Structural and functional insights into the interaction between a PP01 phage gp38 tail fiber tip and an Escherichia coli OmpC receptor

TL;DR

This study reveals how phage PP01 specifically binds to E. coli O157 by analyzing the structure and function of its tail fiber protein and its interaction with a bacterial surface protein.

Contribution

The paper identifies specific molecular contacts and structural features that govern phage PP01's host specificity through experimental and computational methods.

Findings

The crystal structure of gp38PP01's receptor-binding domain shows a unique architecture with receptor recognition loops.

Two key interaction sites were identified between gp38PP01 and OmpCO157 using photo-crosslinking and mass spectrometry.

A structural model of the phage–bacterial receptor complex was validated through mutagenesis experiments.

Abstract

Bacteriophages exhibit strict host specificity, primarily determined by adsorption to bacterial surface receptors. However, the molecular basis underlying this specificity remains incompletely understood. Here, we investigate the interaction between outer membrane protein C (OmpC) of Escherichia coli O157 and gp38, the receptor-binding protein located at the tip of the long tail fibers of phage PP01. We determined the crystal structure of the receptor-binding domain (RBD) of gp38PP01 at 2.1 Å resolution. The structure reveals a lattice of poly-glycine type II helices with protruding receptor recognition loops, resembling that of gp38 from Salmonella phage S16. To identify interaction sites, we performed site-specific photo-crosslinking using p-benzoyl-L-phenylalanine (pBPA), followed by liquid chromatography–tandem mass spectrometry. Two critical contacts were identified: Gly208 in loop-D of gp38PP01 crosslinked to Ser225 and Pro226 in extracellular loop-5 of OmpCO157, and Tyr230 in loop-E of gp38PP01 to the Val304–Arg308 region in loop-7 of OmpCO157. A structural model of the gp38PP01–OmpCO157 complex was constructed using distance-constrained prediction and validated by targeted mutagenesis. Our findings demonstrate that PP01 phage specificity is governed by loop-E of gp38PP01 engaging a cleft formed by loops -5 and -7 of OmpCO157. These structural and functional insights enhance our understanding of phage–host recognition and may inform the rational design of engineered bacteriophages with altered host ranges.

Bacteriophages must precisely recognize and bind to specific molecules on the surface of their bacterial hosts to initiate infection, but the details of these interactions are often unclear. In this study, we examined how phage PP01 targets Escherichia coli O157. Using structural analysis of the phage tail fiber and a technique to capture contact points between the phage and a bacterial surface protein, we mapped the molecular basis of host recognition. We also developed a simple test system using a modified phage to identify which parts of the tail fiber are essential for binding. These methods can be broadly applied to other phages to better understand how they select their hosts. This work provides valuable insights and tools that could aid the design of phages with customized host specificity for therapeutic or biotechnological applications.

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