Pulse Dipolar EPR Reveals Double-Histidine Motif Spin-labelling is Robust Against Competitor Ions

. Pulse-dipolar EPR is an appealing strategy for structural characterization of complex systems in solution that complements other biophysical techniques. Significantly, the emergence of genetically encoded self-assembling spin labels exploiting exogenously introduced double-histidine motifs in conjunction with Cu II -chelates offers high precision distance determination in systems non-permissive to thiol-directed spin labelling. However, the non-covalency of this interaction exposes potential vulnerabilities to competition from adventitious divalent metal ions, and pH sensitivity. Herein, a combination of room-temperature isothermal titration calorimetry (ITC) and cryogenic relaxation-induced dipolar modulation enhancement (RIDME) measurements are applied to the model protein Streptococcus sp. group G. protein G, B1 domain (GB1). Results demonstrate double-histidine motif spin labelling using Cu II nitrilotriacetic acid (Cu II -NTA) is robust against the competitor ligand Zn II -NTA at >1000-fold molar excess, and high nM binding affinity is surprisingly retained under acidic and basic conditions even though room temperature affinity shows a stronger pH dependence. This indicates the strategy is well-suited for diverse biological applications, particularly metalloproteins with divalent metal ion cofactors.

As the complexity of biomolecular assemblies implicated in health and disease has increased, so too has interest in pulse-dipolar EPR (PD-EPR) as a robust strategy for solution-state structural characterization of proteins 1,2 and nucleic acids 3,4 in the nanometer distance regime. 5,6 PD-EPR is a powerful tool that complements X-ray crystallography, NMR, cryo-EM and Förster Resonance Energy Transfer (FRET) data by providing structural insight outwith crystallization, sizelimitation, or structurally perturbative labels. Hence, PD-EPR has been applied to study conformational equilibria, 7 oligomerization degree, 8,9 complexation events, [10][11][12] and competing structural models. 13 Pairs of paramagnetic moieties are commonly introduced into diamagnetic systems of interest using thiol-based site-directed spin labelling. 14 Cysteine residues are typically covalently modified, as for the nitroxide R1 sidechain (figure 1a top). This strategy is suboptimal in systems containing essential cysteine residues, non-permissive to post-translational reduction. However, Cu II -based genetically-encodable self-assembling spin labels using double-histidine motifs have emerged as an alternative labelling strategy. 15,16 Additionally, the bipedal mode of Cu II -chelate attachment at the double-histidine motif (figure 1a bottom) results in significantly improved precision and accuracy in the distance domain. Cu II -nitrilotriacetic acid (Cu II -NTA) spin labelling of double histidine motifs for PD-EPR has been applied successfully to enzymes, 17 metalloproteins, 18 and nucleoprotein complexes. 19 Despite this success, optimization of the spin labelling approach is non-trivial, because the noncovalency of the interaction predisposes sensitivity to variations in binding affinity. For instance, different buffer conditions influence the double-histidine motif labelling efficiency with Cu II -NTA. 20 Furthermore, while the influence of pH upon formation of Cu II -chelates has been characterized by CW-EPR previously, 21  Measurements were first performed in presence of the model competitor ligand, Zn II -NTA, which was chosen because i) it is a weak ligand for double-histidine motifs compared to Cu II -NTA, and ii) it is diamagnetic, so does not contribute to the detected EPR signal. An EPR silent competitor ligand is desirable because analysis of pseudo-titration data is simplified (see SI section 1.6). Room temperature ITC data (figure 2a) fitted to a one-site model where binding stoichiometry could vary, indicated a binding affinity of 513 μM. Using the determined enthalpy change (ΔH), the binding affinity was extrapolated to 235 K (i.e., the temperature at which the binding equilibrium is found to freeze out in our samples, such that diffusional processes cease, meaning our EPR data reflects equilibria at 235 K), 22  Colour bars represent reliability ranges (green: shape reliable; yellow: mean and width reliable; orange: mean reliable; red: no quantification possible). d) A univariate fit of the competitor dissociation constant (32 μM) is shown in solid black. Experimental points are shown as the blue scatter, and 95% confidence intervals are shown as the red error bars.
The corresponding RIDME pseudo-titration was performed at 1 μM protein concentration in presence of 10 μM Cu II -NTA (to ensure quantitative loading and negligible ligand depletion 24 (see SI section 1.6)) and varying Zn II -NTA concentrations. Importantly, the dipolar evolution functions (figure 2b) and distance distributions (figure 2c) show that in all cases, the expected 7 peak at ~2.5 nm is retrieved as the only significant feature following data validation. The fitted competitor KD value (32 μM) is within 2-fold of that determined from ITC when extrapolated to 235 K (48 μM) (figure 2d). This suggests that Cu II -NTA is robust against adventitious divalent metals in vast excesses, >1000-fold, even at low μM protein concentrations. Additionally, this benchmarks quantitation of Cu II -nitroxide RIDME modulation depths for remotely determining binding affinities of EPR silent ligands, in a competition assay format.
Next, the influence of pH upon double-histidine motif loading efficiency with Cu II -NTA was investigated by measuring ITC and RIDME at pH 6.4. Since only deprotonated histidine residues can coordinate Cu II -NTA, it follows that binding affinity should decrease under acidic conditions. Indeed, room-temperature ITC performed at pH 5, below the approximate pKA of solvent-exposed histidine, 25 shows negligible binding (see SI section 2.3), and measurements at pH 6.4, fitted to a one-site model, indicated a 20-fold reduction in affinity compared to previous work 23 (figure 3a). Extrapolating ΔH to 235 K suggested a binding affinity of ~4 μM. A RIDME pseudo-titration was performed at 5 μM protein concentration to validate the roomtemperature ITC prediction of reduced affinity under PD-EPR conditions. Significantly, the dipolar evolution functions (figure 3b) show Cu II -NTA binding is only marginally reduced at lower pH, with one equivalent Cu II -NTA saturating ~70% of available double-histidine motifs.
This is further borne out by the fitted dissociation constant (figure 3d), 0.31 μM compared to 0.14 μM in previous work at pH 7.4. 23 The affinity reduced by only 2-fold, indicating that the 9 influence of pH upon double-histidine motif loading may be attenuated at lower temperatures. A possible explanation is that histidine protonation is endothermic, 26 driving the equilibrium towards the deprotonated state at lower temperatures, compensating for reduced pH and facilitating double-histidine loading. Importantly, this would also imply significantly tighter binding at higher pH, where histidine deprotonation is already favored. To clarify the disparity between ITC and PD-EPR data at pH 6.4, room-temperature ITC was also performed at pH 8.4 (figure 4a), fitted to a one-site model, where a 20-fold increase in affinity was predicted (via improved thermodynamic favorability of binding) compared to previous work. Another RIDME pseudo-titration was performed at 2 μM protein concentration, with dipolar evolution functions (figure 4b) suggesting modest improvement in binding affinity.
The fitted dissociation constant (figure 4d) of 0.091 μM indicates binding affinity is approximately 2-fold higher than at pH 7.4, consistent with observation at pH 6.4 that the influence of pH upon binding affinity is attenuated with decreasing temperature. While an endothermic protonation process would suggest much tighter binding is to be anticipated at pH 8.4, consider that at this pH <1% of histidine -nitrogen atoms should remain protonated. This may explain why the relative increase in binding affinity is smaller than expected, since the deprotonation is already driven toward completion by the high pH.
While the data suggests that spin-labelling and measurement at pH 8.4 will afford enhanced loading and sensitivity, it should be noted that the stoichiometry of binding is ~2, compared to ~1 at pH 6.4. This may arise from deprotonation of the protein surface that promotes nonspecific binding. This would explain the increased exothermic nature of the binding, if nonspecific or additional binding events contributed to the isotherm and would further inflate the binding affinity when extrapolated to cryogenic temperatures. However, the corresponding distance distributions (figure 4c) do not contain additional peaks to support this hypothesis.
Perhaps most significantly, these results clearly show that Cu II -NTA binding affinity for doublehistidine motifs is not strongly perturbed from the high nM concentration regime by fluctuations of pH between 6.4-8.4. Coupled with measurements in presence of competitor ligand Zn II -NTA, findings support that Cu II -NTA is a highly robust spin label when combined with α-helical double-histidine motifs. This is encouraging for the widespread application of double-histidine motifs in metalloproteins, or in systems where divalent metal cofactors are necessary.
Additionally, the benchmarking of a competition assay using PD-EPR is particularly exciting because it allows remote detection of binding interactions with diamagnetic ligands. This will be promising in cases where paramagnetic ligand analogues are not available or cause structural perturbation. PD-EPR also has greater sensitivity than ITC and the coupling of thermodynamic and structural information allows for the facile monitoring of non-specific and competitor ligand interactions. 27 Traditionally, monitoring competitive ligand binding has required expensive radio-labelling and judicious selection of appropriate isotopes. 24,28 PD-EPR may complement these strategies, while obviating potential cost and safety considerations.
ASSOCIATED CONTENT Supporting Information.
The following files are available free of charge.

Notes
The authors declare no competing financial interests.