Structural Basis of Ca2+-Dependent Self-Processing Activity of Repeat-in-Toxin Proteins.

Vojtech Kuban,Peter Sebo,Ladislav Bumba,Martin Faldyna,Pavel Macek,Katerina Nechvatalova,Jozef Hritz,Katerina Nedbalcova,Lukas Zidek,Carmen Buchrieser

doi:10.1128/mbio.00226-20

Abstract

The posttranslational Ca2+-dependent "clip-and-link" activity of large repeat-in-toxin (RTX) proteins starts by Ca2+-dependent structural rearrangement of a highly conserved self-processing module (SPM). Subsequently, an internal aspartate-proline (Asp-Pro) peptide bond at the N-terminal end of SPM breaks, and the liberated C-terminal aspartyl residue can react with a free ε-amino group of an adjacent lysine residue to form a new isopeptide bond. Here, we report a solution structure of the calcium-loaded SPM (Ca-SPM) derived from the FrpC protein of Neisseria meningitidis The Ca-SPM structure defines a unique protein architecture and provides structural insight into the autocatalytic cleavage of the Asp-Pro peptide bond through a "twisted-amide" activation. Furthermore, in-frame deletion of the SPM domain from the ApxIVA protein of Actinobacillus pleuropneumoniae attenuated the virulence of this porcine pathogen in a pig respiratory challenge model. We hypothesize that the Ca2+-dependent clip-and-link activity represents an unconventional strategy for Gram-negative pathogens to adhere to the host target cell surface.IMPORTANCE The Ca2+-dependent clip-and-link activity of large repeat-in-toxin (RTX) proteins is an exceptional posttranslational process in which an internal domain called a self-processing module (SPM) mediates Ca2+-dependent processing of a highly specific aspartate-proline (Asp-Pro) peptide bond and covalent linkage of the released aspartyl to an adjacent lysine residue through an isopeptide bond. Here, we report the solution structures of the Ca2+-loaded SPM (Ca-SPM) defining the mechanism of the autocatalytic cleavage of the Asp414-Pro415 peptide bond of the Neisseria meningitidis FrpC exoprotein. Moreover, deletion of the SPM domain in the ApxIVA protein, the FrpC homolog of Actinobacillus pleuropneumoniae, resulted in attenuation of virulence of the bacterium in a pig infection model, indicating that the Ca2+-dependent clip-and-link activity plays a role in the virulence of Gram-negative pathogens.

Highlights

The posttranslational Ca2ϩ-dependent “clip-and-link” activity of large repeat-in-toxin (RTX) proteins starts by Ca2ϩ-dependent structural rearrangement of a highly conserved self-processing module (SPM)
It consists of Ca2ϩ-induced autocatalytic cleavage of a specific Asp-Pro peptide bond within a polypeptide precursor that generates a reactive C-terminal Asp anhydride, which can react with an ␧-amino group of an adjacent lysine residue to form a new covalent isopeptide bond [1]
The spectrum did not contain residual peaks typical for the 1H-15N heteronuclear single-quantum coherence (HSQC) spectrum of the Ca2ϩ-depleted SPM (Fig. 1B, black contours), documenting that the calcium-loaded SPM (Ca-SPM) preparation was free of residual unfolded protein

Summary

Introduction

The posttranslational Ca2ϩ-dependent “clip-and-link” activity of large repeat-in-toxin (RTX) proteins starts by Ca2ϩ-dependent structural rearrangement of a highly conserved self-processing module (SPM). IMPORTANCE The Ca2ϩ-dependent clip-and-link activity of large repeat-in-toxin (RTX) proteins is an exceptional posttranslational process in which an internal domain called a self-processing module (SPM) mediates Ca2ϩ-dependent processing of a highly specific aspartate-proline (Asp-Pro) peptide bond and covalent linkage of the released aspartyl to an adjacent lysine residue through an isopeptide bond. The Ca2ϩ-dependent “clip-and-link” activity represents a distinct type of posttranslational autoprocessing of proteins It consists of Ca2ϩ-induced autocatalytic cleavage of a specific Asp-Pro peptide bond within a polypeptide precursor that generates a reactive C-terminal Asp anhydride, which can react with an ␧-amino group of an adjacent lysine residue to form a new covalent isopeptide bond [1]. This is the first demonstration that the Ca2ϩ-dependent clip-and-link activity of large RTX proteins plays a role in the virulence of Gram-negative pathogens

Methods

Results

Conclusion