title: the active site residue V266 of Chlamydial HtrA is critical for substrate binding during both in vitro and in vivo conditions.
abstract: HtrA is a complex, multimeric chaperone and serine protease important for the virulence and survival of many bacteria. Chlamydia trachomatis is an obligate, intracellular bacterial pathogen that is responsible for severe disease pathology. C. trachomatis HtrA (CtHtrA) has been shown to be highly expressed in laboratory models of disease. In this study, molecular modelling of CtHtrA protein active site structure identified putative S1-S3 subsite residues I242, I265, and V266. These residues were altered by site-directed mutagenesis, and these changes were shown to considerably reduce protease activity on known substrates and resulted in a narrower and distinct range of substrates compared to wild type. Bacterial two-hybrid analysis revealed that CtHtrA is able to interact in vivo with a broad range of protein sequences with high affinity. Notably, however, the interaction was significantly altered in 35 out of 69 clones when residue V266 was mutated, indicating that this residue has an important function during substrate binding.
authors.affiliation: Institute of Health and Biomedical Innovation, Queensland University of Technology, Brisbane, Qld., Australia.
authors.forename: Sarina
authors.initials: S
authors.lastname: Gloeckl
authors.forename: Joel D A
authors.initials: JD
authors.lastname: Tyndall
authors.forename: Scott H
authors.initials: SH
authors.lastname: Stansfield
authors.forename: Peter
authors.initials: P
authors.lastname: Timms
authors.forename: Wilhelmina M
authors.initials: WM
authors.lastname: Huston
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D002692:Chlamydia trachomatis / Q000201:enzymology*; D008958:Models, Molecular; D018832:Molecular Chaperones / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D057057:Serine Proteases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020798:Two-Hybrid System Techniques; D037521:Virulence Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D018832:Molecular Chaperones; D050505:Mutant Proteins; D037521:Virulence Factors; D057057:Serine Proteases