To identify ENAH EVH1 binders in the human proteome, we applied a screen called MassTitr. MassTitr is a SORT-SEQ method that is based on fluorescence-activated cell sorting (FACS) of a library of peptide-displaying bacteria and subsequent deconvolution of signals by deep sequencing. As shown in Figure 1A and B, peptide-displaying Escherichia coli cells are sorted into bins according to their binding signals across a range of protein concentrations, and the binding signal for each peptide at each protein concentration is extracted by deep sequencing each bin. Two advantages of this method over phage display are that MassTitr supports screening of long peptides and leads to the identification of binders with a broad range of affinities. MassTitr is similar in concept to yeast-surface-display based methods that have been applied to study the interactions of anti-fluorescein scFvs and SARS-CoV-2 receptor binding domain mutants (Adams et al., 2016; Starr et al., 2020).

Using MassTitr, we screened a library of 416,611 36-mer peptides with seven-residue overlaps (the T7-pep library) (Larman et al., 2011). This library spans the entire protein-coding space of the human genome, and we hypothesized that the long lengths of the encoded peptides would illuminate the impact of the sequence surrounding the FP4 motif in a biologically relevant sequence space. We first prescreened the library for binding to an ENAH EVH1 domain that was tetramerized by fusion to the endogenous ENAH coiled coil, as shown in Figure 1A, generating an input library enriched in binders. We then ran MassTitr on the prescreened library, using eight concentrations of ENAH EVH1 tetramer (Figure 1B). After sorting, sequencing, and filtering based on read counts, 108 unique high-confidence binders were identified and classified as either high-affinity or low-affinity as described in the methods (Figure 1C, Supplementary file 1). Of the 108 hits, 14 may have bound to ENAH EVH1 because a library synthesis error introduced a motif that is not present in the human protein; we did not analyze these sequences further.

We validated the binding of 16 MassTitr peptide hits to monomeric ENAH EVH1 domain by using biolayer interferometry (BLI) to determine dissociation constants that ranged from 0.18 μM to 63 μM (Supplementary file 2). Except for SHROOM3 and TENM1 peptides, binders classified as high-affinity by MassTitr bound to the ENAH EVH1 domain more tightly than peptides classified as low-affinity. Many newly identified peptides bound with affinities similar to or tighter than a well-studied control peptide from Listeria monocytogenes protein ActA, which bound with K_D = 4.9 μM in our BLI assay (Supplementary file 2). Prior to this work, this single FP4-motif-containing sequence from ActA was the tightest known endogenously derived binder of Ena/VASP EVH1 domains (Ball et al., 2000). The highest affinity peptide that we discovered was from photoreceptor cilium actin regulator (PCARE) (K_D = 0.18 μM for 36-residue peptide PCARE^813-848; Supplementary file 2), which contains the FP4 motif LPPPP. Successive truncations of this peptide identified the 23-residue minimal region for high-affinity binding, which extends 14 residues beyond the FP4 motif (PCARE^826-848 K_D = 0.32 μM, Figure 1—figure supplement 1).

Although the majority of MassTitr hits contained FP4 motifs, 40 out of the 108 high-confidence hits did not (Figure 1C and D, Supplementary file 1). Most of the non-canonical binders contained a CXC motif. Although disulfide bond formation between CXC-containing peptides and ENAH may contribute to signal in the cell-surface display assay, we confirmed that this motif, and not just the presence of one or more cysteine residues, was important for the binding of a peptide from OLIG3 to ENAH in the presence of 2 mM DTT (Figure 1—figure supplement 2). Also, a CXC-containing peptide from TRIM1 bound reversibly to the ENAH EVH1 domain at mid-micromolar concentrations (Figure 1—figure supplement 2). Peptides from KIAA1522 and TJAP1 bound to ENAH and lacked either an FP4 or a CXC motif (Supplementary file 2). Our results, therefore, add to increasing evidence that the ENAH EVH1 domain can bind sequences beyond the FP4 motif (Boëda et al., 2007; Chen et al., 2014; Menon et al., 2015).

To highlight putative biologically relevant interaction partners of ENAH, we applied a bioinformatic analysis to identify those motifs that are likely to be accessible and co-localized with ENAH. We filtered our high-confidence hits by disorder propensity (IUPred2A > 0.4) (Mészáros et al., 2018) and cytoplasmic subcellular localization (Binns et al., 2009; Thul et al., 2017). This resulted in 34 peptides that mapped to 33 unique proteins, of which 13 are derived from interaction partners previously known to interact or co-localize with an Ena/VASP protein (Supplementary file 1). The Ena/VASP binding sites of 10 of these hits have been previously mapped. Therefore, MassTitr provided new information about the EVH1-binding sites of 23 novel or previously known interaction partners of the Ena/VASP family. Filtered hits were highly enriched in GO biological process terms including actin filament organization (FDR < 10^–6) and positive regulation of cytoskeleton (FDR < 0.05) (Mi et al., 2019), which align with documented cellular functions of ENAH. Notably, we also identified proteins localized to the Golgi body and cilia, where Ena/VASP function is not well characterized (Figure 1E; Kannan et al., 2014; Tang et al., 2016).

We tested whether putative new ENAH interaction partners bound to full-length proteins in mammalian cells. We overexpressed GFP-tagged full-length ENAH with FLAG-tagged FHOD1, IFT52, or TJAP1 and used an anti-FLAG antibody to precipitate complexes from the cell lysate. Probing with anti-FLAG and anti-GFP antibodies showed robust immunoprecipitation (IP) of GFP-ENAH relative to cells expressing GFP-ENAH and a FLAG-tag-only negative control protein (Figure 1F, Figure 1—figure supplement 3).

We used MassTitr data to identify FP4 SLiM-flanking elements that enhance binding to the ENAH EVH1 domain. A sequence logo made of the high-confidence MassTitr hits shows enrichment of prolines C-terminal to the FP4 motif, and a binomial test confirms that peptides containing FP4 motifs followed by three consecutive prolines are enriched our hit list (p < 10^–11; Figure 1D). A peptide from ENAH interactor ABI1 (Chen et al., 2014; Tani et al., 2003) was among the highest affinity ligands that we validated by BLI, with K_D = 2.6 μM (Supplementary file 2). ABI1 contains an FP4 motif followed by four prolines. Mutating FPPPPPPPP (FP₈) to FPPPPSSSS in the context of the ABI1 36-mer reduced affinity by approximately fourfold (p < 0.05; Supplementary file 3). Although this confirms that the C-terminal prolines enhance affinity, peptide FPPPPPPPP alone binds to the ENAH EVH1 domain with K_D = 28 μM, indicating that additional interactions contribute to the high affinity of the ABI1 36-mer. Previous studies have shown that acidic residues N- and C-terminal to the FP4 motif can enhance affinity (Niebuhr et al., 1997) and we hypothesized that positively charged patches on ENAH could bind acidic residues that flank the FP₈ segment in ABI1 (Figure 2—figure supplement 1). Indeed, truncating the N-terminal or C-terminal acidic flanks of the 36-residue ABI1 peptide further decreased affinity (Supplementary file 3).

We solved a crystal structure of the ENAH EVH1-ABI1 peptide complex at 1.88 Å resolution. Only the FP₈ region was fully resolved in the structure under the crystallization conditions, which included high salt and low pH (Figure 2A). The peptide folds into a PPII helix with prolines 1, 4, and 7 (⁰FPPPPPPPP⁸) contacting the EVH1 surface (Figure 2A and B). The FP4 portion of the peptide binds the canonical FP4 groove, as observed in other structures (Prehoda et al., 1999; Fedorov et al., 1999), whereas the 7th proline docks into a previously uncharacterized site on ENAH composed of Ala12, Gly92, Phe32, and the aliphatic part of the side chain of Asn90 (Figure 2C). We note that residue 12 has diverged in EVL and VASP EVH1 domains (Figure 2C), which may contribute the weaker binding of ABI1 to those paralogs (Supplementary file 4), although we have not isolated the affinity difference to this specific change. Notably, a similar binding site at the analogous location is used by the Homer EVH1 domain to bind the phenylalanine of PPXXF motifs (Beneken et al., 2000). However, this site is relatively shallow in ENAH, and modeling large aromatic acids at this position on the ABI1 peptide using Pymol leads to severe steric clashes. Homer contains a smaller Gly89 at the site of Asn90 in ENAH and can accommodate the bulky Phe of the PPXXF motif (Figure 2C).

Another enriched feature of MassTitr-identified binders, relative to the pre-screened input library, is the presence of multiple FP4 motifs (binomial test, p < 10^–22). Multi-motif hits highlighted preferred spacings of approximately five or 15 residues between FP4 motifs (Figure 3A). Multiple motifs were also enriched in MassTitr high-affinity hits relative to all hits (p < 0.02), supporting the hypothesis that multiple FP4 motifs enhance affinity. We confirmed this experimentally by showing that binding was changed significantly (p < 0.05), with a 2.5- to sixfold reduction in affinity, when 36-mer sequences from LPP and NHSL1, which contain two FP4 motifs, were truncated to leave only one motif (Table 1, Figure 3B).

Zyxin, which contains four clustered FP4 motifs, has been shown to bind to the VASP EVH1 domain by contacting both the canonical FP4 site and a noncanonical site on the opposite side of the EVH1 domain (Acevedo et al., 2017). Interestingly, a crystal structure of the ENAH EVH1 domain bound to a single-FP4-motif peptide at the canonical site also contains a second peptide bound to the region corresponding to the noncanonical binding site in VASP (PDB 5NC7, Barone et al., 2020). To test whether multi-FP4-motif peptides engage this noncanonical site, we designed ENAH EVH1 R47A. In the ActA peptide-bound structure, ENAH Arg47 forms a bidentate hydrogen bond with a carbonyl on the peptide PPII helix backbone in the back-side site. The analogous VASP Arg48 exhibits significant NMR HSQC chemical shifts upon titration with a multi-FP4 motif zyxin peptide (Acevedo et al., 2017). Thus, we predicted that the R47A mutation would disrupt back-site binding. Indeed, while the affinities of single-FP4-motif peptides from ActA and PCARE were minimally affected by this mutation, peptides from zyxin, LPP, and NHSL1 that contain two FP4 motifs showed a 5–15-fold reduction in affinity for ENAH upon mutating Arg47 to Ala (p < 0.01) (Table 1, Figure 3C, Supplementary file 5).

Next, we investigated the stoichiometry and thermodynamics of multi-motif peptide binding to a monomeric ENAH EVH1 domain. Using isothermal titration calorimetry (ITC), we confirmed that dual-FP4 motif peptides from LPP and NHSL1 and the single-FP4 motif peptide from ActA fit well to a 1:1 binding model (Figure 3D). Interestingly, the ITC analysis showed that binding of the ActA-derived single-FP4 motif peptide was driven by favorable entropy, whereas binding of the NHSL1 and LPP dual-motif peptides was enthalpically driven. ActA, LPP, and NHSL1 peptides have similar binding free energies, but the entropic contribution to the dual-motif interactions is ~10 fold less favorable (Figure 3E). These data are consistent with a model in which long, disordered dual-motif peptides pay an entropic penalty to wrap around the EVH1 domain and engage two sites but gain enthalpic binding energy from additional interactions. Duplication of the linkers between the two motifs in LPP and NHSL1 led to a very modest, ~ twofold reduction in affinity for the ENAH EVH1 domain compared to the WT LPP and NHSL1 peptides (p < 0.05 for NHSL1 duplicated, n.s. for LPP duplicated; Table 1). This is consistent with a less favorable conformational entropy of binding when the motifs are separated by a greater linker (Table 1). However, our data do not establish whether or not the two motifs bind the same way when the linker is duplicated. We observed that the interaction of truncated dual-motif peptides, which contain only a single motif plus the surrounding linker sequence, is weakened by mutation R47A (FP41 and FP42 peptides in Table 1). This suggests that the linker residues themselves may be able to make favorable interactions with the back-side site.

Finally, we examined the minimal motif-spacing requirements for bivalent binding. We used Rosetta to build a peptide chain to connect single FP4-motif peptides bound to the canonical and noncanonical sites of the ENAH EVH1 domain. There are two orientations of the chain that preserve the directionalities of the bound FP4 peptides observed in structure 5NC7. Ten residues were required to span the two motifs in orientation 1, whereas nine residues were sufficient in orientation 2 (Figure 3—figure supplement 1). This indicates that the ~15-residue spacing that was enriched in our hits is more than enough to span the two binding sites and implies that the chain is not taught between these two sites.

For many protein domains beyond EVH1, degenerate SLiMs have been cataloged in the Eukaryotic Linear Motif (ELM) database to describe their interaction preferences (Kumar et al., 2022). The ELM listing implies that there is a relatively simple recognition code for many key domain interactions. However, the short sequences of most SLiMs are likely insufficient for biological specificity in many or most cases. Here we showed how defining the EVH1 binding motif as [FWYL]PXΦP is an over-simplification and how, by systematically examining the role of flanking sequences for just one EVH1 domain, we readily uncovered numerous examples in which the binding is modulated via additional extra-motif residues. Added to prior reports from investigations of individual interactions (Stein and Aloy, 2008; Li et al., 2018a; Aitio et al., 2010), our work definitively demonstrates the importance of sequence context on SLiM behavior by illustrating specific mechanisms, including an unusual conformational specificity mechanism that is documented in our companion paper (Hwang et al., 2021). MassTitr provides a versatile experimental platform for uncovering context effects on domain-peptide interactions and will surely lead to similar insights into the recognition strategies of other domains.

BLI was carried out as described in Hwang et al., 2021. Briefly, biotinylated 6x-His-SUMO-peptide fusions were immobilized on streptavidin-coated tips and immersed into a concentration series of monomeric ENAH EVH1 domain diluted in BLI buffer (PBS pH 7.4, 1% BSA, 0.1% Tween-20, 1 mM DTT). Data were collected until the binding signal plateaued. Tips were then placed into 200 μL of BLI buffer, to allow dissociation, and data were collected until the signal plateaued. Quantification of the steady-state binding signal was performed to obtain K_D values. Association curves were fit to a one-phase association model in Prism as given below:Y= Yo+Plateau- Yo*(1-e-K*X)

and equilibrium-bound signal values (Plateau) were plotted against ENAH concentration and fit to a single-site binding model:Y= Bmax*XKD+X

to obtain dissociation constants. The kinetics of association and dissociation were too fast to fit accurately. Errors are reported as the standard deviation of two to three replicates (see source data, which includes confidence intervals; we always collected three replicates for cases where we made quantitative comparisons). For the mean K_D of replicates, 95% confidence intervals were calculated by assuming a t-distribution.

Fold-change errors given in Figure 3B and C were calculated through error propagation using the formula:(SDaμa)2+(SDbμb)2

An unpaired, two-tailed t-test was used to calculate whether the difference between two K_D values was statistically significant.

Sequences for proteins used in this study are given in Supplementary file 6. Human ENAH EVH1 domain, followed by a 6x-Gly linker and ENAH mouse coiled coil (for tetramerization), were cloned into a pDW363 biotinylation vector that includes a C-terminal biotin acceptor peptide (BAP) tag and a 6x-His tag. This ENAH tetramer construct was expressed in Rosetta2(DE3) (Novagen) cells in Terrific Broth (TB) with 100 μg/mL ampicillin, 25 μg/mL chloramphenicol, and 0.05 mM D-(+)-biotin (for in vivo biotinylation). Cells were grown at 37 °C with shaking to an optical density at 600 nm (O.D. 600) of 0.5–0.7 and then induced with 1 mM IPTG and grown at 37 °C for 5 hr. One L of cells were then spun down and resuspended in 25 mL of binding buffer (20 mM Tris pH 8.0, 500 mM NaCl, 5 mM imidazole), and frozen at –80 °C overnight. The next day, pellets were thawed and supplemented with 0.2 mM phenylmethylsulfonyl fluoride (PMSF) protease inhibitor. Cells were sonicated ten times for 30 s followed by 30 s of rest on ice and then centrifuged. The clarified lysate was filtered through a 0.2 μm filter and applied to 2 mL of Ni-nitrilotriacetic (Ni-NTA) acid agarose resin (GoldBio) equilibrated in wash buffer (20 mM Tris pH 8.0, 500 mM NaCl, 20 mM imidazole). The resin was then washed three times with 8 mL wash buffer and eluted with 10 mL of elution buffer (20 mM Tris pH 8.0, 500 mM NaCl, 300 mM imidazole). The elution was run through a S75 26/60 size exclusion column equilibrated in gel filtration buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1 mM DTT, 10% glycerol). Purity was verified by SDS-PAGE, and the fractions were pooled, concentrated, and flash-frozen at –80 °C.

SUMO-peptide fusions were cloned into a pDW363 vector that appends a BAP sequence and 6x-His tag to the N-terminus of the protein and transformed into Rosetta2(DE3) cells. For ITC experiments, these cells were expressed in TB supplemented with 100 μg/mL ampicillin, grown to an O.D. 600 of 0.5–0.7, and induced with 1 mM IPTG. Induced cultures were purified as described above for the pMCGS7 constructs, except for the TEV cleavage step. Instead, after elution with elution buffer, the sample was directly applied to the S75 26/60 column equilibrated in gel filtration buffer. Fractions were pooled, concentrated, and flash frozen at –80 °C.

Monomeric EVH1 domain and small-scale preparations of biotinylated SUMO peptides used for BLI were purified as described in Hwang et al., 2021.

Control peptides for display were expressed at the C-terminus of eCPX in a vector designed by the Daughtery group (Rice and Daugherty, 2008). This construct was modified to include a FLAG tag at the N-terminus of the peptide and a c-Myc tag at the C-terminus. The T7-pep library plasmids (gift from Elledge lab, Harvard University and Brigham and Women’s Hospital), were transformed into Pir1 cells (ThermoFisher), grown, and miniprepped (Qiagen) to isolate the library plasmid. Plasmids were then cut with EcoRI (NEB) and XhoI (NEB), and the inserts were gel purified, combined, and concentrated with a Zymo Clean and Concentrate column and eluted with 50 μL of sterile MilliQ Water. To clone the T7-pep library into the eCPX vector, we first grew up 200 mL of DH5a cells containing an empty eCPX vector at 37 °C overnight. This culture was miniprepped (Qiagen) and then digested with EcoI and XhoI at a ratio of 10 units of enzyme:1 μg of vector at 37 °C for 2 hr. The resulting digest was PCR purified and eluted with 40 μL water. This cut vector was then dephosphorylated with Antarctic phosphatase (NEB) at a ratio of 1 μL/1 μg of DNA at 37 °C for 2 hr, followed by 10 min at 65 °C for enzyme inactivation. T7-pep library insert was ligated into the cut eCPX vector using T4 ligase (NEB) at 14 °C overnight. Ligase was subsequently deactivated for 10 min at 70 °C, and the ligation reaction was concentrated with Zymo Clean and Concentrate columns (Zymo Research). Each column was eluted with 12.5 μL elution buffer (from kit). The resulting elutions were desalted on a 0.025 μm filter (Millipore) for 15–20 min and pooled on ice. Electrocompetent MC1061 cells and 10–20 μL DNA were then mixed and transferred to a cold 2 mm cuvette (BioRad). Each cuvette was pulsed at 2.5 kV, 50 μF, 100 ohms on an electroporator (BioRad), immediately rinsed out with 3 × 1 mL of warm SOC and transferred to a culture tube containing 7 mL warm SOC. Cells were incubated at 37 °C for 1 hr and then combined. Serial dilutions of the library were plated on LB/chloramphenicol plates to assess transformation efficiency, and the leftover cells were added to 500 mL of LB + 25 μg/mL chloramphenicol + 0.2% w/v sterile-filtered glucose. The library was grown at 37 °C until it reached an O.D. 600 of 2.0 and then frozen as glycerol stocks to use for FACS analysis and sorting.

The protocol for sample preparation for FACS analysis and MassTitr sorting was adapted from Foight and Keating, 2016 and was as follows: 5 mL cell cultures of eCPX plasmid expressing either library or control peptide were grown overnight at 37 °C in LB + 25 μg/ml chloramphenicol + 0.2% w/v glucose. The next day, cells were inoculated into fresh TB + 25 μg/mL chloramphenicol and grown at 37 °C. Upon reaching an O.D. 600 of 0.5–0.6, cells were induced with 0.04% w/v arabinose for 1.5 hr at 37 °C. The O.D. 600 was then remeasured, and enough cells were pelleted for analysis (1 × 10⁷ cells per FACS analysis sample, 7 × 10⁷ cells for library sorting). Cells were resuspended to a concentration of 4 × 10⁸ cells/mL, washed in PBS + 0.1% BSA, and then incubated with anti-FLAG antibody conjugated to APC (αFLAG-APC; PerkinElmer) diluted 1:100 in PBS + 0.1% BSA at a ratio of 30 μL labeled antibody:10⁷ cells. Tubes wrapped in foil were incubated at 4 °C for 15 min, then cells were washed with PBS + 0.1% BSA and pelleted. For each FACS analysis sample, 25 μL of 1 × 10⁷ cells in PBS were mixed with 25 μL of a 2 x concentration of ENAH tetramer in PBS + 1% BSA + 4 mM DTT (final concentration of 2 mM DTT) and then incubated at 4 °C for 1 hr in foil. After incubation, 50 μL of the mixture was added per well to a 96-well Multi-Screen HTS GV sterile filtration plate (Millipore), buffer was removed by vacuum, and then cells were washed twice with 200 μL of PBS + 0.5% BSA. Each well containing 1 × 10⁷ cells was then resuspended in 30 μL of streptavidin-PE (SAV-PE; ThermoFisher Scientific) diluted 1:100 in PBS + 0.1% BSA, and incubated for 15 minutes at 4 °C, washed with 200 μL of PBS + 0.1% BSA, and resuspended in 250 μL of PBS + 0.1% BSA for subsequent FACS analysis or sorting. For cell sorting, see supplementary methods.

The T7-pep library was prepared as described above. A total of 7 × 10⁷ cells expressing the T7-pep library were incubated with a final concentration of 20 μM ENAH monomers assembled as tetramers (i.e. 20 µM monomer). Cells were sorted on a BDFACS Aria machine. Prior to sorting, a positive (ActA) control and a negative (empty) control were analyzed. A gate to collect cells expressing peptide binders was set that included 0.3% of the negative control and all cells with a greater binding signal, allowing us to enrich moderate-affinity binders. This process was repeated three times on the same day to collect a total of 300,000 cells. These cells were added to warm SOC + 25 μg/mL chloramphenicol and grown at 37 °C overnight. The next day, cells were frozen at –80 °C as glycerol stocks for MassTitr sorting.

The pre-enriched T7-Pep Library was used, as described in the section FACS sample preparation and analysis, above. A total of 7 × 10⁷ cells stained with anti-FLAG APC were incubated with increasing concentrations of biotinylated ENAH EVH1 tetramer (monomer concentrations: 30, 12, 4.8, 1.9, 0.77, 0.31, 0.12, 0.049 μM), and then incubated with streptavidin-PE. Labeled cells were sorted on a BDFACS Aria. Cells were collected in four gates that were drawn with boundaries roughly parallel to the binding vs. expression signal slope for a series of positive controls (ActA, SHIP2, Vinculin). The gates were drawn to evenly sample the binding signal range, in log space, for low-, medium-, and high-affinity clones across the FACS window, as shown for gates labeled A-D in Figure 1—figure supplement 4. A no-peptide negative control (empty) was run to assess the degree of nonspecific binding at the time of sorting. At each concentration of ENAH EVH1 domain, cells were sorted for approximately 20 min and the number of cells that were collected in each gate was recorded. Enough cells were collected per gate to oversample the library at least 10-fold. Cells were collected in LB + 0.2% w/v glucose and then sorted cells were transferred into 10 mL of LB + 0.2% w/v glucose, grown at 37 °C overnight, and plasmid DNA was isolated by miniprep the next day. Three replicate MassTitr experiments were performed.

As described above, sorted pools were grown overnight at 37 °C in LB + 0.2% glucose and then miniprepped (Qiagen). Samples collected at a certain gate per concentration of ENAH tetramer were assigned a unique barcode/index combination. First, we PCR amplified the variable region of the library with a forward primer (Ngsfwd_1) and a corresponding reverse primer that contained one of 5 6-nucleotide (nt) index sequences for multiplexing (Ngsrev_1_i); the amplicon preparation scheme and all primers are listed in Supplementary file 7. Fourteen cycles of amplification were carried out with Phusion polymerase (NEB) using an annealing temperature of 66 °C. The resulting reaction was PCR purified with the Zymo Clean and Concentrate kit (Zymo Research) and eluted with 20 μL milliQ water. 200 ng of each reaction was cut with MmeI (NEB) at 37 °C for 1 hr and then the enzyme was heat inactivated at 65 °C for 20 min. To the 5’ end of 15 μL of digested fragment we ligated a double stranded adapter with matching overhangs using T4 DNA ligase (NEB) at 25 °C for 30 mins, followed by heat inactivation at 65 °C for 10 min. This adapter contained one of 24 5-nt barcodes and the standard Illumina forward primer. The expected ~200 bp band was gel purified with the Zymoclean Gel DNA recovery kit (Zymo Research) and eluted in 17 μL of MilliQ water. Three μL of this reaction was PCR amplified for 10 cycles with primers Ngsfwd_2 and Ngsrev_2 in a 50 μL reaction at an annealing temperature of 66 °C. The 5’ and 3’ Illumina adapter sequences and the reverse priming sequence were included in this step. The final product was PCR purified and eluted in MilliQ water. The DNA concentration of each pool was measured with the Qubit assay and samples were combined and run in one lane. In total, the multiplexed sample included 32 pools (corresponding to cells collected at each of eight concentrations in four gates) for each of three replicates, as well as the pre-enriched input library, for a total of 97 samples distinguished by their barcode/index combination. At each stage of the preparation process, the quality and homogeneity of the amplicon were assessed through Sanger sequencing (Genewiz) and Bioanalyzer. The multiplexed sample was submitted for sequencing on a NextSeq500 instrument using paired-end reads. Supplementary file 7 gives an overview of the procedures used to (1) label DNA from each pool of cells with a barcode/index indicating the ENAH EVH1 tetramer concentration and gate number for the sample and (2) to prepare the library for sequencing; primer sequences are listed, as well as the barcode/index key used for each sample.

Sequences were demultiplexed and processed with an in-house script. . We used the sequencing data to determine the number of clonal cells (i.e., cells displaying the same peptide) that were found in each gate at each concentration. We calculated the clone read frequency in gate j at concentration k as R_ijk/T_jk, where R_ijk is the number of raw reads for sequence i in gate j at concentration k, and T_jk is the total sequencing reads for all clones obtained for gate j at concentration k, i.e., Tjk=∑iRijk . The clone read frequency was multiped by the total number of cells collected in each gate (which was recorded during the sorting) to calculate the total number of cells displaying sequence i, C_ijk, in gate j at each ENAH concentration k. This can be used to compute an effective gate position using the following:∑jCijk•Fj/∑jCijk =Feffi,k

Where F_j values are the mid y-axis fluorescence values for each gate, j = 1–4 (with values of 700, 1500, 2500, 4,000 respectively). Feffi,k was calculated for each clone across all ENAH tetramer concentrations. If this experiment is performed in such a way such that every peptide-expressing cell is collected and sequenced, then the clones-per-gate information can be used to extract a concentration-dependent signal-vs.-concentration curve that can be fit to give an apparent dissociation constant, as done by Adams, et al. (Adams et al., 2016). In our experiment, we collected and sequenced fewer cells, focusing on cells that displayed above-background binding signal, and obtained information about concentration-dependent binding but not complete titration curves.

Our data processing was focused on identifying a subset of well-behaved clones, of varying affinities, that we judged to be good candidates for subsequent analysis of binding features. We first removed clones for which we obtained < 100 reads across all bins at all concentrations and also clones that did not have a total cell count (C_ijk summed over all j) of at least 25 cells in at least four concentrations k. A sequence was assigned as an ENAH binder if (1) the clonal population of cells showed a concentration-dependent increase in binding signal in at least two of three replicate experiments, or (2) the displayed peptide contained an FP4 motif and showed concentration-dependent binding in at least one replicate. Among binders identified in this way, clones were further tagged as “high affinity” if they met additional requirements: (1) total cell count of greater than or equal to 80 cells for at least four concentrations, (2) greater than or equal to 10 cells found in binding gates at the lowest three concentrations (0.31 μM, 0.12 μM, 0.049 μM), (3) cells in more than one gate at the highest two concentrations (30 μM, 12 μM), and (4) clone found in at least two replicates. These filters are were chosen, based on benchmarking against validated binding clones from the screen, to extract a subset of well-behaved clones that we judged likely to be true binders (as proved to be true). Complete data corresponding to the MassTitr read counts per gate, for each sequence at each concentration, are provided have been deposited at GEO with the accession number GSE166938.

A key providing information on which barcode/index pairs correspond to which sample and cell counts for each replicate are provided in Supplementary file 7.

The T7-pep library contains many point mutations and frameshifts, in addition to full-length human 36-mers. Consequently, for many hits we identified multiple closely related but non-identical sequences (Supplementary file 1). To analyze trends in our dataset, we collapsed hits that we judged to be variants of the same sequence into one representative sequence, leading to a total of 108 non-redundant sequences. If one of the sequence variants matched exactly with a sequence from the human proteome (UniprotKB/Swiss-Prot release 2020_01), that sequence was chosen as the representative sequence (UniProt Consortium, 2019). Otherwise, the sequence with the most combined total cell counts was chosen for analysis. To compare these sequences to sequences from the pre-enriched input library, we clustered amino-acid sequences from the input library reads using CD-HIT (Huang et al., 2010) with a sequence identity cut-off of 0.7, which effectively collapsed proteomic sequence variants into one representative sequence.

To identify putative biological interaction partners among our MassTitr hits we first removed peptides that mapped to a human protein but contained an unnatural FP4 or CXC motif due to frameshift or point mutations (highlighted in yellow in Supplementary file 1). Then we identified those peptides predicted to be intrinsically disordered, using an IUPred2A (Mészáros et al., 2018) cutoff of >0.4. Finally, we assessed the cytoplasmic localization of hits using cellular component terms from QuickGO (Binns et al., 2009). Two proteins from our list (NHSL1 and KIAA1522) did not have any associated terms with our search criteria. For these proteins, we manually curated subcellular localizations from the literature (Brooks et al., 2010) and the Human Protein Atlas (Thul et al., 2017). For proteins reported to be membrane-bound, such as MIA3 (Reynolds et al., 2019), we confirmed that the regions we pulled out as hits were cytoplasmic as annotated in Uniprot (Uniprot Consortium, 2019). GO term enrichments were performed using PANTHER with a Fisher’s Exact Test (Mi et al., 2019). GO biological process terms with an FDR < 0.05 were designated as enriched.

ITC experiments were performed with two replicates using a VP-ITC microcalorimeter (MicroCal LLC). To prepare samples for ITC, 2.5 mL of 100 μM ENAH EVH1 domain and 1 mL of 800 μM-1.2 mM of SUMO-peptide fusions were dialyzed against 2 L of ITC Buffer (20 mM HEPES pH 7.6, 150 mM NaCl, 1 mM TCEP) at 4 °C overnight. The concentrations of proteins were remeasured after dialysis on the day of the experiment. SUMO-peptide was titrated into the ENAH EVH1 domain at 25 °C. Data analysis and curve fitting were performed with the Origin 7.0 software (OriginLab). The error reported is the fitting error.

Crystals of ENAH fused at the C-terminus to ABI1 were grown in hanging drops over a reservoir containing 0.1 M sodium acetate pH 4.5 and 2.90 M NaCl. 1 μL of ENAH-ABI1 (250 μM in 20 mM HEPES, 150 mM NaCl, 1 mM DTT) was mixed with 1 μL of reservoir solution, and 3D crystals appeared within two weeks at 18 °C. Diffraction data were collected at the Advanced Photon Source at Argonne National Laboratory, NE-CAT beamline 24-IDE. The ENAH-ABI1 dataset was integrated and scaled to 1.88 Å with AIMLESS and the structure was solved with molecular replacement using ENAH EVH1 structure 5NC7 as a search model. The structure was refined using iterative rounds of model rebuilding with PHENIX and COOT (Liebschner et al., 2019; Emsley and Cowtan, 2004). Supplementary file 8 reports refinement statistics. The structure is deposited in the PDB with identifier 7LXE. Note that the ABI1 FP₈ peptide is numbered 120–129 in accordance with the ENAH-ABI1 fusion protein numbering.

Mammalian expression plasmids for 3xFLAG-FHOD1 and 3xFLAG-IFT52 were generated by Gateway recombination of destination and entry vectors using LR Clonase II (Thermo, 11791020) per the manufacturer’s instructions. Destination vector pEZYflag was a gift from Yu-Zhu Zhang (Addgene plasmid #18700; RRID:Addgene_18700). Entry vectors for FHOD1 (HsCD00516058), IFT52 (HsCD00399318), and TJAP1 (HsCD00861157) were obtained from DNASU. The mammalian expression plasmid for GFP-mouse ENAH was a gift from Frank Gertler, MIT. Note that mouse and human ENAH have identical EVH1 domains.

Six-cm plates of 60% confluent HEK293T (confirmed mycoplasma negative and validated through STR testing) were transfected with 2 µg of GFP-ENAH plasmid and either 3xFLAG-FHOD1 or 3xFLAG-IFT52 plasmid, and 12 µg of polyethyleneimine (PEI, linear MW 25,000; Polysciences, 23966). Twenty-four hours post-transfection, cells were lysed with ice cold immunoprecipitation (IP) buffer (10% glycerol, 1% NP-40, 50 mM Tris pH 7.5, 200 mM NaCl, 2 mM MgCl₂) supplemented with protease and phosphatase inhibitor cocktails (MilliporeSigma, 539134; Boston BioProducts, BP-479). Crude lysates were incubated on ice for 10 min and clarified by centrifugation at 21,000 g for 10 min at 4 °C. Supernatant protein concentration was determined using a Bradford assay, and 2 mg of protein was loaded into each IP reaction, with 5% input set aside. Lysates were pre-cleared in 5 µL of magnetic Protein A/G bead slurry (Thermo, 88802), and incubated overnight at 4 °C with orbital rotation with mouse monoclonal anti-FLAG (ProteinTech Group 66008–3, RRID:AB_2749837; 5 µg antibody per mg protein). 25 µL magnetic Protein A + G bead slurry was added to each immunoprecipitation assay, and incubated for 2 hr at 4 °C with orbital rotation. Beads were washed three times in ice cold IP buffer with 0.1% Triton X-100 and eluted by boiling at 95 °C for 5 min in 1:1 2 X Laemmeli buffer:IP buffer.

Samples were resolved using SDS-PAGE and 8% acrylamide gels and transferred to Immobilon-FL PVDF membranes (Millipore-Sigma, IPFL00005). Membranes were blocked in Intercept Blocking Buffer (LI-COR, 927–70001) for 1 hr rocking at room temperature and were probed overnight at 4 °C with primary antibodies diluted in blocking buffer and 0.2% Tween-20. Blots were probed sequentially with rabbit polyclonal anti-FLAG (ProteinTech Group 20543–1, RRID:AB_11232216; 1:1000) and the following day with mouse monoclonal anti-GFP (ProteinTech Group 66002–1, RRID:AB_11182611; 1:1000). Membranes were probed with near-infrared fluorescent secondary antibodies goat anti-mouse Alexa Fluor 680 and goat anti-rabbit Alexa Fluor 790 (Thermo, A21057 and A11367 1:20,000), diluted in blocking buffer and 0.2% Tween-20 for 1 hr at room temperature, and then scanned on an Odyssey DLx (LICOR).