AbstractsBiology & Animal Science

Structural analysis of Helicobacter pylori motility protein B: implications for stator assembly into the flagellar motor

by Daniel Alexander Andrews

Institution: Monash University
Year: 2016
Keywords: Flagellar motor; Motility protein B; Protein engineering; Stator assembly
Posted: 02/05/2017
Record ID: 2120033
Full text PDF: http://arrow.monash.edu.au/hdl/1959.1/1282741


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… Advisors/Committee Members: Principal Supervisor: Anna Roujeinikova, Supervisor: Ashley Buckle.