The structural analysis of the periplasmic domain of Sinorhizobium meliloti chemoreceptor McpZ reveals a novel fold and suggests a complex mechanism of transmembrane signaling

Abstract

Chemotaxis is a fundamental process whereby bacteria seek out nutrient sources and avoid harmful chemicals. For the symbiotic soil bacterium Sinorhizobium meliloti, the chemotaxis system also plays an essential role in the interaction with its legume host. The chemotactic signaling cascade is initiated through interactions of an attractant or repellent compound with chemoreceptors or methyl-accepting chemotaxis proteins (MCPs). S. meliloti possesses eight chemoreceptors to mediate chemotaxis. Six of these receptors are transmembrane proteins with periplasmic ligand-binding domains (LBDs). The specific functions of McpW and McpZ are still unknown. Here, we report the crystal structure of the periplasmic domain of McpZ (McpZPD) at 2.7 Å resolution. McpZPD assumes a novel fold consisting of three concatenated four-helix bundle modules. Through phylogenetic analyses, we discovered that this helical tri-modular domain fold arose within the Rhizobiaceae family and is still evolving rapidly. The structure, offering a rare view of a ligand-free dimeric MCP-LBD, reveals a novel dimerization interface. Molecular dynamics calculations suggest ligand binding will induce conformational changes that result in large horizontal helix movements within the membrane-proximal domains of the McpZPD dimer that are accompanied by a 5 Å vertical shift of the terminal helix toward the inner cell membrane. These results suggest a mechanism of transmembrane signaling for this family of MCPs that entails both piston-type and scissoring movements. The predicted movements terminate in a conformation that closely mirrors those observed in related ligand-bound MCP-LBDs.

Keywords: chemotaxis; helical tri-modular sensor domain; ligand-binding domain; methyl-accepting chemotaxis protein; piston; scissoring; transmembrane signaling.

Figure 1.. Structure of McpZ_PD.

a. Cartoon depiction of McpZ_PD. A single McpZ_PD molecule forms the asymmetric unit of the crystal. The three four-helix bundle subdomains are labeled according to their relative positions to the inner membrane of the cell. The length of the molecule and the angle between the central and distal FHBs are displayed. The figure was generated using Pymol . b. Topology diagram for McpZ_PD. c. Correlation between primary and secondary structure of McpZ_PD.

Figure 2.. Phylogeny and sequence conservation of McpZ

a. Distribution of HTM-containing chemoreceptors in the family Rhizobiaceae. The presence of HTM is marked by red circles on terminal branches of the Rhizobiaceae family genome tree. Branches shown in blue identify genera where chemotaxis systems are present. The likely origin of the HTM domain is marked by a red circle next to the longest internal branch. Numbers in brackets show the number of genomes in each clade. Asterisk indicates that only one genome is available in a given clade. These symbols are automatically generated by AnnoTree b. McpZ_PD colored according to sequence conservation. c. Packing of a single layer of McpZ_PD molecules inside the crystal. A single symmetric dimer is highlighted. d. Cartoon depiction of a McpZ_PD dimer. Molecules are colored according to sequence conservation using the same approach as in 2 b. The large highly conserved patch between the central and membrane-distal FHBs is now buried at the dimer interface. Figures b. and d. were generated with the Consurf server using the subset of McpZ homolog sequences also used in fig. 2a. The conservation scores were generated using the default Bayesian method.

Figure 3.. Structural comparison of McpZ_PD with McpS_PD and TorS_PD.

a. Left panel. Superposition of McpZ_PD and McpS_PD. Right panel. Conservation-colored surface depiction of the region in McpZ_PD, which is equivalent to the succinate binding pocket in the membrane-proximal FHB of McpS_PD. Conserved residues are labeled and their side chains are displayed. The succinate is faintly shown to mark the putative ligand binding pocket. b. Left panel. Superposition of McpZ_PD and TorS_PD. Right panel. TorT molecule (grey) modeled with a McpZ_PD dimer (blue and green). The model was created by superimposing a 1:1 TorS-TorT complex onto the McpZ_PD dimer. The TorS_PD molecules were then hidden to mark potential binding sites for periplasmic binding proteins on McpZ_PD.

Figure 4.. Results of MD calculations.

a. Superposition of the most prevalent conformations obtained the MD calculations with the original structure of the McpZ_PD dimer. RMSD values and percent of simulation time the dimers are observed in these prevalent conformations are indicated below. The final conformations of the three replicate runs are very similar with RMSD values ranging between 1.5 to 2.2 Å. b. Kink angles of helices 1 and 8 of McpZ. Comparison of initial structure (left)and the MD endpoint conformations (right). The hinge angle between the central of distal FHBs straightens, which in turn causes a straightening of the kinked H8 helix. The net results are a ~55⁰ clockwise rotation of the membrane proximal FHBs around and a ~5 Å downward shift of H8.

Figure 5.. Mechanistic implications of the distinctive dimerization interface of McpZ_PD in the membrane-proximal FHB region.

a. Dimerization interfaces of the ligand bound McpS_PD, PcaY_PD (PDB code: 6S3B), and TorS_PD viewed from the perspective of the inner membrane. N- and C-terminal helices, which connect to the transmembrane helices in the full-length proteins are color-coded blue and red, respectively. Numbers indicate the distances between the helices in Å b. MD-predicted scissoring motion. Left, N-terminal and C-terminal helices of the McpZ_PD dimer viewed from the perspective of the inner membrane. Right, MD calculations predict significant horizontal helical shifts of N- and C-terminal helices of the McpZ_PD dimer. We propose that the shifts occur upon ligand binding. Numbers indicate the distances between the helices in Å. c. MD-predicted piston-type motion. Left, side view of the membrane-proximal FHB of the McpZ_PD dimer. The indicated dihedral angle measures the H8-H1-H1-H8 angle between the termini of these helices. Right, MD calculations predict up toa 5.6 Å vertical shift of H8 toward the membrane of the McpZ_PD dimer, whereas H1 remains in the same plane. We propose that the shift occurs upon ligand binding. The vertical motion is also manifested in a nearly sixty degree shift of the H8-H1-H1-H8 dihedral angle between the termini of these helices. Angle values are averaged across the three replicates.