Modulating Liquid–Liquid Phase Separation of Nck Adaptor Protein against Enteropathogenic Escherichia coli Infection

Signaling proteins often form biomolecular condensates through liquid–liquid phase separation (LLPS) during intracellular signal transduction. Modulating the LLPS property of intracellular protein condensates will redirect intracellular signals and provide a potential way to regulate cellular physiology. Phosphorylation of multiple tyrosine residues of the transmembrane receptor nephrin is known to drive the LLPS of the adaptor protein Nck and neuronal Wiskott–Aldrich Syndrome protein (N-WASP) and form the Nck signaling complex. Phosphorylation of the translocated intimin receptor (Tir) in the host cell may recruit this enteropathogenic Escherichia coli (EPEC) virulence factor to the Nck signaling complex and lead to the entry of EPEC into the intestine cell. In this work, we first identified a phosphotyrosine (pY)-containing peptide 3pY based on the sequence similarity of nephrin and Tir; 3pY promoted the LLPS of Nck and N-WASP, mimicking the role of phosphorylated nephrin. Next, we designed a covalent blocker of Nck, peptide p1 based on the selected pY peptides, which site-selectively reacted with the SH2 domain of Nck (Nck-SH2) at Lys331 through a proximity-induced reaction. The covalent reaction of p1 with Nck blocked the protein binding site of Nck-SH2 and disintegrated the 3pY/Nck/N-WASP condensates. In the presence of membrane-translocating peptide L17E, p1 entered Caco-2 cells in the cytosol, reduced the number of Nck puncta, and rendered Caco-2 cells resistant to EPEC infection. Site-selective covalent blockage of Nck thereby disintegrates intracellular Nck condensates, inhibits actin reorganization, and shuts down the entrance pathway of EPEC. This work showcases the promotion or inhibition of protein phase separation by synthetic peptides and the use of reactive peptides as LLPS disruptors and signal modulators.


INTRODUCTION
−10 The mechanism of EPEC infection centers around signal transduction of the adaptor protein Nck.An EPEC bacterium first latches to the surface of an intestinal cell and injects one of the virulence factors, the translocated intimin receptor (Tir), into the cell.Tir is encoded by espE located on the locus of enterocyte effacement (LEE) pathogenicity island in EPEC strains.These steps result in an attaching and effacing (A/E) lesion between the EPEC bacterium and the intestine cell. 6,11Phosphorylation of the Tir protein at Tyr 474 induces its binding with the SH2 domain of Nck (Nck-SH2), which subsequently recruits Nck-binding proteins such as WIP and N-WASP and activates the Arp2/3 complex-dependent actin polymerization pathway in the host cells. 12Actin polymerization forms a pedestal underneath the bacterium and leads to the invasion of EPEC and other microbes, such as vaccinia 13,14 and other vertebrate poxviruses. 15Nck proteins (Nck1 and Nck2, also called Nckβ and Grb4) contain four domains in tandem, including one SH2 domain and three SH3 domains.Nck-SH2 is known to bind to the phosphorylated peptide sequence on the Tir protein in EPEC, the viral membrane protein A36, and nephrin. 12,16,17ecause of the high degree of sequence homology of Nck1 and Nck2, Nck1 (for simplicity, Nck hereinafter) was chosen in this work.Due to the essential role of the interaction between Tir and Nck-SH2 in EPEC infection, we envision that a peptide that blocks the protein-binding site of the Nck-SH2 domain will inhibit EPEC-mediated pedestal formation, which will block the entrance of EPEC and guard the cells against EPEC infection (Figure 1).
Biological molecules, such as proteins or nucleic acids, form biomolecular condensates or coacervates and function as membrane-less organelles in eukaryotic cells. 18,19The condensates are highly concentrated micron-sized liquid droplets formed by liquid−liquid phase separation (LLPS) driven by physical interactions. 20The changes in physical states of biomolecules are associated with the occurrence and prevention of some diseases, such as protein aggregates involved in neurodegenerative diseases, protein accumulation 21 or transcription abnormal in cancer, 22 and viral replication condensates in SARS-CoV-2 infection. 23−29 Nck phase transition is associated with the activity of an actin nucleation factor, the Arp2/3 complex, and is governed by the degree of nephrin phosphorylation.However, the role of Nck-mediated LLPS in infectious diseases and whether LLPS can derive druggable targets for treating bacterial infections remain largely untapped.
Because Nck-SH2 binds to phosphorylated Tir, and this interaction drives EPEC invasion, we reason that blocking Nck-SH2 will disintegrate the Nck signaling complex and shut down the Nck-mediated actin rearrangement.However, the SH2 domains in mammalian cells are highly promiscuous, so selective blocking of the Nck-SH2 domain among ∼120 SH2 domains in the proteome is difficult. 30Because the phosphorylated tyrosine (pY) residue provides approximately half of the total binding energy in the pY-peptide−SH2 complex, it is challenging to design high-affinity and highselectivity blockers specifically for the Nck-SH2 domain while sparing other SH2 domains or pY-binding proteins in the cytoplasm.In previous work, we developed proximity-driven cysteine-selective reactions between pY-containing peptides with SH2 domains using the reaction between an αchloroacetyl group and a cysteine.This allowed us to differentiate different SH2 domains and achieve high-affinity covalent inhibition of specific SH2 domains selectively. 30The site-selective cysteine reactions were also brought inside cells, leading to intracellular domain-specific irreversible inhibition of a PDZ domain and an SH3 domain and effective blockage of signal transduction inside cells and even in animals. 31,32−36 Here, we first validate that Nck forms phaseseparated condensates with N-WASP and a pY peptide trimer (Figure 1a).Then, a structure-guided design leads to a reactive peptide that can covalently react with the Nck-SH2 domain at a selected lysine residue to interfere with the Nck/N-WASP LLPS (Figure 1b).We also report the intracellular delivery of this reactive peptide, the disintegration of Nck coacervates, and the blockage of EPEC infection in intestine cells (Figure 1c).

RESULTS AND DISCUSSION
A. Peptide Promoter of Nck Phase Separation in Vitro.Nck, N-WASP, and p-nephrin are known to undergo liquid−liquid phase separation (LLPS). 24−29 N-WASP consists of 5 domains: WH1 (WASP-homology 1), BR1 (a highly basic region), GBD (GTPase-binding domain), and proline-rich and VVCA (verprolin homology, central hydrophobic region, acidic region) domains.Here, the GBD-P-VCA tridomain of N-WASP was expressed and purified, and it was called N-WASP thereafter in this work. 27In the absence of p-nephrin, Nck and N-WASP formed micrometer-sized microdroplets in aqueous PBS buffer at very high concentrations, i.e., 40 μM of Nck and 60 μM of N-WASP (Figure 2a).The droplets showed liquid-like characteristics based on a fluorescence recovery assay after photobleaching (FRAP) assay (Figure 2b), proving to be coacervates driven by LLPS.Phosphorylated Tir is known to bind to Nck-SH2.A 12residue pY-containing Tir peptide EEHIpYDEVAADP was identified as a strong binder to Nck-SH2, and the crystal structure of the complex was reported. 17Also, the sequence of nephrin contains three Tyr-containing sequences that are similar to those of the Tir peptide: HLYDEV, PLYDEV, and GIYDQV (the potential phosphorylation sites are underscored), with a hydrophobic residue at the pY-1 site, and negatively charged residues at pY+1 and/or pY+2 (Figure 2c).Therefore, we reason that phosphorylation at these Tyr turns nephrin into p-nephrin, a multivalent Nck-SH2 binder, which drives the LLPS of Nck-SH2 and other binding proteins. 27To prove this hypothesis, we designed a trimeric phosphopeptide 3pY containing three LpYDEV linked through PEG linkers� all with L at pY-1 and D and E at pY+1 and pY+2 sites, to mimic the multivalent phosphorylated nephrin or Tir (Figure 2c).A nonphosphorylated version 3Y was also synthesized as a control.Adding 3pY to Nck and N-WASP turned the solution to opalescent, and micron-sized spherical droplets were visible under the microscope at significantly lower protein concentrations (Figure 2d).Under a time-lapse microscope, we observed that the microdroplets could fuse into larger condensates (Figure 2e).A phase diagram revealed that the minimal phase-forming concentrations of Nck, N-WASP, and 3pY were 10 μM, 5 μM, and 1 μM, respectively (Figure 2f).Next, we fluorescently labeled N-WASP with Cy3 and Nck with Cy5.The Cy3 and Cy5 signals colocalized in the Nck/N-WASP/3pY system under the fluorescent microscope (Figure S1 in the Supporting Information).The fluorescence recovery after photobleaching (FRAP) assay showed rapid fluorescence recovery after photobleaching the fluorescent signal within 30 s (Figure 2g).Taken together, these results show that 3pY promotes the condensation of Nck and N-WASP through the interaction with Nck-SH2 similar to that of p-nephrin, and the condensates formed in the Nck/N-WASP/3pY system have liquid-like properties, meeting the properties, of liquid−liquid phase separation (LLPS).We reason that the multivalent feature and two flexible PEG 4 linkers are essential for 3pY to promote the LLPS of the Nck/N-WASP system. 27. Peptide Inhibitor of Nck/N-WASP/3pY Coacervation.Next, we explored the intervention of Nck/N-WASP/ 3pY LLPS using pY peptides (Figure 3a).Three pY-containing peptides extracted from different Tir sequences were synthesized, namely, peptides Y474, Y751, and Y147 (Figure 3b).When peptide Y474 was added to the Nck/N-WASP/3pY system at a high concentration of 50 μM, protein microdroplets disappeared; such an effect was not observed when peptides Y751 and Y147 of the same concentration were added to the coacervates (Figure 3c).Using solution turbidity as a measure of coacervate formation, peptide Y474 caused a rapid decrease in the turbidity of the Nck/N-WASP/3pY solution.In contrast, the effect of the other two peptides was significantly less pronounced (Figure 3d).The inhibitory effect of peptide Y474 was also found to be concentration dependent: 50 μM Y474 caused more turbidity decrease than 5 μM y474 (Figure 3e).Also, peptide Y474 was found to bind with Nck-SH2 with a K D of 70 nM, based on the measurement by microscale thermophoresis (MST) (Figure S2 in the Supporting Information).These data show that monovalent Nck-SH2 binding peptides disrupt Nck LLPS at high concentrations by competing with multivalent binders for Nck-SH2 binding sites.
C. Reactive Peptides Based on Proximity-Induced Lysine Reaction.Although peptide Y474 can block Nckmediated LLPS, the requirement of a high concentration of the blocker peptide is hard to achieve inside the cell.We then sought to design covalent, irreversible blockers, anticipating that, at a significantly lower concentration, the covalent blocker can effectively inhibit Nck coacervation in the cell.The cocrystal structure of the Tir peptide and Nck-SH2 (PDB ID: 2CI9) shows that several lysine residues of Nck (including Lys328, Lys331, and Lys369) form a positively charged surface to host negatively charged Glu and Asp at pY+1 and pY+2 sites of the ligand EEHIpYDEVAAD (shown in red) (Figure 4a).The side chains of the Nck lysine residues are pointing toward the residues at pY+1 and pY+2 positions with distances of only 4 to 6 Å.This unique feature gives us an opportunity to design covalent blockers for Nck-SH2 by installing electrophiles at the pY+1 or pY+2 positions to enable a proximal conjugation reaction with one of the lysine residues.Covalent blockage through the characteristic lysine residues will maximize the specificity of the inhibitor toward Nck-SH2.As sulfonyl fluoride has been reported to react with proteinaceous nucleophiles such as lysine, histidine, or cysteine, 34 we installed a sulfonyl fluoride group on the side chain of diaminopropionic acid, giving an unnatural amino acid X 1 at different positions such as pY+1 (Asp6) and pY+3 (Val8) of peptide EEHIpYDEVAAD.Besides the amino acid X 1 , we also included α-chloroacetyl-carrying amino acids X 2 and X 3 , which may also react with proteineous nucleophiles.Electrophile-containing reactive peptides p1 to p11 were synthesized with a biotin tag at the N-termini (Figure 4b).After the reactive peptides were incubated with recombinant Nck protein at a 5:1 ratio at 37 °C in PBS for 1 h, the formation of covalently linked Nck−peptide conjugates could be observed, shown as new protein bands with molecular weights higher than that of Nck based on denatured SDS-PAGE and Western blotting analysis against biotin (Figure 4c).The SDS-PAGE gel showed that among all the peptides, p1 gave the highest cross-linking efficiency with Nck, with >90% of Nck protein converted to conjugates.Even though the ratio of p1 to Nck is 5 fold in excess, only one Nck−peptide conjugate band was observed in the SDS-PAGE; the 1:1 reaction ratio suggests that the reaction most likely happens at the peptide-binding groove of Nck-SH2, i.e., with the projected lysine residues.All the peptides carrying sulfonyl fluoride (p1, p5, p7, p10, and p11) showed noticeable reactivity with Nck, whereas among the chloroacetyl-containing peptides, only p2 and p9 showed reactivity.Microscale thermophoresis (MST) analysis also showed that p1 outperformed p7 (as one example), giving a significantly lower EC 50 value, 0.75 vs 6.2 μM respectively (Figure S3 in the Supporting Information).At 37 °C in PBS buffer, the 1:1 reaction efficiency reached 50% in 2 min (Figure S4 in the Supporting Information).These data show that peptides with side-chain-modified sulfonyl fluoride are viable reactive peptides for Nck, and the Nck-peptide binding induces a covalent cross-linking reaction between Nck and the irreversible blocker.
D. Mass Spectrometric Evidence for a Single-Site Lysine Reaction in Nck-SH2.We next identified the residues in Nck-SH2 that are involved in the reaction with p1 by mass spectrometry.LC-MS analysis first confirmed that only one p1 was conjugated with Nck (Figure S5 in the Supporting Information).The Nck−p1 conjugate was digested with trypsin, and peptide fragments were analyzed with an in-line EASY-spray source and nano-LC UltiMate 3000 HPLC system interfaced with an Elite mass spectrometer, operated in the data-dependent acquisition (DDA) mode with one full MS scan at R = 60,000 (m/z = 200) mass followed by HCD MS/ MS scans.A new peak corresponding to the precursor mass (Mr + 3H) 3+ of biotinylated p1 peptide (α-chain) conjugated with Nck-SH2 (β-chain) was observed at 2631.1402 Da after searching by pLink v2.3.11. 37The fragmentation spectral analysis of [EEHIpYXEVAAD(α)] + [HFKVQLK(β)] extracted by pLabel v2.4.3.0 (Figure 5a and Figure S6 in the Supporting Information) 38,39 confirmed the ligand-directed conjugation of Nck-SH2 protein with p1 peptide.The underlined lysine residue in the β-chain sequence indicated the cross-linked site on the predicted Nck Lys331 residue.Next, we mutated Nck Lys331 to Ala, expressed and purified the Nck-SH2 K331A mutant.In contrast to wild-type Nck-SH2 and Nck, Nck-SH2 K331A did not show noticeable reactivity with peptide p1 (Figure 5b).These data identify Lys331 as a reaction site for p1 on Nck-SH2, suggesting that the proximitydriven lysine reaction occurred in a residue-selective manner.
In addition, we performed the reaction between p1 and Nck in a cell lysate, representing a complex solution.We incubated peptide p1 with the lysate of Caco-2 cells overexpressing Nck, and the reaction mixture was resolved on SDS-PAGE and analyzed with Western blotting against an anti-Nck antibody.A complete band shift was observed in the reaction system with p1, whereas for other peptides only a partial band shift was observed (Figure S7 in the Supporting Information).Next, we incubated biotinylated peptide Y474 and Nck solutions with or without p1 peptide, and pulled down proteins that bound with biotinylated Y474.In the absence of p1, we clearly observed that Nck bound with Y474.However, in the presence of p1, the Nck protein did not bind with Y474, suggesting that p1 competitively blocked the peptide-binding site of Nck-SH2 (Figure S8 in the Supporting Information).These data show that p1 competitively blocks the protein binding site of Nck-SH2.
E. Nck-p1 Reaction Disrupts LLPS.To explore whether intracellularly delivered peptide p1 could disrupt the Nckmediated phase separation in mammalian cells (Figure 6a), we first examined whether Nck can form coacervates with Nck in mammalian cells.Confocal microscopic images of HeLa cells overexpressing fluorescently tagged Dsred-Nck and GFP-N-WASP revealed the formation of micrometer-sized puncta with colocalized Dsred and GFP signals (Figure 6b).An intracellular FRAP analysis using a home-built two-photon fluorescence microscope at room temperature revealed the fluidlike property of the puncta, showing they are not precipitates (Figure 6c).After photobleaching the GFP fluorescence in puncta to 40% of its original fluorescence intensity, gradual fluorescence recovery to 70% in 50 s was observed.This shows that the Nck/N-WASP condensates are dynamic phase-separated droplets.
Delivering p1 into mammalian cells across the plasma membrane is a challenge, as the negatively charged p1 can not spontaneously enter mammalian cells.After exploring several delivery systems, we finally selected the L17E peptide to deliver p1 into mammalian cells.The lipid-sensitive endosomolytic peptide L17E (IWLTALKFLGKHAAKHE-AKQQLSKL-amide) was discovered based on peptide screening and was found to bring cargo into the cytosol through endosomal escape by sampling mixing with the cargo. 40uorescently labeled p1 was efficiently delivered into Caco-2 cells by mixing with L17E, and the fluorescence signal was distributed in the cytosol (Figure S9 in the Supporting Information).We also confirmed that L17E-delivered p1 did not cause noticeable cytotoxicity based on the CCK8 assay (Figure S10 in the Supporting Information).Western blotting using an Nck antibody identified an Nck complex with higher molecular weight in the lysate of L17E/p1-treated cells, showing L17E-delivered p1 reacted with the endogenous Nck in the cytosol of Caco-2 cells (Figure S11 in the Supporting Information).The extent of the molecular weight increase in this case was lower than that in vitro, likely due to the intracellular degradation of the p1 peptide.Despite the fact that p1 may react with a range of nucleophiles in the cells, here we provide evidence of the reaction between endogenous Nck and p1, which paves the way for the following function-based experiments.When Caco-2 cells overexpressing dsRed-Nck were treated with the p1 peptide (1 μM) and L17E (40 μM) for 2 h, we found a 50% decline in puncta numbers in the cells, showing a significant decrease of phase-separated Nck condensates (Figure 6d).This data show that Nck and N-WASP form phase-separated condensates in cotransfected HeLa cells, and L17E-delivered p1 peptide may disrupt the Nck/N-WASP coacervates.
F. L17E/p1 Protects Cells against EPEC Infection.Lastly, to examine whether pretreatment of Caco-2 cells with L17E/p1 could prevent EPEC infection, we first established a bacterial infection assay (Figure 7a).Caco-2 cells were pretreated with L17E/p1 for 2 h.After excess peptides were removed, Caco-2 cells were then infected with EPEC at different multiplicity of infection (MOI) values (MOI is commonly defined as the ratio of infectious bacteria to cells in a culture) for 3 h, and extra bacterial cells were removed from the culture.Next, gentamicin was added to the cell culture to remove EPEC that are merely attached to the surface of Caco-2 cells, leaving only engulfed EPEC inside Caco2 cells to be counted (gentamicin treatment will not kill intracellular bacteria due to its cellular impermeability).After gentamicin was washed away, cells were lysed to release EPEC cells inside, and the cell lysates were diluted to count the number of EPEC cells as colony-forming units (cfu) on agar plates.L17E/p1 treatment reduced the CFU numbers in a dose-dependent manner, with 5 μM of p1 reducing about 50% of the EPEC infection (Figure 7b).Comparing the covalent blocker p1 and the noncovalent blocker Y474 peptide, both blockers can disintegrate phase-separated Nck droplets and reduce the turbidity of the solution (Figure S12 and S13 in the Supporting Information).Although both peptides, in conjunction with L17E-mediated delivery, can block EPEC invasion to Caco-2 cells, covalent blocker p1 showed significantly higher efficacy than Y474 at lower concentrations (Figure 7c).
Lastly, to confirm that the inhibitors conferred resistance through actin rearrangement and pedestal formation, Tir and actin were visualized in inhibitor-treated cells.Briefly, infected Caco-2 cells were fixed with paraformaldehyde, stained with immunofluorescent anti-Tir antibody, and treated with phalloidin labels actin.EPEC cells were found to be located on the surface of Caco-2 cells and supported by the actin network, showing that EPEC infection induced actin pedestal formation (Figure 7d). 36Treatment with L17E/p1 significantly reduced the number of EPEC cells that attach to the surface of Caco-2: nearly no noticeable EPEC cells can be found under the microscope.Also, actin is evenly distributed on the surface of Caco-2 cells without signs of forming pedestals.These data show that L17E-delivered p1 prevented the invasion of EPEC into Caco-2 cells by inhibiting Nckmediated pedestal formation.

DISCUSSION
Nck bridges the externally introduced EPEC virulence factor Tir with a downstream actin rearrangement signal in an intestine cell and makes the cell a recipient of EPEC.To do so, Nck utilizes its four protein-binding domains, three SH3 and one SH2, to bind with designated proteins upstream and downstream and form a signaling complex.Our work shows that the signaling complex formed between Nck and N-WASP may exist as phase-separated condensates, where phosphorylated nephrin (mimicked by a synthetic peptide 3pY here) promotes the Nck/N-WASP phase separation and reduces the minimal protein concentrations required for phase separation.Based on this observation, we designed noncovalent and covalently reactive peptides to block the pY-binding site of Nck-SH2.A sulfonyl fluoride-containing pY peptide p1 covalently reacted with Nck-SH2 at Lys331 and effectively blocked the Nck-mediated phase separation.Through an L17E delivery system, the covalent blocker p1 reversed the phase separation of Nck and N-WASP and disallowed EPEC from entering Caco-2 cells.Unlike 3pY, which promoted the LLPS of Nck and N-WASP by providing multivalent interaction sites and flexible internal PEG 4 linkers, Y474-derived monovalent binders at high concentrations competed with the multivalent binders and disrupted the LLPS.
Blockage of a specific SH2 domain is challenging because the binding of pY to the pY-binding pockets on the SH2 domains contributes to about 50% of the binding energy, which makes SH2−peptide binding to be promiscuous.In a previous report, we identified SH2 domains that have special cysteine residues at the peptide-binding site and developed reactive peptides containing an α-chloroacetyl group at selected sites that are adjacent to the cysteine residue.Proximity-driven reactivity enables selective reaction at the cysteine. 30Here, we show a proximity-driven lysine reaction at Lys331 of the Nck-SH2 domain.Covalently reactive peptide p1 showed high reactivity with Nck-SH2 both in the cell lysate in vitro and in the cytosol.Admittedly, the sulfonyl fluoride on p1 also reacts with other nucleophiles inside cells, but these side reactions did not cause noticeable changes in the cell physiology or toxicity.On another note, delivery of the reactive peptide p1 to the cytosol while retaining its reactivity is not trivial.Covalent linkage of p1 with positively charged cellpenetrating peptides invalidated the activity of p1 possibly because of shielding of the pY residue (data not shown).Importantly, the L17E delivery system does not need a covalent linkage with p1, and the endosomolytic activity of L17E allows p1 to escape endosome entrapment and be distributed in the cytosol.However, the drawback of this delivery method is that it is not economical due to the high concentration of L17E peptide required in the delivery: For example, for the delivery of the 1 μM p1 peptide, 40 μM L17E is required.
Targeting the phase separation property of the signaling protein complex is emerging as a possible way to interfere with cellular signal transduction.Condensate-modifying therapeutics (c-mods), including peptides and small molecules, have been designed to alter condensate behaviors with functional consequences in cell-based studies.For example, Zhou and coworkers identified a peptide targeting the dimerization domain of the SARS-CoV-2 nucleocapsid protein (SARS2-NP) and found it disrupted the SARS2-NP LLPS and inhibited virus replication. 41Instead of directly acting on bacterial proteins, the inhibitory blocker in our approach targets an adaptor protein inside the cytosol of the host cells.Blockage of the Nck-SH2 binding site inhibits the Nck LLPS, shuts down Nckmediated signal transduction, and inhibits bacterial infection.The inhibition of the Nck-SH2 binding site directly results in two events: the disintegration of Nck coacervates and the blockage of EPEC infection.According to Banjade and Rosen, p-nephrin/Nck/N-WASP condensates promote Arp2/3 complex-dependent actin assembly. 28Arp2/3-dependent actin polymerization is also known to play a critical role in EPEC infection. 16Therefore, disruption of the Nck condensates may affect Arp2/3-dependent actin polymerization and subsequently inhibit EPEC infection.Although direct evidence for the linkage between these events is yet to be acquired, modulating the LLPS of adaptor proteins by blocking essential protein−protein interactions in the host cells may provide a new antibacterial mechanism against the infection of EPEC.
On another note, unlike p-nephrin, the role of phosphorylated Tir (p-Tir) in the Nck LLPS is yet unclear, as neither 3pY nor peptide Y474 represents the membrane-bound protein p-Tir.Because peptide Y474 at low concentrations did not disintegrate the Nck/N-WASP/3pY coacervates, it is possible that the membrane-bound p-Tir (with the C-terminal Y474 phosphorylated) at low concentrations may join the Nck/N-WASP/p-nephrin coacervates.Banjade and Rosen's work suggests that intimin on the surface of EPEC may cluster p-Tir and promote the formation of Nck coacervates, 28 which consequently leads to pedestal formation during the EPEC infection.The role of p-Tir clearly needs more investigation.

■ ASSOCIATED CONTENT
* sı Supporting Information The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acscentsci.3c01068.Methods and experimental procedures; colocalization of Cy3-N-WASP and Cy5-Nck in Nck/N-WASP/3pY microdroplets; microscale thermophoresis (MST) experiment to measure the binding of Y474 to Nck; MST experiment measured the EC50 of peptide 1 and 7; reaction kinetics; LC-MS analysis of Nck and Nck-p1 conjugate; mass data of the selected peaks in Figure 5a; reaction of biotin-p1 to cell lysate; a competitive pulldown experiment to show Nck-p1 complex failed to bind to peptide Y474; L17E delivers p1 peptide into the cytosol of Caco-2 cells; cell viability analysis by CCK8 assay; in cell reaction; peptide p1 reverses the phase separation of Nck/N-WASP/3pY; peptide Y474 reverses the phase separation of Nck/N-WASP/3pY; chemical structure of all the peptides and the mass spectrum data (PDF) ■ AUTHOR INFORMATION

Figure 1 .
Figure 1.Schematic illustration summarizing the main discoveries of this work.(a) Schematic illustration showing that a trimeric pY-containing peptide promotes LLPS of Nck and N-WASP, and covalent and noncovalent Nck-SH2 blockers reverse the coacervate formation.(b) Proximityinduced lysine reaction of Nck-SH2 yields covalent SH2 blockers with a selected lysine.(c) Covalent and noncovalent Nck-SH2 inhibitors disintegrate Nck coacervates in cells and prevent EPEC invasion.

Figure 4 .
Figure 4. Covalently reactive peptides designed based on a Tir peptide.(a) Crystal structure of Nck-SH2 in complex with a peptide EEHIpYDEVAAD from EPEC protein Tir (PDB: 2CI9).(b) List of synthetic reactive peptides.(c) Unnatural amino acids in the peptides.(d) SDS-PAGE (top) and Western blotting (bottom) analyses showing covalent conjugation of the peptides with Nck.Briefly, biotin-labeled peptides were mixed with recombinant Nck protein at a peptide-to-protein ratio of 5:1 at 37 °C for 1 h before denaturation and resolution by SDS-PAGE.

Figure 5 .
Figure 5. Peptide p1 reacts with Lys 331.(a) MS/MS analysis of the conjugated site between p1 peptide and Nck-SH2 at Lys331.The corresponding mass data are shown in Figure S6 in the Supporting Information.(b) Mutation of Lys331 to Ala abolished the covalent reaction of Nck with p1.Purified recombinant proteins were incubated with peptides in PBS buffer at 37 °C for 1 min (left) or 10 min (right).Molecular weight markers are shown in the middle.Red arrows indicate the covalent peptide−protein complex.

Figure 6 .
Figure 6.Peptide p1 inhibits the formation of the Nck/N-WASP condensate in mammalian cells.(a) Schematic illustration showing inhibition of intracellular Nck coacervation by p1.(b) Confocal microscopic images showing the formation of Nck/N-WASP puncta in HeLa cells that overexpress GFP-N-WASP and dsRed-Nck.Scale bar: 10 μm.(c) FRAP analysis showing the Nck/N-WASP condensates in HeLa cells have fluidity properties based on the recovery of the GFP fluorescent signal after photobleaching in a two-photon microscope with excitation at 920 nm.Scale bar: 5 μm.(d) Confocal microscopic images showing that L17E/p1-treatment reduced the numbers of Nck punta in Caco-2 cells transfected to overexpress dsRed−Nck.L17E, 40 μM; p1, 1 μM.Statistical analyses of puncta number per cell in the control and experiment (L17E/p1).N = 20 cells.

Figure 7 .
Figure 7. L17E/p1 treatment safeguards Caco-2 cells against EPEC infection.(a) Schematic illustration showing the procedure of the infection assay.(b) Anti-infection efficacy of L17E/p1 treatment at different concentrations of p1 using CFU as an indicator for infection.(c) Comparison of p1 and Y474 peptides in the anti-infection assay.(d) Representative microscopic images of L17E/p1-treated Caco-2 cells showing the absence of actin pedestals, whereas in the absence of p1 peptide, EPEC induces actin pedestal formation to attach to Caco-2 cells.The concentration of L17E in all of the experiments was 40 μM.