Structural analysis of Helicobacter pylori motility protein B: implications for stator assembly into the flagellar motor
Creators: name: Andrews, Daniel Alexander
Publisher: name: Monash University
Year: 2017
Subjects: Uncategorized

description: Molecular dynamics study of the C-terminal domain of motility protein B (MotB) The C-terminal domain of MotB, part of each stator unit (MotA₄MotB₂, heterohexamer) that assembles into a ring around the rotor (at least 11 in Escherichia coli) to power the rotation of the flagellar motor, is believed to be required for peptidoglycan (PG)-binding, as it contains the LSX₂RAX₂VX₃L motif that is conserved across Outer membrane protein A (OmpA)-like proteins. However, the previously solved fragment structures of Helicobacter pylori and Salmonella MotB showed that the conserved PG-binding residues are buried. For this reason, a molecular dynamics simulation of the H. pylori C-terminal domain structure (Hp-MotB-C, residues 125-250) was employed to assess the hypothesis that PG-binding is preceded, or accompanied by, structural rearrangements that result in solvent exposure of MotB's conserved PG-binding residues. Assessment of the Hp-MotB-C structure and its MD simulation revealed that the petal-like loops within the C-terminal domain were mobile, as evidenced by their high crystallographic and theoretical (calculated from MD simulation) temperature factors, and the high Cα root mean square fluctuation values (RMSFs) of many petal-like loop residues across the MD trajectory. In addition, a combination of correlated (loops move together) and anti-correlated motions (loops move in opposite directions) of the petal-like loops, identified through principal component analysis (PCA) of the MD trajectory, were found to promote concurrent unmasking and masking of the conserved PG-binding residues. Taken together, this suggested that populations of MotB coexist where the PG-binding site is either solvent exposed or buried. Therefore, PG may bind to the C-terminal domain of MotB by selecting a conformation where the conserved PG-binding residues are available to form interactions with the peptide stem of PG. The PG-binding mechanism identified for H. pylori MotB may be generally applicable to other species including Salmonella and E. coli, as the petal-like loops are semi-conserved. Overall, it appears that the intrinsic conformational heterogeneity of the petal-like loops is essential for the C-terminal domain of MotB to embed into the PG layer, which stabilises the assembly of the stator around the rotor. Structural characterisation of a soluble chimeric variant of H. pylori MotB (Hp-chimMotB) The peptidoglycan (PG)-anchored stator complexes that assemble around the rotor within proton-motive force-driven flagellar motors are comprised of integral membrane proteins, specifically a MotB dimer surrounded by four MotA subunits. At present, our understanding of the mechanism of stator assembly and torque generation is limited by the lack of full-length MotA and MotB X-ray structures. To avoid difficulties associated with the instability of detergent-solubilised MotB and its low level of expression, a chimera (Hp-chimMotB) was engineered where the periplasmic structure of MotB (plug, linker, and C-terminal domain) was directly attached to a leucine zipper (Saccharomyces cerevisiae GCN4 zipper), which replaced the two native TM helices of the MotB dimer. Biophysical and biochemical analysis revealed that Hp-chimMotB was highly expressed, folded and dimeric at low pH. It had a similar hydrodynamic radius to the H. pylori MotB variant that lacked the N-terminal 63 residues, but showed a significantly improved stability against proteolytic degradation. These results suggested that the periplasmic structure of MotB within Hp-chimMotB is stabilised by the leucine zipper dimeric coiled coil, that likely extends into the plug coiled coil (as the heptad repeat pattern continues through the leucine zipper into the plug), and that Hp-chimMotB adopts a native-like conformation where its well-structured domains are connected by a largely compact linker. The distance between the fluorescent donor and acceptor labels attached to the C146 residues within respective subunits of the Hp-chimMotB dimer, calculated using time-resolved fluorescence resonance energy transfer (TR-FRET) measurements (46.6 ± 6.2 Å), agreed well with the equivalent distance between the fluorescent labels modelled in silico using the crystal structure of the C-terminal domain of H. pylori MotB (Hp-MotB-C, residues 119-251, PDB ID: 3CYP) (44 Å). An in vitro PG-binding assay showed that Hp-chimMotB and Hp-MotB-C bound to PG more strongly than the MotB variant that contained part of the linker (Hp-MotB-C/L, residues 90-256). Furthermore, BUNCH modelling of the Hp-chimMotB structure with the C-terminal domain in Hp-MotB-C conformation fit the experimental small angle X-ray scattering (SAXS) data better than with the C-terminal domain in the Hp-MotB-C/L conformation. Taken together, this suggested that the C-terminal of Hp-chimMotB adopted the PG-binding conformation, previously observed in the crystal structure of the C-terminal domain that lacked the linker (Hp-MotB-C). Furthermore, SAXS data supports a model in which the linker is not folded against the C-terminal domain; instead it forms a subdomain that occupies the region between the plug and the C-terminal domain, as well as the area around the plug. The groups of the selected ensemble optimisation method (EOM) models that fit the Hp-chimMotB SAXS data best showed a variety of mostly compact but some elongated conformations, suggesting that Hp-chimMotB's linker adopts multiple conformations in solution. Together, the structural characterisation of Hp-chimMotB culminated in a model that described the conformational rearrangements required for the stator to assemble around the rotor. According to this model, in the inactive MotB-containing precomplexes that diffuse in the membrane, the linker is folded against the C-terminal domain of MotB retaining it in the conformation not competent for binding to PG. Upon assembly of the stator complex into the motor, association of the two plug helices into the dimeric coiled coil occurs concurrently with the dissociation of the linker from the C-terminal domain and reorientation of the two C-terminal domains with respect to each other to align their PG-binding sites. This rearrangement of the linker causes elongation of the periplasmic region of MotB, allowing it to span the periplasmic gap (100 Å) and position the C-terminal domain to embed into the PG layer and anchor the stator next to the rotor. In this conformation, part of the linker subdomain ‘clamps’ the coiled coil of the plug, stabilising the stator’s proton conducting channel (PCC) in an open state.