Solution structure of the EsxG·EsxH complex. A, a best-fit superposition of the protein backbone for the family of 30 converged structures obtained for EsxG·EsxH, with EsxH shown in blue and EsxG shown in red. B, ribbon representations of the backbone topology of the EsxG·EsxH complex based on the converged structure closest to the mean, with EsxG shown in red and EsxH shown in blue. Panels A and B clearly show the two helix-turn-helix hairpin structures formed by the individual proteins and the flexible N and C termini of both proteins within the complex.

Mapping of the Zn²⁺ binding site on EsxG·EsxH. A, an overlay of two ¹⁵N/¹H HSQC spectra of uniformly ¹⁵N-labeled EsxG bound to unlabeled EsxH (100 μm) acquired in the presence (red) and absence (black) of equimolar amounts of Zn²⁺. B, an overlay of ¹⁵N-labeled EsxH bound to unlabeled EsxG in the presence (red) and absence (black) of Zn²⁺. For clarity, the region containing the four tryptophan indolic signals from EsxH is not shown. C and D, histograms of the minimal chemical shift changes observed for backbone amide groups of EsxG (C) and EsxH (D) on binding of Zn²⁺ to the EsxG·EsxH complex. The positions of the helices in the EsxG·EsxH complex are highlighted above the histograms. Residues that showed significant line broadening of backbone amide signals upon the addition of Zn²⁺ are shown as light gray bars. For clarity, the minimal shift shown for Gly-45 of EsxG has been truncated (minimal shift 0.8 ppm). E, surface views of the EsxG·EsxH complex where residues are colored according to the perturbation of the backbone amide signals induced by Zn²⁺ binding. Residues that showed a minimal shift of less than 0.02 ppm are shown in white, residues with a shift over 0.1 ppm are in red, and residues with a shift between 0.02 and 0.1 ppm are colored according to the magnitude of the shift on a linear gradient between white and red. Residues for which no data were obtained are shown in yellow. The structures in E are rotated by 180° about the x axis and −45°/135° about the y axis in comparison with the ribbon representation shown in Fig. 1. F, positions of key amino acid side chains, in the structure closest to the mean, which contribute to the formation of the Zn²⁺ binding site on the EsxG·EsxH complex. The three histidine residues (His-14, His-70, and His-76) are colored blue, and the glutamic acid residue (Glu-77) is shown in yellow. The family of NMR structures indicates that the side chains of residues His-14, His-76, and Glu-77 are fairly flexible; however, upon binding Zn²⁺, these side chains are likely to adopt a single orientation and become less flexible.

Surface view of the EsxG·EsxH complex, including identification and conservation of amino acids found within the EsxG·EsxH cleft. A, the surface of the complex is colored according to electrostatic potential (40), with areas of significant negative charge shown in red, areas of significant positive charge are shown in blue and neutral areas are shown in white. An interesting feature of the EsxG·EsxH complex is the cleft that is formed between the loop linking the helices of EsxG and the C-terminal arm of EsxH. The structures shown here are rotated by approximately −45°/135° about the y axis in comparison with the ribbon representation shown in Fig. 1. B, orientation of key amino acid side chains that contribute to the formation of the cleft on the surface of the EsxG·EsxH complex, where aliphatic residues with hydrophobic side chains (Ala, Ile, and Met) are shown in cyan, aromatic residues (His and Phe) are colored blue, and the polar residue (Ser) is shown in red. C and D show multiple sequence alignments highlighting the conservation of amino acids in EsxG (C) and EsxH (D) from M. tuberculosis, M. bovis, M. marinum, M. ulcerans, M. leprae, and Mycobacterium smegmatis. Residues forming the cleft are highlighted by light gray triangles, and conserved aromatic residues at the C-terminal of EsxG are highlighted by light gray circles. Dark gray triangles indicate residues from EsxH that form the Zn²⁺ binding site.

Comparison of EsxG·EsxH with family members EsxA·EsxB and EsxR·EsxS. A–C, equivalent views of ribbon representations of the solution structures of the heterodimeric EsxA·EsxB (A) and EsxG·EsxH (B) complexes, alongside the crystal structure of the heterotetrameric form of the EsxR·EsxS (C) complex. In EsxA·EsxB and EsxG·EsxH, the individual proteins form helix-turn-helix hairpin motifs, which are arranged antiparallel to each other, forming a four-helix bundle. In comparison, the domain-swapped heterotetramer of EsxR·EsxS is composed of two molecules of EsxR, which form helix-turn-helix hairpin motifs, and two molecules of EsxS, which form long antiparallel α-helices (8). The helices within the EsxG·EsxH complex, particularly the N-terminal helix of EsxH, are significantly shorter than the equivalent helices in the EsxA·EsxB complex. Among the most striking features of the EsxA·EsxB and EsxG·EsxH complexes are the flexible N and C termini of both proteins, which are not present in the EsxR·EsxS crystal structure. D and E show optimized sequence alignments of EsxG and EsxH with EsxA/B and EsxR/S, respectively. D, EsxG aligned with EsxB (32% identity) and EsxS (95% identity). E, EsxH aligned with EsxA (19% identity) and EsxR (85% identity). The α-helical regions observed in the solved complex structures are indicated by dark gray bars, and regions of 3₁₀ helix are indicated by light gray bars. Dark blue triangles indicate residues involved in intermolecular salt bridges, and light blue triangles show residues involved in intramolecular salt bridges. Residues are highlighted as follows: aliphatic residues with hydrophobic side chains (Ala, Ile, Leu, Met, and Val) are in red, and aromatic residues (Phe, Trp, and Tyr) are in yellow.