Peptide‐based ligand antagonists block a Vibrio cholerae adhesin
Mingyu Wang, Grace Du, Charity Yongo‐Luwawa, Angelina Lu, Brett Kinrade, Kim Munro, Karl E. Klose, William D. Lubell, Peter Davies, Shuaiqi Guo
TL;DR
Researchers designed peptides that block Vibrio cholerae adhesion, offering a new way to treat cholera without promoting drug resistance.
Contribution
A structure-guided design of D-amino acid-containing tripeptides with nanomolar affinity for blocking Vibrio cholerae adhesion.
Findings
X-ray crystallography revealed how AGYTD binds tightly to the FrhA-PBD Ca2+ pocket.
D-amino acid-containing tripeptides showed higher affinity and metabolic stability compared to AGYTD.
The findings provide a structural blueprint for anti-adhesion therapeutics against cholera.
Abstract
Vibrio cholerae, the causative agent of cholera, uses surface proteins such as the repeats‐in‐toxin (RTX) adhesin FrhA to colonize hosts and initiate infection. Blocking bacterial adhesion represents a promising therapeutic strategy to treat infections without promoting drug resistance. FrhA contains a peptide‐binding domain (PBD) that is key for hemagglutination, human epithelial cell binding, and V. cholerae biofilm formation. Previous studies identified a lead pentapeptide ligand with the sequence Ala‐Gly‐Tyr‐Thr‐Asp (AGYTD) that blocks V. cholerae colonization of the mouse small intestine at high micromolar concentrations. In this study, a structure‐guided approach identified a minimal D‐amino acid‐containing tripeptide motif with higher affinity for the FrhA‐PBD and predicted metabolic stability. Our results contribute to the development of anti‐adhesion strategies to combat…
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Fig. 1
Fig. 2
Fig. 3
Fig. 4| Peptides |
|
|---|---|
| AGYTd (D‐Asp) | 3.0 |
| GYTD | 1.11 |
| AGYTD | 0.503 |
| AGWTD | 0.381 |
| GyTD (D‐Tyr) | 0.217 |
| WTD | 0.196 |
| YTD | 0.187 |
| wTD (D‐Trp) | 0.122 |
| yTD (D‐Tyr) | 0.071 |
- —Canadian Antimicrobial Resistance Network studentship
- —McGill Centre for Structural Biology Research Bluesky award
- —NSERC Undergraduate Student Research Award
- —McGill Faculty of Medicine and Health Sciences
- —McGill Centre for Structural Biology Research Studentship
- —Canadian Institutes of Health Research10.13039/501100000024
- —Fonds de Recherche du Québec ‐ Santé10.13039/501100000156
- —Natural Sciences and Engineering Research Council of Canada10.13039/501100000038
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Taxonomy
TopicsEscherichia coli research studies · Vibrio bacteria research studies · Biochemical and Structural Characterization
Abbreviations
AGYTD, pentapeptide with the sequence Ala‐Gly‐Tyr‐Thr‐Asp
CBM, carbohydrate‐binding module
FrhA, Flagellar‐regulated hemagglutinin A (an adhesin of Vibrio cholerae)
FrhASplit‐PBD, split‐domain construct of the FrhA peptide‐binding domain
ITC, isothermal titration calorimetry
K _ d _, dissociation constant
LBR, ligand‐binding region of FrhA
MST, microscale thermophoresis
** MpIBP**, Marinomonas primoryensis ice‐binding protein
PBD, peptide‐binding domain
RTX, repeats‐in‐toxin (protein family)
** V. cholerae **, Vibrio cholerae (bacterium)
Bacterial adhesion to host tissues is a critical first step in establishing infections [1] as stable attachment is required for subsequent colonization and virulence [2, 3, 4]. This process is mediated by surface‐exposed adhesins that recognize and bind to host molecules such as glycans and proteins [3, 5, 6]. In contrast, non‐adherent bacteria are rapidly removed by fluid flow [7, 8].
The Gram‐negative bacterium Vibrio cholerae is the causative agent of the diarrheal disease cholera, which is responsible for over 100 000 deaths globally each year [9]. Multiple adhesins in V. cholerae contribute to attachment to the intestinal epithelium [10]. For example, the repeats‐in‐toxin (RTX) adhesin FrhA promotes binding to chitin, erythrocytes, and epithelial cells [5, 11] and assists in biofilm formation, which facilitates virulence [12, 13, 14, 15]. FrhA is localized on the outer membrane of V. cholerae by the type I secretion system [5, 11, 16]. The retention of FrhA on the surface is regulated by a periplasmic proteolysis system through the signaling molecule c‐di‐GMP [17, 18, 19, 20, 21]. The N terminus of FrhA helps anchor the adhesin to the bacterial surface [22]. The C‐terminal ligand‐binding region (LBR) projects away from the bacterial surface for binding other cells and surfaces (Fig. 1A) [11]. The LBR of FrhA includes a carbohydrate‐binding module (CBM) [23] and a peptide‐binding domain (PBD) [12]. While the recombinant FrhA‐CBM demonstrates hemolytic activity against human type O erythrocytes [23], FrhA‐PBD is required for hemagglutination by V. cholerae O395 and enhances biofilm formation [12, 21]. The two ligand‐binding domains engage distinct host receptors and may act synergistically to enhance adhesion and virulence [12, 23, 24].
X‐ray crystal structure of FrhASplit‐PBD revealed interactions with AGYTD. (A) The linear domain maps of full‐length FrhA and the FrhASplit‐PBD construct. The PBD is shown in orange (residues 1139–1333), and the split domain is shown in dark gray (residues 1127–1138 and 1334–1439). Residue numbers marking domain boundaries are indicated in bold. (B) Isothermal titration calorimetry (ITC) measurement of the peptide AGYTD binding to FrhASplit‐PBD (n value = 1). Fitting of the binding isotherm yielded an N value of 1.09 and a dissociation constant (K d) of 370 ± 10 nm. (C) The 2.5‐Å X‐ray crystal structure of FrhASplit‐PBD bound to AGYTD (PDB ID: 9YBQ). The PBD is colored orange, and the companion split domain is colored gray. Ca2+ ions are depicted as green spheres, oxygen atoms in red, and nitrogen atoms in blue. The experimental electron density for AGYTD is shown as a light blue mesh. Protein residues are labeled in black; peptide residues are labeled in red and underlined. (D) A close‐up view of the ligand‐binding pocket of FrhASplit‐PBD, shown in orange, bound to AGYTD. The peptide AGYTD is shown in white, with oxygen atoms in red and nitrogen atoms in blue. Hydrogen bonds are shown as gray dashed lines, and ionic bonds between Ca2+ ions (green) and oxygen atoms are shown as black dashed lines. Atomic models of amino acid residue hydrogen bonds to AGYTD were overlaid on the ribbon model. The N‐terminal alanine residue of AGYTD was not clearly resolved. Protein residues are labeled with one‐letter codes and sequence positions (black), while peptide residues are labeled with three‐letter codes (red). (E) Back view of the binding pocket shown in (D) following a 180° rotation around the vertical axis.
Anti‐adhesion therapies can target pathogen–host attachment by using inhibitors that block specific bacterial adhesins [25, 26]. By preventing host tissue engagement, the inhibitors can impede initial colonization that leads to pathogenesis [25, 27], promote the dissociation of bacteria that are already attached to the host tissues, and attenuate the expression of virulence factors [1, 3, 27, 28, 29]. The anti‐adhesion strategy has shown efficacy in multiple models against diverse bacterial infections and may impose less selection pressure for antimicrobial resistance than conventional antibiotics [4, 25, 30].
In V. cholerae, several lines of evidence support the feasibility of the adhesin‐targeted inhibition. Soluble chitooligosaccharides [31], human milk oligosaccharides [32, 33], and synthetic L‐fucose derivatives [34] have all been shown to inhibit V. cholerae adhesion to intestinal tissues. The inhibitory effects of L‐fucose on adhesion in vivo are supported and explained by the mechanistic finding that the CBM of FrhA specifically binds to fucosylated glycans [23]. Collectively, carbohydrate adhesin inhibitors have shown potential for reducing adhesion, but do not completely abolish attachment. Targeting additional adhesins and their ligand‐binding domains, such as FrhA‐PBD, could improve the therapeutic effectiveness of anti‐adhesion therapies against V. cholerae.
In designing the first inhibitors of FrhA‐PBD, we leveraged knowledge of the homologous adhesin MpIBP that binds the Antarctic bacterium Marinomonas primoryensis to diatoms and ice [12, 35]. Structure‐guided design and iterative screening demonstrated that the peptide‐binding domain of MpIBP (MpIBP‐PBD), which shares over 60% sequence identity with FrhA‐PBD, preferentially binds to the C‐terminal residues of pentapeptides: Ala‐Gly‐Tyr‐Thr‐Asp (AGYTD, dissociation constant K d ≈ 30 nm), Ala‐Gly‐Tyr‐Thr‐Ser (AGYTS, K d ≈ 26 nm), and Ala‐Gly‐Trp‐Thr‐Asp (AGWTD, K d ≈ 23 nm) [35]. Co‐crystal structures of MpIBP‐PBD bound to AGYTD revealed that the C‐terminal aspartate residue is coordinated by two Ca^2+^ ions in the binding pocket through the terminal α‐carboxylate oxygens [35]. The penultimate threonine (the second residue from the C terminus) and the pre‐penultimate tyrosine residues (the third residue from the C terminus) of the peptide engage MpIBP‐PBD via hydrogen‐bond networks. Additionally, the side chain of the peptide pre‐penultimate tyrosine interacts with the MpIBP‐PBD tyrosine (Y294) via hydrophobic packing [35]. Considering FrhA‐PBD shares high overall sequence identity with MpIBP‐PBD with conserved residues lining the binding pocket (Fig. S1), AGYTD was repurposed to target FrhA‐PBD [12, 36, 37]. Binding assays in vitro confirmed that AGYTD inhibited FrhA‐mediated adhesion to human red‐blood cells and epithelial (HEp2) cells and disrupted biofilm formation of V. cholerae [12]. Furthermore, in the infant mouse competition assay, 500 μm AGYTD showed moderate inhibitory effects on colonization of the small intestine by V. cholerae [12].
The relatively low efficacy and poor biological stability of short peptide inhibitors such as AGYTD pose major challenges for developing pharmacological candidates. Maintaining the high concentrations required for effective inhibition would be challenging in the human intestinal tract, which is rich in proteases and peptidases that rapidly degrade peptides composed of natural L‐amino acids [12, 38]. To enable the rational design of inhibitors with improved affinity and enzymatic stability, we determined the molecular basis of adhesin‐ligand interactions by solving the FrhA‐PBD–peptide crystal structures. Guided by these structural insights, we designed shorter peptides incorporating D‐amino acids that exhibit enhanced binding affinity and are predicted to resist enzymatic degradation [39]. Our results thus provide a foundation for developing potent inhibitors to block V. cholerae adhesion for attenuating virulence while lowering the risk of developing antimicrobial resistance [4, 25, 30].
Materials and methods
Expression and purification of the FrhASplit
‐PBD construct
The gene encoding the FrhA_Split‐PBD_ construct was cloned between the NdeI and XhoI sites of a pET28a expression vector posessing an N‐terminal 6× His‐tag. Transformed Escherichia coli BL21DE3 (ThermoFisher) cells were grown in LB medium at 37 °C and subsequently at 23 °C in the presence of 1 mm IPTG (Bioshop) for protein overexpression as previously described [23, 40]. The protein was purified based on previously published protocols [23, 40], using a Ni‐NTA affinity chromatography column and a Superdex™ 200 Increase 10/300 GL (Cytiva) size‐exclusion column.
Peptide synthesis
All peptides used in X‐ray crystallography, ITC measurements, and MST experiments were purchased from GenicBio (Shanghai, China). [35] Representative data from mass spectrometry performed by the vendor support purity > 96% (Fig. S2).
Isothermal titration calorimetry
Isothermal titration calorimetry (ITC) measurements were performed at 20 °C using an iTC_200_ microcalorimeter (Malvern Instruments, Northampton, MA). FrhA_Split‐PBD_ was dialyzed overnight against a buffer (50 mm Tris/HCl, pH 9, 150 mm NaCl, 5 mm CaCl_2_) and diluted to a concentration of 20 μm. Lyophilized AGYTD was weighed and solubilized in the same buffer to a concentration of 200 μm.
Each titration consisted of 29 injections. For each injection, 1.3 μL of 200 μm peptide AGYTD solution was added into 20 μm FrhA_Split‐PBD_, using 700 RPM stirring and a 180‐s interval between injections. The resulting binding isotherm was fitted using Origin 7.0 software (OriginLab Corp., Northampton, MA) to determine the binding parameters for the interaction.
Microscale thermophoresis
Microscale thermophoresis (MST) was performed in the presence of the fluorescently labeled FrhA_Split‐PBD_ using unlabeled ligands such as AGYTD. FrhA_Split‐PBD_ was labeled using the primary amine‐based labeling kit of Red‐NHS 2nd Generation (NanoTemper Technologies, San Francisco, CA, USA). The labeling reaction was performed in MST buffer (50 mm HEPES pH 8, 150 mm NaCl, 5 mm CaCl_2_) with a protein concentration of 20 μm (molar ratio dye:protein ≈ 3:1). The reaction mixture was incubated at room temperature for 1 h in a dark environment for protection from photobleaching. Subsequently, the unreacted dye was removed from the labeled protein using the supplied desalting column, which was equilibrated with MST buffer. The degree of labeling (DOL) was determined using UV/Vis spectrophotometry at 650 and 280 nm—a DOL of 0.5 was typically achieved.
After dialysis of the labeled protein against a buffer containing 50 mm Tris/HCl pH 9, 150 mm NaCl, 5 mm CaCl_2_, and 0.05% v/v Tween‐20, the post‐dialysis buffer was filtered and used to solubilize the ligands. A series of sixteen 1:1 dilutions of the ligands was prepared, resulting in ligand concentrations ranging from 440 μm to 13.4 nm. After 1 h incubation, the samples were loaded into standard monolith NT.115 capillaries from NanoTemper Technologies. The MST measurements were conducted using a Monolith NT.115 instrument at an ambient temperature of ~22 °C, with instrument parameters set to 20% excitation power and 40% MST power. Three independently prepared replicates were performed for each ligand. The NanoTemper MO.Affinity Analysis software (version 2.3, NanoTemper Technologies) was used to analyze the fluorescence data, fit sigmoidal binding models, and provide the dissociation constants. In the software, the K d values were determined by plotting the change in normalized fluorescence [ΔF_norm_(‰) = F_1_/F_0_] against the logarithm of the concentrations of peptide ligands, where F_1_ and F_0_ respectively correspond to the heated and baseline regions of the thermophoresis traces [41]. The time between F_1_ and F_0_, was set to 20 s for analysis.
X‐ray crystallography and model building
The FrhA_Split‐PBD_ protein construct was respectively incubated with ligand AGYTD and AGWTD in 0.16 M calcium acetate, 0.08 m sodium cacodylate, and 24.4% (w/v) PEG 8000. The protein construct produced thick, triangular crystals after one week. Diffraction data were collected at the CMCF‐IM beamline of the Canadian Light Source synchrotron. The phase problem was solved using molecular replacement with the MpIBP‐PBD domains (PDB ID: 6X5W). The initial models were improved by rounds of refinement in Phenix [42, 43] and manual model building in Coot [44, 45]. Residues S1127 and F1439 were modeled in the final X‐ray crystal structures [12]. Figures relevant to structural studies were prepared using ChimeraX 1.9 [46, 47].
Results
The pentapeptide AGYTD binds the PBD of FrhA with nanomolar affinity
Previously, we demonstrated that the pentapeptide AGYTD inhibits V. cholerae hemagglutination [35] and intestinal colonization, albeit at relatively high micromolar concentrations [12]. To determine whether AGYTD directly binds the PBD of FrhA, in vitro characterization of the FrhA‐PBD is required. Because the isolated single‐domain FrhA‐PBD exhibited a strong tendency to aggregate, we recombinantly produced a 38‐kDa two‐domain construct (FrhA_Split‐PBD_) containing the PBD and the adjacent “split” domain to reduce the propensity for aggregation (Fig. 1A). The N‐terminally His‐tagged FrhA_Split‐PBD_ was purified to homogeneity through a combination of Ni^2+^‐NTA affinity chromatography and size‐exclusion chromatography.
To validate the direct binding of AGYTD to FrhA_Split‐PBD_, we performed isothermal titration calorimetry (ITC). The peptide bound FrhA_Split‐PBD_ with submicromolar affinity (K d of ~370 ± 10 nm) in a 1:1 stoichiometry (Fig. 1B). The stoichiometry is consistent with the structural data from the homologous MpIBP, which contains a single Ca^2+^‐dependent peptide‐binding site.
The structure of FrhASplit
‐PBD ‐AGYTD complex reveals the molecular basis of protein‐peptide interactions
Although AGYTD binds FrhA_Split‐PBD_ with submicromolar affinity, high micromolar concentrations of the peptide are required in vivo to modestly reduce V. cholerae colonization in the mouse intestine [12]. This limitation underscores the need for inhibitors with higher potency as well as greater metabolic stability to withstand the peptidases and proteases in intestinal fluid. To gain structural insights into the PBD‐peptide interaction, we crystallized FrhA_Split‐PBD_ in complex with AGYTD and analog AGWTD, respectively, using under‐oil microbatch methods (Table S1) [35]. We solved the structures of these FrhA_Split‐PBD_‐peptide complexes using molecular replacement with MpIBP domains as search models. The FrhA_Split‐PBD_‐AGYTD complex was resolved to 2.4 Å resolution in the space group of P1, with six symmetry‐related molecules in the unit cell (Fig. 1C). The split and PBD domains adopted the same overall fold as those in MpIBP, as both formed oblong β‐sandwich structures. The cores of the domains were composed mainly of antiparallel β‐strands with flexible loops at the extremities. Each protein chain contained six Ca^2+^ ions: two in the ligand‐binding pocket, three at another site in the PBD, and one buried in the split domain.
The overall mode of peptide engagement was conserved between the homologous MpIBP‐PBD and FrhA‐PBD binding pockets. Key interactions included ionic coordination between the two PBD Ca^2+^ ions and the carboxylate group of the C‐terminal aspartate (AGYT D , Fig. 1C–E) as well as hydrogen bonding from the penultimate threonine hydroxyl group (AGY T D) [35]. However, notable differences were observed at the pre‐penultimate position: the MpIBP‐PBD*–*AGYTD structure showed that the pre‐penultimate tyrosine residue (AG Y TD) projected upward to form a stabilizing hydrophobic contact with a conserved tyrosine (Y294) near the binding pocket (Fig. S3) [35]. In contrast, in FrhA_Split‐PBD_, the peptide tyrosine residue points downward and does not participate in hydrophobic packing (Fig. 1C, Fig. S4). Furthermore, the glycine residue at the fourth position (A G YTD) remains solvent‐exposed and did not interact with the protein. The alanine residue at the fifth position ( A GYTD) was not resolved, suggesting that it has a high degree of flexibility (Fig. 1C). Together, these observations indicated that the two N‐terminal residues of AGYTD may be dispensable for binding, and that shorter peptide fragments may retain affinity while reducing molecular complexity.
Tripeptide YTD is a stronger binder than the pentapeptide AGYTD
Considering that structural analysis indicated that the binding pocket engaged only the three C‐terminal residues of AGYTD, we tested shorter peptide variants (GYTD, YTD, and TD; GenicBio) for binding affinity to FrhA_Split‐PBD_. To circumvent the requirement for large quantities of pure protein and ligands and the labor‐intensive setup of ITC [41], we used microscale thermophoresis (MST) as a higher‐throughput alternative. MST determines binding by detecting fluorescence changes in a temperature gradient and requires only one binding partner to be fluorescently labeled. In our assay, FrhA_Split‐PBD_ was fluorescently labeled on the side‐chain primary amines of lysine residues that are located away from the PBD binding site.
To validate this workflow, we first measured the binding of AGYTD, which yielded a K_d_ of ~500 ± 140 nm, consistent with the ITC results (370 ± 10 nm). Among the truncated variants, the tetrapeptide GYTD bound weaker (K d ≈ 1.1 ± 0.37 μm), but the tripeptide YTD bound significantly more tightly (K d ≈ 190 ± 62 nm) than AGYTD (Fig. 2A,B, Fig. S5). In contrast, the dipeptide TD showed no detectable binding, underscoring the critical role of the tyrosine residue in PBD recognition. The tripeptide motif YTD retains high affinity while minimizing molecular complexity, providing a promising template for small‐molecule mimics.
Binding of FrhASplit‐PBD to the pentapeptide AGYTD and variants measured by MST. (A–H) Thermophoretic mobility traces and dose–response binding curves for reactions of FrhASplit‐PBD binding to various groups of ligands: AGYTD (dark red), GYTD (dark green), YTD (yellow), TD (blue), AGYTd (purple), GyTD (light green), and yTD (orange). (A, C, E, G) Thermophoretic mobility traces of the microscale thermophoresis (MST) reactions depict individual traces for each ligand concentration and highlight the F0 (blue) and F1 (red) regions used to calculate binding. (B, D, F, H) Dose–response binding curves for fluorescently labeled FrhASplit‐PBD with the ligands. Data points are the mean from three independent experiments, plotted as ΔFnorm (F1/F0) versus ligand concentration (in M). Error bars show standard deviation. Solid lines were fitted using the law of mass action to derive K d values (n value = 3).
Replacement of L‐tyrosine with its D‐enantiomer at the pre‐penultimate position enhances ligand affinity for the PBD
Having established that the tripeptide YTD improved binding affinity, we sought to enhance peptide stability against enzymatic degradation in the gastrointestinal tract. To resist hydrolysis by carboxypeptidases [48], which cleave peptides from the C termini, we substituted the terminal L‐aspartic acid with its D enantiomer. This modification yielded the peptide Ala‐Gly‐Tyr‐Thr‐D‐Asp (AGYT d ; lowercase letter indicates residues in D configuration), which bound FrhA_Split‐PBD_ with significantly reduced affinity compared to AGYTD (K d ≈ 3.0 ± 1.6 μm vs 500 ± 140 nm; Fig. 2C,D). This loss of affinity was expected, because the stereochemical inversion reorientated the terminal aspartate side chain and likely disrupted coordinating hydrogen bonds in the binding pocket.
We then tested whether stereochemical modification at the pre‐penultimate residue could enhance binding. Substituting L‐ with D‐tyrosine in the tetrapeptide GyTD was hypothesized to reorient the aromatic side chain, potentially enabling contact between the peptide tyrosine and Y1229 in FrhA_Split‐PBD_. Indeed, this substitution improved binding by approximately sevenfold with a K d of ~220 ± 69 nm from GYTD (K d ≈ 1.1 ± 0.37 μm; Fig. 2E,F).
Encouraged by this result, we next examined the tripeptide yTD by removing the N‐terminal glycine residue (Fig. 2G,H). MST measurements showed that yTD binds with a K d of ~71 ± 19 nm, greater than two‐fold stronger than that of YTD (K d ≈ 190 ± 62 nm) and greater than sevenfold stronger than the initial pentapeptide lead AGYTD. This single modification conferred two key advantages: enhanced inhibitory potency and predicted resistance to degradation by aminopeptidases and related enzymes due to the D‐amino acid at the N‐terminus [49].
Having established the affinity‐enhancing effects of truncation to a tripeptide and stereochemical inversion of the aromatic amino acid residue, we next asked whether similar modifications could enhance binding in a different ligand. To explore this, we turned to AGWTD (K d ≈ 380 ± 100 nm to FrhA_Split‐PBD_), another high‐affinity pentapeptide binder of MpIBP‐PBD, for truncation and D‐amino acid substitution. Structural analysis of the FrhA_Split‐PBD_‐AGWTD complex revealed a peptide conformation like that of AGYTD (Fig. 3A,B). Notably, no interaction was observed between the peptide tryptophan and Y1229 of the protein. Truncating AGWTD to the tripeptide WTD increased binding affinity to a K d of ~200 ± 46 nm compared to the parent pentapeptide (K d ≈ 380 ± 100 nm, Fig. 3C,D). Substituting the tryptophan in the tripeptide with its D‐enantiomer further strengthened binding, improving the affinity to a K d of ~120 ± 41 nm.
Binding of FrhASplit‐PBD to the pentapeptide AGWTD and variants measured by MST. (A) Zoomed‐in views of the ligand‐binding site of the 2.4‐Å X‐ray crystal structure of the FrhASplit‐PBD–AGWTD complex (PDB ID: 9Y9W). The PBD is shown in orange, Ca2+ ions in green, and the peptide in white. The same color scheme was used as shown in in Fig. 1. (B) Back view of the binding pocket shown in (A) after a 180° rotation around the vertical axis. The experimental electron density of AGWTD is shown as a light blue mesh. (C, D) Thermophoretic mobility traces and dose–response binding curves for reactions of FrhASplit‐PBD binding AGWTD (dark blue), WTD (light blue), and wTD (red). (C) Thermophoretic mobility traces of the microscale thermophoresis (MST) reactions depict individual traces for each ligand concentration and highlight the F0 (blue) and F1 (red) regions used to calculate binding. (D) Dose–response binding curves for fluorescently labeled FrhASplit‐PBD with representative ligands. Data points are the mean from three independent experiments, plotted as ΔFnorm (F1/F0) versus ligand concentration (in M). Error bars show standard deviation. Solid lines were fitted using the law of mass action to derive K d values (n value = 3).
Together, these results establish that D‐amino acid substitution at the pre‐penultimate position can enhance both affinity and potential proteolytic stability across multiple peptide series (Fig. 4A,B; Table 1). The identification of compact tripeptide motifs such as D‐Tyr‐Thr‐Asp and D‐Trp‐Thr‐Asp highlights a general strategy for creating small scaffolds that bind the FrhA_Split‐PBD_ with high affinity and lays a foundation for developing peptidomimetics and non‐peptide inhibitors of the PBD with improved pharmacokinetic properties.
Tripeptide yTD showed a sevenfold increase in affinity compared to the parent pentapeptide AGYTD. (A) Dose–response binding curves for fluorescently labeled FrhASplit‐PBD with the ligands AGYTD (dark red), GyTD (light green), YTD (yellow), and yTD (orange), as shown in previous figures. Data points are the mean from three independent experiments, plotted as ΔFnorm (F1/F0) versus ligand concentration (in M). Error bars show standard deviation (n value = 3). Solid lines were fitted using the law of mass action to derive K d values. (B) Summary of the binding affinities of peptidyl ligands to FrhASplit‐PBD. The peptides were shown with decreasing numbers of amino acid residues from left to right. Error bars show K d confidence interval (1 standard deviation). The bracket with an asterisk indicates the statistically significant difference in the binding affinities of AGYTD and yTD determined by an unpaired t‐test with a false discovery rate set below 0.01.
Discussion
Despite decades of progress, the development of anti‐adhesion treatments remains challenging [26, 27]. To have clinical utility, an inhibitor must target the adhesin with high specificity and binding affinity, as well as remain active at physiologically relevant concentrations by resisting degradation in the protease‐rich intestinal environment [25, 38]. Using a recombinant FrhA_Split‐PBD_ construct, we validated direct peptide binding in vitro, complementing previous in vivo evidence from the infant mouse model [12]. X‐ray crystal structures of FrhA_Split‐PBD_ in complex with ligands guided the rational design of shorter derivatives with improved affinity. In parallel, the selective incorporation of D‐amino acids further enhanced potency and provided a strategy to improve predicted inhibitor stability against proteases [39].
After measuring the affinity of the starting peptide AGYTD by ITC, we prioritized MST because it requires far less protein than ITC and enabled rapid and reproducible results for screening the relative potency of peptide variants [41]. Systematic truncation of AGYTD into GYTD, YTD, and TD revealed a non‐linear relationship between peptide length and binding affinity; GYTD exhibited weaker binding than AGYTD (Fig. 2A,B). Tripeptide YTD exhibited strong binding to FrhA_Split‐PBD_. Removal of the pre‐penultimate tyrosine residue abolished binding in the dipeptide TD (Fig. 2A,B). This loss can be explained by the absence of hydrophobic interactions provided by the tyrosine side chain and the elimination of hydrogen bonds from its main‐chain carbonyl oxygen [50]. More importantly, the more polar peptide TD potentially has an unfavorable desolation energy associated with displacing structured water from the peptide and pocket, abolishing binding [51, 52, 53].
Having established the contributions of peptide length for binding through truncation, we next examined how stereochemical changes at specific positions impact peptide–PBD interactions. The effects of D‐amino acid substitution on peptide binding affinity were position‐dependent, reflecting the local structural environment within the binding pocket. Substitution of L‐ for D‐aspartic acid at the C terminus greatly reduced binding affinity. In the X‐ray crystal structure of the FrhA_Split‐PBD_–AGYTD complex, the C‐terminal carboxylate group of the aspartic acid coordinates two Ca^2+^ ions in the binding pocket; one carboxylate oxygen and the backbone carbonyl oxygen of the aspartic acid form hydrogen bonds with the protein residues V1228 and S1230, respectively (Fig. 2D,E). Inverting stereochemistry at this critical Asp residue alters the orientation of both the terminal and side‐chain carboxylates, disrupting the geometry required for the bonding network. In contrast, D‐tyrosine substitution at the pre‐penultimate position ( y TD) was well tolerated, because inverting the stereochemistry of this residue minimally affected Ca^2+^ coordination as well as the hydrogen‐bond network.
Future work is merited to optimize the minimal yTD scaffold as a structural template that captures the essential interactions for strong PBD binding. Incorporation of D‐amino acids may offer a promising route to enhance proteolytic stability, because antimicrobial peptides possessing D‐residues are known to resist cleavage by intestinal proteases such as trypsin and chymotrypsin [54, 55]. The peptide yTD is expected to be more resistant to intestinal enzymes and to persist longer in the intestinal lumen [38, 49]. Future studies could test this prediction using in vitro enzyme digestion assays in simulated intestinal fluids coupled with mass spectrometric analysis to quantify degradation and confirm stability.
Looking ahead, future therapeutic strategies may employ a cocktail of adhesin‐specific inhibitors to achieve broad‐spectrum anti‐adhesion coverage across V. cholerae biotypes [26, 27]. The epidemiologically relevant El Tor biotype, responsible for the seventh and ongoing cholera pandemic, potentially expresses different adhesins than the classical biotype [56, 57]. The role of FrhA in adhesion in the El Tor biotype remains poorly defined, whereas other adhesins, such as the mannose‐sensitive hemagglutinin (MSHA) pilus [56, 58, 59] and the toxin‐coregulated pilus (TCP) [60, 61, 62], are more prominent in this strain and contribute to virulence. In this context, the inhibitors of FrhA‐PBD may serve as one component of a broader anti‐adhesion strategy that also includes inhibitors of FrhA‐CBM and other adhesins like MSHA, to achieve broad‐spectrum anti‐adhesion coverage across V. cholerae biotypes [26]. Overall, this multi‐inhibitor strategy could offer a viable adjunct or alternative to treatment by conventional antibiotics in clinically relevant settings.
Author contributions
SG, WDL, PLD, and MW conceived the research; MW, BK, GD, KM, AL, CY, and SG performed the experiments; MW and SG wrote the manuscript. All authors contributed to the editing and revision of the manuscript.
Supporting information
Fig. S1. Protein sequence alignment of the PBD and split domains of FrhA with their homologs in MpIBP. Fig. S2. Mass spectrum of AGYTD. Fig. S3. Binding pockets of X‐ray crystal structures of MpIBP‐PBD and FrhA_Split‐PBD_ binding to AGYTD. Fig. S4. Alternative views of the electron density of AGYTS in complex with FrhA_Split‐PBD_. Fig. S5. ANOVA of binding affinities of ligands to FrhA‐PBD. Table S1. X‐ray and refinement statistics.
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