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Title: PilN binding modulates the structure and binding partners of the Pseudomonas aeruginosa type IVa pilus protein PilM

Pseudomonas aeruginosa is an opportunistic bacterial pathogen that expresses type IVa pili. The pilus assembly system, which promotes surface-associated twitching motility and virulence, is composed of inner and outer membrane subcomplexes, connected by an alignment subcomplex composed of PilMNOP. PilM binds to the N terminus of PilN, and we hypothesize that this interaction causes functionally significant structural changes in PilM. To characterize this interaction, we determined the crystal structures of PilM and a PilM chimera where PilM was fused to the first 12 residues of PilN (PilM·PilN(1–12)). Structural analysis, multiangle light scattering coupled with size exclusion chromatography, and bacterial two-hybrid data revealed that PilM forms dimers mediated by the binding of a novel conserved motif in the N terminus of PilM, and binding PilN abrogates this binding interface, resulting in PilM monomerization. Structural comparison of PilM with PilM·PilN(1–12) revealed that upon PilN binding, there is a large domain closure in PilM that alters its ATP binding site. Using biolayer interferometry, we found that the association rate of PilN with PilM is higher in the presence of ATP compared with ADP. Bacterial two-hybrid data suggested the connectivity of the cytoplasmic and inner membrane components of the type IVa pilus machinery inmore » P. aeruginosa, with PilM binding to PilB, PilT, and PilC in addition to PilN. Pull-down experiments demonstrated direct interactions of PilM with PilB and PilT. As a result, we propose a working model in which dynamic binding of PilN facilitates functionally relevant structural changes in PilM.« less
Authors:
 [1] ;  [2] ;  [1] ;  [3] ;  [2] ;  [4] ;  [4] ;  [4] ;  [5] ;  [1]
  1. Univ. of Toronto, Toronto, ON (Canada); Hospital for Sick Children, Toronto, ON (Canada)
  2. Hospital for Sick Children, Toronto, ON (Canada)
  3. Brookhaven National Lab. (BNL), Upton, NY (United States)
  4. Univ. of Toronto, Toronto, ON (Canada)
  5. McMaster Univ., Hamilton, ON (Canada); Hospital for Sick Children, Toronto, ON (Canada)
Publication Date:
Report Number(s):
BNL-112064-2016-JA
Journal ID: ISSN 0021-9258
Grant/Contract Number:
SC00112704
Type:
Accepted Manuscript
Journal Name:
Journal of Biological Chemistry
Additional Journal Information:
Journal Volume: 291; Journal Issue: 21; Journal ID: ISSN 0021-9258
Publisher:
American Society for Biochemistry and Molecular Biology
Research Org:
Brookhaven National Laboratory (BNL), Upton, NY (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; ligand-binding protein; protein chimera; Pseudomonas aeruginosa (P. aeruginosa); type IV pili; x-ray crystallography; PilM; PilN
OSTI Identifier:
1340346