Mycobacterium tuberculosis EsxA and EsxB proteins are founding members of the WXG100 (WXG) protein family, characterized by their small size (ϳ100 amino acids) and conserved WXG amino acid motif. M. tuberculosis contains 11 tandem pairs of WXG genes; each gene pair is thought to be coexpressed to form a heterodimer. The precise role of these proteins in the biology of M. tuberculosis is unknown, but several of the heterodimers are secreted, which is important for virulence. However, WXG proteins are not simply virulence factors, since nonpathogenic mycobacteria also express and secrete these proteins. Here we show that three WXG heterodimers have structures and properties similar to those of the M. tuberculosis EsxBA (MtbEsxBA) heterodimer, regardless of their host species and apparent biological function. Biophysical studies indicate that the WXG proteins from M. tuberculosis (EsxG and EsxH), Mycobacterium smegmatis (EsxA and EsxB), and Corynebacterium diphtheriae (EsxA and EsxB) are heterodimers and fold into a predominately ␣-helical structure. An in vivo protein-protein interaction assay was modified to identify proteins that interact specifically with the native WXG100 heterodimer. MtbEsxA and MtbEsxB were fused into a single polypeptide, MtbEsxBA, to create a biomimetic bait for the native heterodimer. The MtbEsxBA bait showed specific association with several esx-1-encoded proteins and EspA, a virulence protein secreted by ESX-1. The MtbEsxBA fusion peptide was also utilized to identify residues in both EsxA and EsxB that are important for establishing protein interactions with Rv3871 and EspA. Together, the results are consistent with a model in which WXG proteins perform similar biological roles in virulent and nonvirulent species.The WXG100 (WXG; pfam06013) proteins are a class of effector molecules found in gram-positive bacteria (26). WXG proteins are characterized by their small size (ϳ 100 amino acids [aa]) and the presence of a WXG motif, or its structural equivalent, near the midpoint of their primary sequence (26). Bioinformatic analyses have shown that one WXG gene is frequently positioned near, or directly adjacent to, a second, related, WXG gene (14). The gene pairs characterized thus far encode proteins that associate to form 1:1 complexes (20, 31). The WXG proteins were once thought to be restricted to the mycobacteria, but homologues have now been detected in species of Bacillus, Listeria, Streptomyces, and Corynebacterium, among others, and the Pfam server lists Ͼ89 distinct WXGencoding species and strains (10).The identification of WXG proteins encoded by the pathogens Mycobacterium tuberculosis (15,17,19,36), Mycobacterium marinum (13), and Staphylococcus aureus (5) has created significant interest in the proteins' biological activity. Nevertheless, these proteins are not a priori virulence factors (39), since organisms expressing WXG proteins are not necessarily capable of causing disease. In addition to pathogenesis, the WXG proteins are associated with processes as disparate as zinc ...