the dependence of the selectivity parameter αR on the receptor surface density and cofactor concentration (eqs 1–4; nL = 8, cPa3NA = 0.001, Kd,R–cf = 100Kd,L–cf). (B) Schematic of AnxA5 (PDB code 1AVR (21)) binding to supported lipid bilayers presenting PS lipids in a background of PC lipids. (C) Experimental dependence of AnxA5 (nonoligomerizing mutant at cP = 0.56 μM) binding on PS density at different Ca2+ concentrations (symbols; error bars represent experimental precision) is well reproduced by the theory (solid lines in matching colors) that explicitly models binding to the two types of lipids and membrane fluidity (see Supporting Information). (D) The sets of data at different Ca2+ concentration collapse onto a master curve when plotted as a function of fPS × [Ca2+]. Slopes with α values are included in (C) and (D) for reference.

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    Figure 3

    Figure 3. Effect of monovalent competitors. (A) Illustrative example of the dependence of the selectivity parameter αR on the receptor surface density and competitor concentration (eqs 1–3, 5; nL = 8, cPa3NA = 0.001). (B) Schematic of HA binding to CD44 obtained from a crystal structure. (24) (C) Competition of HA polysaccharides (HApoly) with octasaccharides (HA8) binding CD44 monovalently: experimental data from ref (19) (blue symbols), analytical fit (blue line), and the competitor selectivity αmc (red line).

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  • References

    This article references 29 other publications.

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      Mammen, M.; Choi, S.-K.; Whitesides, G. M. Polyvalent interactions in biological systems: Implications for design and use of multivalent ligands and inhibitors. Angew. Chem., Int. Ed. 1998, 37, 2754– 94,  DOI: 10.1002/(SICI)1521-3773(19981102)37:20<2754::AID-ANIE2754>3.0.CO;2-3
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      Polyvalent Interactions in Biological Systems: Implications for Design and Use of Multivalent Ligands and Inhibitors
      Mammen Mathai; Choi Seok-Ki; Whitesides George M
      Angewandte Chemie (International ed. in English) (1998), 37 (20), 2754-2794 ISSN:.
      Found throughout biology, polyvalent interactions are characterized by the simultaneous binding of multiple ligands on one biological entity to multiple receptors on another (top part of the illustration) and have a number of characteristics that monovalent interactions do not (bottom). In particular, polyvalent interactions can be collectively much stronger than corresponding monovalent interactions, and they can provide the basis for mechanisms of both agonizing and antagonizing biological interactions that are fundamentally different from those available in monovalent systems.
    2. 2
      Dubacheva, G. V.; Curk, T.; Auzely-Velty, R.; Frenkel, D.; Richter, R. P. Designing multivalent probes for tunable superselective targeting. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 5579– 84,  DOI: 10.1073/pnas.1500622112
      2
      Designing multivalent probes for tunable superselective targeting
      Dubacheva, Galina V.; Curk, Tine; Auzely-Velty, Rachel; Frenkel, Daan; Richter, Ralf P.
      Proceedings of the National Academy of Sciences of the United States of America (2015), 112 (18), 5579-5584CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Specific targeting is common in biol. and is a key challenge in nanomedicine. It was recently demonstrated that multivalent probes can selectively target surfaces with a defined d. of surface binding sites. Here we show, using a combination of expts. and simulations on multivalent polymers, that such "superselective" binding can be tuned through the design of the multivalent probe, to target a desired d. of binding sites. We develop an anal. model that provides simple yet quant. predictions to tune the polymer's superselective binding properties by its mol. characteristics such as size, valency, and affinity. This work opens up a route toward the rational design of multivalent probes with defined superselective targeting properties for practical applications, and provides mechanistic insight into the regulation of multivalent interactions in biol. To illustrate this, we show how the superselective targeting of the extracellular matrix polysaccharide hyaluronan to its main cell surface receptor CD44 is controlled by the affinity of individual CD44-hyaluronan interactions.
    3. 3
      Dubacheva, G. V.; Curk, T.; Mognetti, B. M.; Auzély-Velty, R.; Frenkel, D.; Richter, R. P. Superselective targeting using multivalent polymers. J. Am. Chem. Soc. 2014, 136, 1722– 5,  DOI: 10.1021/ja411138s
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      Superselective Targeting Using Multivalent Polymers
      Dubacheva, Galina V.; Curk, Tine; Mognetti, Bortolo M.; Auzely-Velty, Rachel; Frenkel, Daan; Richter, Ralf P.
      Journal of the American Chemical Society (2014), 136 (5), 1722-1725CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      Despite their importance for material and life sciences, multivalent interactions between polymers and surfaces remain poorly understood. Combining recent achievements of synthetic chem. and surface characterization, we have developed a well-defined and highly specific model system based on host/guest interactions. We use this model to study the binding of hyaluronic acid functionalized with host mols. to tunable surfaces displaying different densities of guest mols. Remarkably, we find that the surface d. of bound polymer increases faster than linearly with the surface d. of binding sites. Based on predictions from a simple anal. model, we propose that this superselective behavior arises from a combination of enthalpic and entropic effects upon binding of nanoobjects to surfaces, accentuated by the ability of polymer chains to interpenetrate.
    4. 4
      Carlson, C. B.; Mowery, P.; Owen, R. M.; Dykhuizen, E. C.; Kiessling, L. L. Selective tumor cell targeting using low-affinity, multivalent interactions. ACS Chem. Biol. 2007, 2, 119– 27,  DOI: 10.1021/cb6003788
      4
      Selective Tumor Cell Targeting Using Low-Affinity, Multivalent Interactions
      Carlson, Coby B.; Mowery, Patricia; Owen, Robert M.; Dykhuizen, Emily C.; Kiessling, Laura L.
      ACS Chemical Biology (2007), 2 (2), 119-127CODEN: ACBCCT; ISSN:1554-8929. (American Chemical Society)
      This report highlights the advantages of low-affinity, multivalent interactions to recognize one cell type over another. Our goal was to devise a strategy to mediate selective killing of tumor cells, which are often distinguished from normal cells by their higher levels of particular cell surface receptors. XTo test whether multivalent interactions could lead to highly specific cell targeting, we used a chem. synthesized small-mol. ligand composed of two distinct motifs: (1) an Arg-Gly-Asp (RGD) peptidomimetic that binds tightly (Kd ≈ 10-9 M) to ανβ3 integrins and (2) the galactosyl-α(1-3)galactose(α-Gal epitope), which is recognized by human anti-α-galactosyl antibodies (anti-Gal). Importantly, anti-Gal binding requires a multivalent presentation of carbohydrate residues; anti-Gal antibodies interact weakly with the monovalent oligosaccharide (Kd ≈ 10-5 M) but bind tightly (Kd ≈ 10-11 M) to multivalent displays of α-Gal epitopes. Such a display is generated when the bifunctional conjugate decorates a cell possessing a high level of ανβ3 integrin; the resulting cell surface, which presents many α-Gal epitopes, can recruit anti-Gal, thereby triggering complement-mediated lysis. Only those cells with high levels of the integrin receptor are killed. In contrast, doxorubicin tethered to the RGD-based ligand affords indiscriminate cell death. These results highlight the advantages of exploiting the type of the multivalent recognition processes used by physiol. systems to discriminate between cells. The selectivity of this strategy is superior to traditional, abiotic, high-affinity targeting methods. Our results have implications for the treatment of cancer and other diseases characterized by the presence of deleterious cells.
    5. 5
      Curk, T.; Dobnikar, J.; Frenkel, D. Design principles for super selectivity using multivalent interactions. In Multivalency: Concepts, Research & Applications; Haag, R.; Huskens, J.; Prins, L.; Ravoo, B. J., Eds.; John Wiley & Sons: Oxford, 2018.
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      Martinez-Veracoechea, F. J.; Frenkel, D. Designing super selectivity in multivalent nano-particle binding. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 10963– 8,  DOI: 10.1073/pnas.1105351108
      6
      Designing super selectivity in multivalent nano-particle binding
      Martinez-Veracoechea, Francisco J.; Frenkel, Daan
      Proceedings of the National Academy of Sciences of the United States of America (2011), 108 (27), 10963-10968, S10963/1-S10963/4CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      A key challenge in nano-science is to design ligand-coated nano-particles that can bind selectively to surfaces that display the cognate receptors above a threshold (surface) concn. Nano-particles that bind monovalently to a target surface do not discriminate sharply between surfaces with high and low receptor coverage. In contrast, "multivalent" nano-particles that can bind to a larger no. of ligands simultaneously, display regimes of "super selectivity" where the fraction of bound particles varies sharply with the receptor concn. We present numerical simulations that show that multivalent nano-particles can be designed such that they approach the 'on-off" binding behavior ideal for receptor-concn. selective targeting. We propose a simple anal. model that accounts for the super selective behavior of multivalent nano-particles. The model shows that the super selectivity is due to the fact that the no. of distinct ligand-receptor binding arrangements increases in a highly nonlinear way with receptor coverage. Somewhat counterintuitively, our study shows that selectivity can be improved by making the individual ligand-receptor bonds weaker. We propose a simple rule of thumb to predict the conditions under which super selectivity can be achieved. We validate our model predictions against the Monte Carlo simulations.
    7. 7
      Richter, R. P.; Lai Kee Him, J.; Tessier, B.; Tessier, C.; Brisson, A. On the kinetics of adsorption and two-dimensional self-assembly of annexin A5 on supported lipid bilayers. Biophys. J. 2005, 89, 3372– 85,  DOI: 10.1529/biophysj.105.064337
      7
      On the kinetics of adsorption and two-dimensional self-assembly of annexin A5 on supported lipid bilayers
      Richter, Ralf P.; Him, Josephine Lai Kee; Tessier, Beatrice; Tessier, Celine; Brisson, Alain R.
      Biophysical Journal (2005), 89 (5), 3372-3385CODEN: BIOJAU; ISSN:0006-3495. (Biophysical Society)
      Annexin A5 is a protein that binds to membranes contg. neg. charged phospholipids in a calcium-dependent manner. We previously found that annexin A5 self-assembles into two-dimensional (2D) crystals on supported lipid bilayers (SLBs) formed on mica while a monolayer of disordered trimers is formed on SLBs on silica. Here, we investigated in detail and correlated the adsorption kinetics of annexin A5 on SLBs, supported on silica and on mica, with the protein's 2D self-assembly behavior. For this study, quartz crystal microbalance with dissipation monitoring and ellipsometry were combined with at. force microscopy. We find, in agreement with previous studies, that the adsorption behavior is strongly dependent on the concn. of dioleoylphosphatidylserine (DOPS) in the SLB and the calcium concn. in soln. The adsorption kinetics of annexin A5 are similar on silica-SLBs and on mica-SLBs, when taking into account the difference in accessible DOPS between silica-SLBs and mica-SLBs. In contrast, 2D crystals of annexin A5 form readily on mica-SLBs, even at low protein coverage (≤10%), whereas they are not found on silica-SLBs, except in a narrow range close to maximal coverage. These results enable us to construct the phase diagram for the membrane binding and the states of 2D organization of annexin A5. The protein binds to the membrane in two different fractions, one reversible and the other irreversible, at a given calcium concn. The adsorption is detd. by the interaction of protein monomers with the membrane. We propose that the local membrane environment, as defined by the presence of DOPS, DOPC, and calcium ions, controls the adsorption and reversibility of protein binding.
    8. 8
      Nores, G. A.; Dohi, T.; Taniguchi, M.; Hakomori, S. Density-dependent recognition of cell surface GM3 by a certain anti-melanoma antibody, and GM3 lactone as a possible immunogen: Requirements for tumor-associated antigen and immunogen. J. Immunol. 1987, 139, 3171– 6
      8
      Density-dependent recognition of cell surface GM3 by a certain anti-melanoma antibody, and GM3 lactone as a possible immunogen: requirements for tumor-associated antigen and immunogen
      Nores, Gustavo A.; Dohi, Taeko; Taniguchi, Masaru; Hakomori, Sen Itiroh
      Journal of Immunology (1987), 139 (9), 3171-6CODEN: JOIMA3; ISSN:0022-1767.
      A murine melanoma-specific monoclonal antibody, M2590, was shown to be directed to ganglioside GM3. Since GM3 is widely distributed in essentially all types of animal cells, there is a conflict with the concept of a tumor-assocd. antigen and immunogen. Studies on the reactivity of M2590 antibody with various cells having different GM3 d. at their cell surface indicated that 1) reactivity of the antibody M2590 depends greatly on the d. of GM3 exposed at the cell surface, on liposomes, or on solid phase; and 2) there is a threshold d. that is recognized by the antibody in all-or-none fashion. In addn., the antibody M2590 reacts not only with GM3 but also with GM3 lactone, and the binding affinity of the antibody to GM3 lactone is strikingly higher than to GM3; however, the antibody does not react with GM3 Et ester. GM3 lactone was detected in melanoma as 3H-labeled GM3 gangliosidol after melanoma cells were directly treated with NaB[3H]4. A comparative immunization of BALB/c mice with GM3 and GM3 lactone showed that GM3 lactone is a much stronger immunogen than GM3, although the antibody elicited reacts with both GM3 and its lactone. Thus, the real immunogen could be GM3 lactone, although it is a minor membrane component.
    9. 9
      English, N. M.; Lesley, J. F.; Hyman, R. Site-specific de-n-glycosylation of CD44 can activate hyaluronan binding, and CD44 activation states show distinct threshold densities for hyaluronan binding. Cancer Res. 1998, 59, 3736– 42
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    10. 10
      Lawrance, W.; Banerji, S.; Day, A. J.; Bhattacharjee, S.; Jackson, D. G. Binding of hyaluronan to the native lymphatic vessel endothelial receptor LYVE-1 is critically dependent on receptor clustering and hyaluronan organization. J. Biol. Chem. 2016, 291, 8014– 30,  DOI: 10.1074/jbc.M115.708305
      10
      Binding of hyaluronan to the native lymphatic vessel endothelial receptor LYVE-1 is critically dependent on receptor clustering and hyaluronan organization
      Lawrance, William; Banerji, Suneale; Day, Anthony J.; Bhattacharjee, Shaumick; Jackson, David G.
      Journal of Biological Chemistry (2016), 291 (15), 8014-8030CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The lymphatic endothelial receptor LYVE-1 has been implicated in both uptake of hyaluronan (HA) from tissue matrix and in facilitating transit of leukocytes and tumor cells through lymphatic vessels based largely on in vitro studies with recombinant receptor in transfected fibroblasts. Curiously, however, LYVE-1 in lymphatic endothelium displays little if any binding to HA in vitro, and this has led to the conclusion that the native receptor is functionally silenced, a feature that is difficult to reconcile with its proposed in vivo functions. Nonetheless, as we reported recently, LYVE-1 can function as a receptor for HA-encapsulated Group A streptococci and mediate lymphatic dissemination in mice. Here we resolve these paradoxical findings and show that the capacity of LYVE-1 to bind HA is strictly dependent on avidity, demanding appropriate receptor self-assocn. and/or HA multimerization. In particular, we demonstrate the prerequisite of a crit. LYVE-1 threshold d. and show that HA binding may be elicited in lymphatic endothelium by surface clustering with divalent LYVE-1 mAbs. In addn., we show that crosslinking of biotinylated HA in streptavidin multimers or supramol. complexes with the inflammation-induced protein TSG-6 enables binding even in the absence of LYVE-1 crosslinking. Finally, we show that endogenous HA on the surface of macrophages can engage LYVE-1, facilitating their adhesion and transit across lymphatic endothelium. These results reveal LYVE-1 as a low affinity receptor tuned to discriminate between different HA configurations through avidity and establish a new mechanistic basis for the functions ascribed to LYVE-1 in matrix HA binding and leukocyte trafficking in vivo.
    11. 11
      Vachieri, S. G.; Xiong, X.; Collins, P. J.; Walker, P. A.; Martin, S. R.; Haire, L. F.; Zhang, Y.; McCauley, J. W.; Gamblin, S. J.; Skehel, J. J. Receptor binding by H10 influenza viruses. Nature 2014, 511, 475– 7,  DOI: 10.1038/nature13443
      11
      Receptor binding by H10 influenza viruses
      Vachieri, Sebastien G.; Xiong, Xiaoli; Collins, Patrick J.; Walker, Philip A.; Martin, Stephen R.; Haire, Lesley F.; Zhang, Ying; McCauley, John W.; Gamblin, Steven J.; Skehel, John J.
      Nature (London, United Kingdom) (2014), 511 (7510), 475-477CODEN: NATUAS; ISSN:0028-0836. (Nature Publishing Group)
      H10N8 follows H7N9 and H5N1 as the latest in a line of avian influenza viruses that cause serious disease in humans and have become a threat to public health. Since Dec. 2013, three human cases of H10N8 infection have been reported, two of whom are known to have died. To gather evidence relating to the epidemic potential of H10 we have detd. the structure of the hemagglutinin of a previously isolated avian H10 virus and we present here results relating esp. to its receptor-binding properties, as these are likely to be major determinants of virus transmissibility. Our results show, first, that the H10 virus possesses high avidity for human receptors and second, from the crystal structure of the complex formed by avian H10 hemagglutinin with human receptor, it is clear that the conformation of the bound receptor has characteristics of both the 1918 H1N1 pandemic virus and the human H7 viruses isolated from patients in 2013 (ref. 3). We conclude that avian H10N8 virus has sufficient avidity for human receptors to account for its infection of humans but that its preference for avian receptors should make avian-receptor-rich human airway mucins an effective block to widespread infection. In terms of surveillance, particular attention will be paid to the detection of mutations in the receptor-binding site of the H10 hemagglutinin that decrease its avidity for avian receptor, and could enable it to be more readily transmitted between humans.
    12. 12
      Overeem, N. J.; Hamming, P. H. E.; Grant, O. C.; Di Iorio, D.; Tieke, M.; Bertolino, M. C.; Li, Z.; Vos, G.; de Vries, R. P.; Woods, R. J.; Tito, N. B.; Boons, G. P. H.; van der Vries, E.; Huskens, J. Hierarchical multivalent effects control influenza host specificity. ACS Cent Sci. 2020, 6, 2311– 8,  DOI: 10.1021/acscentsci.0c01175
      12
      Hierarchical Multivalent Effects Control Influenza Host Specificity
      Overeem, Nico J.; Hamming, P. H. Erik; Grant, Oliver C.; Di Iorio, Daniele; Tieke, Malte; Bertolino, M. Candelaria; Li, Zeshi; Vos, Gael; de Vries, Robert P.; Woods, Robert J.; Tito, Nicholas B.; Boons, Geert-Jan P. H.; van der Vries, Erhard; Huskens, Jurriaan
      ACS Central Science (2020), 6 (12), 2311-2318CODEN: ACSCII; ISSN:2374-7951. (American Chemical Society)
      Understanding how emerging influenza viruses recognize host cells is crit. in evaluating their zoonotic potential, pathogenicity, and transmissibility between humans. The surface of the influenza virus is covered with hemagglutinin (HA) proteins that can form multiple interactions with sialic acid-terminated glycans on the host cell surface. This multivalent binding affects the selectivity of the virus in ways that cannot be predicted from the individual receptor-ligand interactions alone. Here, the intrinsic structural and energetic differences between the interactions of avian- or human-type receptors with influenza HA translate from individual site affinity and orientation through receptor length and d. on the surface into virus avidity and specificity. The authors introduce a method to measure virus avidity using receptor d. gradients. Influenza viruses attached stably to a surface at receptor densities that correspond to a min. no. of ∼8 HA-glycan interactions, but more interactions were required if the receptors were short and human-type. Thus, the avidity and specificity of influenza viruses for a host cell depend not on the sialic acid linkage alone but on a combination of linkage and the length and d. of receptors on the cell surface. The authors' findings suggest that threshold receptor densities play a key role in virus tropism, which is a predicting factor for both their virulence and zoonotic potential. Virus binding to receptor d. gradients at cell surface mimics shows that the avidity and selectivity of influenza A virus are governed by receptor structure and d.
    13. 13
      Overeem, N. J.; Hamming, P. H. E.; Tieke, M.; van der Vries, E.; Huskens, J. Multivalent affinity profiling: Direct visualization of the superselective binding of influenza viruses. ACS Nano 2021, 15, 8525– 36,  DOI: 10.1021/acsnano.1c00166
      13
      Multivalent Affinity Profiling: Direct Visualization of the Superselective Binding of Influenza Viruses
      Overeem, Nico J.; Hamming, P. H.; Tieke, Malte; van der Vries, Erhard; Huskens, Jurriaan
      ACS Nano (2021), 15 (5), 8525-8536CODEN: ANCAC3; ISSN:1936-0851. (American Chemical Society)
      The influenza A virus (IAV) interacts with the glycocalyx of host cells through its surface proteins hemagglutinin (HA) and neuraminidase (NA). Quant. biophys. measurements of these interactions may help to understand these interactions at the mol. level with the long-term aim to predict influenza infectivity and answer other biol. questions. We developed a method, called multivalent affinity profiling (MAP), to measure virus binding profiles on receptor d. gradients to det. the threshold receptor d., which is a quant. measure of virus avidity toward a receptor. Here, we show that imaging of IAVs on receptor d. gradients allows the direct visualization and efficient assessment of their superselective binding. We show how the multivalent binding of IAVs can be quant. assessed using MAP if the receptor d. gradients are prepd. around the threshold receptor d. without crowding at the higher densities. The threshold receptor d. increases strongly with increasing flow rate, showing that the superselective binding of IAV is influenced by shear force. This method of visualization and quant. assessment of superselective binding allows not only comparative studies of IAV-receptor interactions, but also more fundamental studies of how superselectivity arises and is influenced by exptl. conditions.
    14. 14
      Scheepers, M. R. W.; van Ijzendoorn, L. J.; Prins, M. W. J. Multivalent weak interactions enhance selectivity of interparticle binding. Proc. Natl. Acad. Sci. U. S. A. 2020, 117, 22690– 7,  DOI: 10.1073/pnas.2003968117
      14
      Multivalent weak interactions enhance selectivity of interparticle binding
      Scheepers, M. R. W.; van IJzendoorn, L. J.; Prins, M. W. J.
      Proceedings of the National Academy of Sciences of the United States of America (2020), 117 (37), 22690-22697CODEN: PNASA6; ISSN:0027-8424. (National Academy of Sciences)
      Targeted drug delivery critically depends on the binding selectivity of cargo-transporting colloidal particles. Extensive theor. work has shown that two factors are necessary to achieve high selectivity for a threshold receptor d.: multivalency and weak interactions. Here, we study a model system of DNA-coated particles with multivalent and weak interactions that mimics ligand-receptor interactions between particles and cells. Using an optomagnetic cluster expt., particle aggregation rates are measured as a function of ligand and receptor densities. The measured aggregation rates show that the binding becomes more selective for shorter DNA ligand-receptor pairs, proving that multivalent weak interactions lead to enhanced selectivity in interparticle binding. Simulations confirm the exptl. findings and show the role of ligand-receptor dissocn. in the selectivity of the weak multivalent binding.
    15. 15
      Linne, C.; Visco, D.; Angioletti-Uberti, S.; Laan, L.; Kraft, D. J. Direct visualization of superselective colloid-surface binding mediated by multivalent interactions. Proc. Natl. Acad. Sci. U. S. A. 2021, 118, e210603611  DOI: 10.1073/pnas.2106036118
      There is no corresponding record for this reference.
    16. 16
      Schroeder, A.; Heller, D. A.; Winslow, M. M.; Dahlman, J. E.; Pratt, G. W.; Langer, R.; Jacks, T.; Anderson, D. G. Treating metastatic cancer with nanotechnology. Nat. Rev. Cancer 2012, 12, 39– 50,  DOI: 10.1038/nrc3180
      16
      Treating metastatic cancer with nanotechnology
      Schroeder, Avi; Heller, Daniel A.; Winslow, Monte M.; Dahlman, James E.; Pratt, George W.; Langer, Robert; Jacks, Tyler; Anderson, Daniel G.
      Nature Reviews Cancer (2012), 12 (1), 39-50CODEN: NRCAC4; ISSN:1474-175X. (Nature Publishing Group)
      A review. Metastasis accounts for the vast majority of cancer deaths. The unique challenges for treating metastases include their small size, high multiplicity and dispersion to diverse organ environments. Nanoparticles have many potential benefits for diagnosing and treating metastatic cancer, including the ability to transport complex mol. cargoes to the major sites of metastasis, such as the lungs, liver and lymph nodes, as well as targeting to specific cell populations within these organs. This Review highlights the research, opportunities and challenges for integrating engineering sciences with cancer biol. and medicine to develop nanotechnol.-based tools for treating metastatic disease.
    17. 17
      Dubacheva, G. V.; Curk, T.; Frenkel, D.; Richter, R. P. Multivalent recognition at fluid surfaces: The interplay of receptor clustering and superselectivity. J. Am. Chem. Soc. 2019, 141, 2577– 88,  DOI: 10.1021/jacs.8b12553
      17
      Multivalent recognition at fluid surfaces: The interplay of receptor clustering and superselectivity
      Dubacheva, Galina V.; Curk, Tine; Frenkel, Daan; Richter, Ralf P.
      Journal of the American Chemical Society (2019), 141 (6), 2577-2588CODEN: JACSAT; ISSN:0002-7863. (American Chemical Society)
      The interaction between a biol. membrane and its environment is a complex process, as it involves multivalent binding between ligand/receptor pairs, which can self-organize in patches. Any description of the specific binding of biomols. to membranes must account for the key characteristics of multivalent binding, namely, its unique ability to discriminate sharply between high and low receptor densities (superselectivity), but also for the effect of the lateral mobility of membrane-bound receptors to cluster upon binding. Here, we present an exptl. model system that allows us to compare systematically the effects of multivalent interactions on fluid and immobile surfaces. A crucial feature of our model system is that it allows us to control the membrane surface chem., the properties of the multivalent binder, and the binding affinity. We found that multivalent probes retained their superselective binding behavior at fluid interfaces. Supported by numerical simulations, we demonstrated that, as a consequence of receptor clustering, superselective binding was enhanced and shifted to lower receptor densities at fluid interfaces. To translate our findings into a simple, predictive tool, we propose an anal. model that enables rapid predictions of how the superselective binding behavior is affected by the lateral receptor mobility as a function of the physicochem. characteristics of the multivalent probe. We believe that our model, which captures the key phys. mechanisms underpinning multivalent binding to biol. membranes, will greatly facilitate the rational design of nanoprobes for the superselective targeting of cells.
    18. 18
      Jeppesen, B.; Smith, C.; Gibson, D. F.; Tait, J. F. Entropic and enthalpic contributions to annexin V-membrane binding: A comprehensive quantitative model. J. Biol. Chem. 2008, 283, 6126– 35,  DOI: 10.1074/jbc.M707637200
      18
      Entropic and Enthalpic Contributions to Annexin V-Membrane Binding: A Comprehensive Quantitative Model
      Jeppesen, Brian; Smith, Christina; Gibson, Donald F.; Tait, Jonathan F.
      Journal of Biological Chemistry (2008), 283 (10), 6126-6135CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      Annexin V binds to membranes with very high affinity, but the factors responsible remain to be quant. elucidated. Anal. by isothermal microcalorimetry and calcium titrn. under conditions of low membrane occupancy showed that there was a strongly pos. entropy change upon binding. For vesicles contg. 25% phosphatidylserine at 0.15 M ionic strength, the free energy of binding was -53 kcal/mol protein, whereas the enthalpy of binding was -38 kcal/mol. Addn. of 4 M urea decreased the free energy of binding by about 30% without denaturing the protein, suggesting that hydrophobic forces make a significant contribution to binding affinity. This was confirmed by mutagenesis studies that showed that binding affinity was modulated by the hydrophobicity of surface residues that are likely to enter the interfacial region upon protein-membrane binding. The change in free energy was quant. consistent with predictions from the Wimley-White scale of interfacial hydrophobicity. In contrast, binding affinity was not increased by making the protein surface more pos. charged, nor decreased by making it more neg. charged, ruling out general ionic interactions as major contributors to binding affinity. The affinity of annexin V was the same regardless of the head group present on the anionic phospholipids tested (phosphatidylserine, phosphatidylglycerol, phosphatidylmethanol, and cardiolipin), ruling out specific interactions between the protein and non-phosphate moieties of the head group as a significant contributor to binding affinity. Anal. by fluorescence resonance energy transfer showed that multimers did not form on phosphatidylserine membranes at low occupancy, indicating that annexin-annexin interactions did not contribute to binding affinity. In summary, binding of annexin V to membranes is driven by both enthalpic and entropic forces. Dehydration of hydrophobic regions of the protein surface as they enter the interfacial region makes an important contribution to overall binding affinity, supplementing the role of protein-calcium-phosphate chelates.
    19. 19
      Lesley, J.; Hascall, V. C.; Tammi, M.; Hyman, R. Hyaluronan binding by cell surface CD44. J. Biol. Chem. 2000, 275, 26967– 75,  DOI: 10.1016/S0021-9258(19)61467-5
      19
      Hyaluronan binding by cell surface CD44
      Lesley, Jayne; Hascall, Vincent C.; Tammi, Markku; Hyman, Robert
      Journal of Biological Chemistry (2000), 275 (35), 26967-26975CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      CD44 is the primary cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan. Here the authors detd. the relative avidities of unlabeled hyaluronan prepns. for cell surface CD44 by their ability to block the binding of fluorescein-conjugated hyaluronan to a variety of cells. The authors show that hyaluronan binding at the cell surface is a complex interplay of multivalent binding events affected by the size of the multivalent hyaluronan ligand, the quantity and d. of cell surface CD44, and the activation state of CD44 as detd. by cell-specific factors and/or treatment with CD44-specific monoclonal antibody (mAb). Using low Mr hyaluronan oligomers of defined sizes, the authors obsd. monovalent binding between 6 and 18 sugars. At ∼20 to ∼38 sugars, there was an increase in avidity (∼3x), suggesting that divalent binding was occurring. In the presence of the inducing mAb IRAWB14, monovalent binding avidity was similar to that of noninduced CD44, but beginning at ∼20 residues, there was a dramatic and progressive increase in avidity with increasing oligomer size (∼22 < 26 < 30 < 34 < 38 sugars). Kinetic studies of binding and dissocn. of fluorescein-conjugated hyaluronan indicated that inducing mAb treatment had little effect on the binding kinetics, but dissocn. from the cell surface was greatly delayed by inducing mAb.
    20. 20
      Ercolani, G.; Schiaffino, L. Allosteric, chelate, and interannular cooperativity: A mise au point. Angew. Chem., Int. Ed. Engl. 2011, 50, 1762– 8,  DOI: 10.1002/anie.201004201
      20
      Allosteric, chelate, and interannular cooperativity: a mise au point
      Ercolani Gianfranco; Schiaffino Luca
      Angewandte Chemie (International ed. in English) (2011), 50 (8), 1762-8 ISSN:.
      There is no expanded citation for this reference.
    21. 21
      Huber, R.; Berendes, R.; Burger, A.; Schneider, M.; Karshikov, A.; Luecke, H.; Romisch, J.; Paques, E. Crystal and molecular structure of human annexin V after refinement. Implications for structure, membrane binding and ion channel formation of the annexin family of proteins. J. Mol. Biol. 1992, 223, 683– 704,  DOI: 10.1016/0022-2836(92)90984-R
      21
      Crystal and molecular structure of human annexin V after refinement. Implications for structure, membrane binding and ion channel formation of the annexin family of proteins
      Huber, Robert; Berendes, Robert; Burger, Alexander; Schneider, Monika; Karshikov, Andrej; Luecke, Hartmut; Roemisch, Juergen; Paques, Eric
      Journal of Molecular Biology (1992), 223 (3), 683-704CODEN: JMOBAK; ISSN:0022-2836.
      Two crystal forms (P63 and R3) of human annexin V have been crystallog. refined at 2.3 and 2.0 Å resoln. to R-values of 0.184 and 0.174, resp., applying very tight stereochem. restraints with deviations from ideal geometry of 0.01 Å and 2°. The three independent mols. (2 in P63, 1 in R3) are similar, with deviations in Cα positions of 0.6 Å. The polypeptide chain of 320 amino acid residues is folded into a planar cyclic arrangement of four repeats. The repeats have similar structures of five α-helical segments wound into a right-handed compact superhelix. Three calcium ion sites in repeats I, II and IV and two lanthanum ion sites in repeat I have been found in the R3 crystals. They are located at the convex face of the mol. opposite the N terminus. Repeat III has a different conformation at this site and no calcium bound. The calcium sites are similar to the phospholinase A2 calcium-binding site, suggesting analogy also in phospholipid interaction. The center of the mol. is formed by a channel of polar charged residues and also harbors a chain of ordered water mols. conserved in the different crystal forms. Comparison with amino acid sequences of other annexins shows a high degree of similarity between them. Long insertions are found only at the N termini. Most conserved are the residues forming the metal-binding sites and the polar channel. Annexins V and VII form voltage-gated calcium ion channels when bound to membranes in vitro. It is suggested that annexins bind with their convex face to membranes, causing local disorder and permeability of the phospholipid bilayers. Annexins are Janus-faced proteins that face phospholipid and water and mediate calcium transport.
    22. 22
      Bouter, A.; Gounou, C.; Berat, R.; Tan, S.; Gallois, B.; Granier, T.; d’Estaintot, B. L.; Poschl, E.; Brachvogel, B.; Brisson, A. R. Annexin-A5 assembled into two-dimensional arrays promotes cell membrane repair. Nat. Commun. 2011, 2, 270,  DOI: 10.1038/ncomms1270
      22
      Annexin-A5 assembled into two-dimensional arrays promotes cell membrane repair
      Bouter Anthony; Gounou Celine; Berat Remi; Tan Sisareuth; Gallois Bernard; Granier Thierry; d'Estaintot Beatrice Langlois; Poschl Ernst; Brachvogel Bent; Brisson Alain R
      Nature communications (2011), 2 (), 270 ISSN:.
      Eukaryotic cells possess a universal repair machinery that ensures rapid resealing of plasma membrane disruptions. Before resealing, the torn membrane is submitted to considerable tension, which functions to expand the disruption. Here we show that annexin-A5 (AnxA5), a protein that self-assembles into two-dimensional (2D) arrays on membranes upon Ca(2+) activation, promotes membrane repair. Compared with wild-type mouse perivascular cells, AnxA5-null cells exhibit a severe membrane repair defect. Membrane repair in AnxA5-null cells is rescued by addition of AnxA5, which binds exclusively to disrupted membrane areas. In contrast, an AnxA5 mutant that lacks the ability of forming 2D arrays is unable to promote membrane repair. We propose that AnxA5 participates in a previously unrecognized step of the membrane repair process: triggered by the local influx of Ca(2+), AnxA5 proteins bind to torn membrane edges and form a 2D array, which prevents wound expansion and promotes membrane resealing.
    23. 23
      Perschl, A.; Lesley, J.; English, N.; Trowbridge, I.; Hyman, R. Role of CD44 cytoplasmic domain in hyaluronan binding. Eur. J. Immunol. 1995, 25, 495– 501,  DOI: 10.1002/eji.1830250228
      23
      Role of CD44 cytoplasmic domain in hyaluronan binding
      Perschl, Astrid; Lesley, Jayne; English, Nicole; Trowbridge, Ian; Hyman, Robert
      European Journal of Immunology (1995), 25 (2), 495-501CODEN: EJIMAF; ISSN:0014-2980. (VCH)
      The hyaluronan (HA) binding activity of mutant CD44 constructs expressed in AKR1 T-lymphoma cells was evaluated by flow cytometry using fluorescein-conjugated HA (Fl-HA). Previous studies showed that wild-type hematopoietic CD44 bound Fl-HA when expressed in AKR1, but that truncated "tailless" CD44, lacking all but six amino acids of the cytoplasmic domain, did not bind. Here, we show that a disulfide-bonded dimer of CD44, formed by substituting the transmembrane region of CD3ζ chain for that of CD44, binds Fl-HA, even when the cytoplasmic domain of the CD44 dimer is absent. We conclude that dimerization of CD44 abrogates the requirement for the cytoplasmic domain, suggesting that the cytoplasmic domain of CD44 may contribute to HA binding by promoting CD44 clustering. These results suggest that changes in the distribution of CD44 on the cell surface, induced by mol. interactions either from within the cell or from outside, may regulate its role as a receptor. Further studies sought to localize the region of the CD44 cytoplasmic domain contributing to HA binding by the construction of a series of cytoplasmic domain truncation mutants and internal deletion mutants. All of the mutant CD44 mols. bound Fl-HA similarly to wild-type CD44. Thus, it was not possible to assign the function mediating HA binding to a specific region of the cytoplasmic domain, suggesting either that multiple regions of the cytoplasmic domain can promote enhancement of HA binding, or that the role of the cytoplasmic domain in mediating this function does not require a specific amino acid sequence.
    24. 24
      Banerji, S.; Wright, A. J.; Noble, M.; Mahoney, D. J.; Campbell, I. D.; Day, A. J.; Jackson, D. G. Structures of the CD44–hyaluronan complex provide insight into a fundamental carbohydrateprotein interaction. Nat. Struct Mol. Biol. 2007, 14, 234– 9,  DOI: 10.1038/nsmb1201
      24
      Structures of the Cd44-hyaluronan complex provide insight into a fundamental carbohydrate-protein interaction
      Banerji, Suneale; Wright, Alan J.; Noble, Martin; Mahoney, David J.; Campbell, Iain D.; Day, Anthony J.; Jackson, David G.
      Nature Structural & Molecular Biology (2007), 14 (3), 234-239CODEN: NSMBCU; ISSN:1545-9993. (Nature Publishing Group)
      Regulation of transient interactions between cells and the ubiquitous matrix glycosaminoglycan hyaluronan is crucial to such fundamental processes as embryonic development and leukocyte homing. Cd44, the primary cell surface receptor for hyaluronan, binds ligand via a lectin-like fold termed the Link module, but only after appropriate functional activation. The mol. details of the Cd44-hyaluronan interaction and hence the structural basis for this activation are unknown. Here we present the first crystal structure of Cd44 complexed with hyaluronan. This reveals that the interaction with hyaluronan is dominated by shape and hydrogen-bonding complementarity and identifies two conformational forms of the receptor that differ in orientation of a crucial hyaluronan-binding residue (Arg45, equiv. to Arg41 in human CD44). Measurements by NMR indicate that the conformational transition can be induced by hyaluronan binding, providing further insight into possible mechanisms for regulation of Cd44.
    25. 25
      Takahashi, R.; Al-Assaf, S.; Williams, P. A.; Kubota, K.; Okamoto, A.; Nishinari, K. Asymmetrical-flow field-flow fractionation with on-line multiangle light scattering detection. 1. Application to wormlike chain analysis of weakly stiff polymer chains. Biomacromolecules 2003, 4, 404– 9,  DOI: 10.1021/bm025706v
      25
      Asymmetrical-Flow Field-Flow Fractionation with On-Line Multiangle Light Scattering Detection. 1. Application to Wormlike Chain Analysis of Weakly Stiff Polymer Chains
      Takahashi, Rheo; Al-Assaf, Saphwan; Williams, Peter A.; Kubota, Kenji; Okamoto, Akio; Nishinari, Katsuyoshi
      Biomacromolecules (2003), 4 (2), 404-409CODEN: BOMAF6; ISSN:1525-7797. (American Chemical Society)
      Four samples of hyaluronan in the sodium form, ranging in wt.-av. mol. wt., Mw, from 6.67 × 105 to 4.23 × 106 were investigated by asym.-flow field-flow fractionation coupled to multiangle light scattering (FlFFF-MALS) in 0.2 M aq. NaCl at 25 °C. Mw and z-av. radii of gyration, RGz, obtained via FlFFF-MALS showed a good agreement with the results obtained by conventional static light scattering. Furthermore, the mol. wt. dependence of the radius of gyration for sodium hyaluronan obtained via FlFFF-MALS was analyzed on the basis of the Kratky-Porod model for unperturbed wormlike chains combined with the Yamakawa theory for radius expansion factor, and a sufficiently good agreement was obsd. between the theor. prediction and exptl. data. These results show the potential usage of FlFFF-MALS regarding size sepn. and mol. characterization even for weakly stiff chains.
    26. 26
      Banerji, S.; Hide, B. R. S.; James, J. R.; Noble, M. E. M.; Jackson, D. G. Distinctive properties of the hyaluronan binding domain in the lymphatic endothelial receptor LYVE-1 and their implications for receptor function. J. Biol. Chem. 2010, 285, 10724– 35,  DOI: 10.1074/jbc.M109.047647
      26
      Distinctive Properties of the Hyaluronan-binding Domain in the Lymphatic Endothelial Receptor Lyve-1 and Their Implications for Receptor Function
      Banerji, Suneale; Hide, Branwen R. S.; James, John R.; Noble, Martin E. M.; Jackson, David G.
      Journal of Biological Chemistry (2010), 285 (14), 10724-10735CODEN: JBCHA3; ISSN:0021-9258. (American Society for Biochemistry and Molecular Biology)
      The lymphatic endothelial hyaluronan (HA) receptor Lyve-1 is a member of the Link protein superfamily most similar to the leukocyte HA receptor CD44. However, the structure of Lyve-1 and the nature of its interaction with ligand are obscure. Here we present new evidence that Lyve-1 is functionally distinct from CD44. Using truncation mutagenesis we confirm that Lyve-1 in common with CD44 contains an extended HA-binding unit, comprising elements flanking the N and C termini of the consensus lectin-like Link module, bridged by a third conserved disulfide linkage that is crit. for HA binding. In addn., we identify six essential residues Tyr-87, Ile-97, Arg-99, Asn-103, Lys-105, and Lys-108 that define a compact HA-binding surface on Lyve-1, encompassing the epitope for an adhesion-blocking monoclonal antibody 3A, in an analogous position to the HA-binding surface in CD44. The overtly electrostatic character of HA binding in Lyve-1 and its sensitivity to ionic strength (IC50 of 150 mM NaCl) contrast markedly with CD44 (IC50 > 2 M NaCl) in which HA binding is mediated by hydrogen bonding and hydrophobic interactions. In addn., unlike the extended Link module in CD44, which binds HA efficiently when expressed as a sol. monomer (Kd = 65.7 μM), that of Lyve-1 requires artificial dimerization, although the full ectodomain is active as a monomer (Kd = 35.6 μM). Finally, full-length Lyve-1 did not form stable dimers in binding-competent 293T transfectants when assessed using bioluminescent resonance energy transfer. These results reveal that elements addnl. to the extended Link module are required to stabilize HA binding in Lyve-1 and indicate important structural and functional differences with CD44.
    27. 27
      Cyphert, J. M.; Trempus, C. S.; Garantziotis, S. Size matters: Molecular weight specificity of hyaluronan effects in cell biology. Int. J. Cell Biol. 2015, 2015, 563818,  DOI: 10.1155/2015/563818
      27
      Size Matters: Molecular Weight Specificity of Hyaluronan Effects in Cell Biology
      Cyphert Jaime M; Trempus Carol S; Garantziotis Stavros
      International journal of cell biology (2015), 2015 (), 563818 ISSN:1687-8876.
      Hyaluronan signaling properties are unique among other biologically active molecules, that they are apparently not influenced by postsynthetic molecular modification, but by hyaluronan fragment size. This review summarizes the current knowledge about the generation of hyaluronan fragments of different size and size-dependent differences in hyaluronan signaling as well as their downstream biological effects.
    28. 28
      Tito, N. B. Multivalent ″attacker and guard″ strategy for targeting surfaces with low receptor density. J. Chem. Phys. 2019, 150, 184907,  DOI: 10.1063/1.5086277
      28
      Multivalent "attacker and guard" strategy for targeting surfaces with low receptor density
      Tito, Nicholas B.
      Journal of Chemical Physics (2019), 150 (18), 184907/1-184907/16CODEN: JCPSA6; ISSN:0021-9606. (American Institute of Physics)
      Multivalent particles, i.e., microscopic constructs having multiple ligands, can be used to target surfaces selectively depending on their receptor d. Typically, there is a sharp onset of multivalent binding as the receptor d. exceeds a given threshold. However, the opposite case, selectively binding to surfaces with a receptor d. below a given threshold, is much harder. Here, the authors present a simple strategy for selectively targeting a surface with a low d. of receptors, within a system also having a surface with a higher d. of the same receptors. The authors' strategy exploits competitive adsorption of two species. The first species, called "guards," are receptor-sized monovalent particles designed to occupy the high-d. surface at equil., while the second multivalent "attacker" species outcompetes the guards for binding onto the low-d. surface. Surprisingly, the recipe for attackers and guards yields more selective binding with stronger ligand-receptor assocn. consts., in contrast to std. multivalency. The authors derive explicit expressions for the attacker and guard mol. design parameters and concns., optimized within bounds of what is exptl. accessible, thereby facilitating implementation of the proposed approach. (c) 2019 American Institute of Physics.
    29. 29
      Berg, J. M.; Tymoczko, J. L.; Gatto, G. J., Jr.; Stryer, L. Biochemistry; Macmillan Learning, 2019.
      There is no corresponding record for this reference.

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    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.2c06942.

    • Theoretical derivations, details of the AnxA5-to-membrane binding experiments and binding model, and details of the HA-to-CD44 binding analysis (PDF)


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