Poster Presentation The 45th Lorne Conference on Protein Structure and Function 2020

Structural characterisation of molybdate acquisition by ModA in Pseudomonas aeruginosa (#340)

Dalton H. Y. Ngu 1 , Zhenyao Luo 1 , Bryan Y. J. Lim 1 , Christopher A. McDevitt 2 , Bostjan Kobe 1
  1. School of Chemistry & Molecular Biosciences, The University of Queensland, Brisbane, Queensland, Australia
  2. Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, The University of Melbourne, Melbourne, Victoria, Australia

Pseudomonas aeruginosa is a clinically significant Gram-negative bacterial pathogen often implicated in hospital-acquired infections. Because P. aeruginosa is able to survive in harsh environmental conditions and is resistant to multiple antibiotics, it is crucial to study the proteins that contribute to its survival and pathogenicity. In most organisms, molybdenum is an essential transition metal required for survival and is used by many proteins as a cofactor for carbon, sulfur and nitrogen metabolism. For P. aeruginosa, the periplasmic solute-binding protein (SBP) ModA is responsible for the binding and transport of molybdenum, in the form of molybdate (MoO42-) in solution, to the cytoplasmic membrane-bound ATP-binding cassette importer complex ModBC. Besides molybdate, ModA has also been found to be able to bind to tungstate (WO42-) due to their similar sizes. Currently, there is no known crystal structure of ModA from P. aeruginosa. Given the critical role ModA plays in molybdate acquisition, the main aim of this study is to structurally characterise ModA in both ligand-free and ligand-bound forms using X-ray crystallography.  

 

Multi-angle laser light scattering coupled with size exclusion chromatography suggests that ModA exists as a monomer in solution. Protein crystals of ligand-free and molybdate-bound ModA diffracted to 1.8 Å and 1.7 Å respectively. The crystal structure of ModA consists of two discontinuous globular domains connected by two short linkers. Each domain consists of a five-stranded β-sheet surrounded by five to six α-helices. Comparison with other bacterial ModA homologs shows that the ModA structure is highly conserved within the Cluster D-III SBPs. Overlay of the ligand-free and molybdate-bound structures shows a 13.9 º movement between both domains, resembling that of the “Venus-flytrap” mechanism. Future directions include obtaining the crystal structure of tungstate-bound ModA, determining the binding affinities of ModA for the two ligands, and site-directed mutagenesis of residues involved in ligand binding.