Structure-Function Analysis of VapB4 Antitoxin Identifies Critical Features of a Minimal VapC4 Toxin-Binding Module

Bacterial toxin-antitoxin systems play a critical role in the regulation of gene expression, leading to developmental changes, re-versible dormancy, and cell death. Type II toxin-antitoxin pairs, composed of protein toxins and antitoxins, exist in nearly all bacteria and are classified into six groups on the basis of the structure of the toxins. The VapBC group comprises the most com-mon type II system and, like other toxin-antitoxin systems, functions to elicit dormancy by inhibiting protein synthesis. Activa-tion of toxin function requires protease degradation of the VapB antitoxin, which frees the VapC toxin from the VapBC com-plex, allowing it to hydrolyze the RNAs required for translation. Generally, type II antitoxins bind with high specificity to their cognate toxins via a toxin-binding domain and endow the complex with DNA-binding specificity via a DNA-binding domain. Despite the ubiquity of VapBC systems and their critical role in the regulation of gene expression, few functional studies have addressed the details of VapB-VapC interactions. Here we report on the results of experiments designed to identify molecular determinants of the specificity of theMycobacterium tuberculosis VapB4 antitoxin for its cognate VapC4 toxin. The results iden-tify the minimal domain of VapB4 required for this interaction as well as the amino acid side chains required for binding to VapC4. These findings have important implications for the evolution of VapBC toxin-antitoxin systems and their potential as targets of small-molecule protein-protein interaction inhibitors. IMPORTANC