The crystal structure of the mycobacterium tuberculosis Rv3019c‐Rv3020c ESX complex reveals a domain‐swapped heterotetramer

Abstract

Mycobacterium tuberculosis encodes five gene clusters (ESX-1 to ESX-5) for Type VII protein secretion systems that are implicated in mycobacterial pathogenicity. Substrates for the secretion apparatus are encoded within the gene clusters and in additional loci that lack the components of the secretion apparatus. The best characterized substrates are the ESX complexes, 1:1 heterodimers of ESAT-6 and CFP-10, the prototypical member that has been shown to be essential for Mycobacterium tuberculosis pathogenesis. We have determined the structure of EsxRS, a homolog of EsxGH of the ESX-3 gene cluster, at 1.91 Å resolution. The EsxRS structure is composed of two four-helix bundles resulting from the 3D domain swapping of the C-terminal domain of EsxS, the CFP-10 homolog. The four-helix bundles at the extremities of the complex have a similar architecture to the structure of ESAT-6ÁCFP-10 (EsxAB) of ESX-1, but in EsxRS a hinge loop linking the a-helical domains of EsxS undergoes a loop-to-helix transition that creates the domain swapped EsxRS tetramer. Based on the atomic structure of EsxRS and existing biochemical data on ESX complexes, we propose that higher order ESX oligomers may increase avidity of ESX binding to host receptor molecules or, alternatively, the conformational change that creates the domain swapped structure may be the basis of ESX complex dissociation that would free ESAT-6 to exert a cytotoxic effect.

Heterologous Expression of Mycobacterial Esx Complexes in Escherichia coli for Structural Studies Is Facilitated by the Use of Maltose Binding Protein Fusions

“…The overall architecture of the complexes is similar to known Esx structures [9], [26], [39], [42] in that each heterodimer forms a four helix bundle with the CFP-10 and ESAT-6 subunit homologs each contributing an α-helical hairpin to the complex (Figure 3). The α-helices of each subunit are connected by a flexible loop containing the canonical ESAT-6/CFP-10 signature motif (Trp-Xaa-Gly; WXG) although the EsxG ms CFP-10 homolog contains a modified motif (His-Xaa-Gly).…”

Dimer recognition and secretion by the ESX secretion system inBacillus subtilis

“…Mycobacterial and staphylococcal substrates of ESX systems tend to dimerize similarly to the heterodimerization of prototypical M. tuberculosis substrates EsxA and EsxB (19)(20)(21)(22)(23)(24)32). In S. aureus both EsxA and EsxB homologs are present, but EsxA was shown to homodimerize similarly to the GBS1074 protein from Streptococcus agalactiae (21,22).…”

“…Two hairpins interact in an antiparallel configuration to form either homo-or heterodimers (Fig. S1B) (19)(20)(21)(22)(23)(24). This arrangement places the N and C termini of one subunit in close proximity with the WXG hairpin of the interacting partner.…”

“…S1A). All characterized WXG proteins share helix-turn-helix hairpin structures where the conserved WXG motif forms a sharp turn between the N-and C-terminal helices (19)(20)(21)(22)(23). Two hairpins interact in an antiparallel configuration to form either homo-or heterodimers (Fig.…”

Comparative Genomics ofesxGenes from Clinical Isolates of Mycobacterium tuberculosis Provides Evidence for Gene Conversion and Epitope Variation

“…Proteins encoded by two other paralogous gene pairs, EsxR-EsxS and EsxH-EsxG, also form 1:1 complexes, suggesting that this may be typical of all Esx protein couplets (8,32). Five of the 11 tandem gene pairs are contained within conserved genetic loci ESX-1 to ESX-5, encoding components of a type VII secretory apparatus (Table 1) (2,26,48).…”