title: Functional role of conserved residues in the characteristic secretion NTPase motifs of the Pseudomonas aeruginosa type IV pilus motor proteins PilB, PilT and PilU.
abstract: Type IV pili are retractable protein fibres used by many bacterial pathogens for adherence, twitching motility, biofilm development and host colonization. In Pseudomonas aeruginosa, PilB and PilT are bipolar proteins belonging to the secretion NTPase superfamily, and power pilus extension and retraction, respectively, while the unipolar PilT paralogue PilU supports pilus retraction in an unknown manner. Assay of purified 6xHis-tagged PilB, PilT and PilU from P. aeruginosa showed that all three proteins have ATPase activities in vitro. Conserved residues in the Walker A (WA), Walker B (WB), Asp Box and His Box motifs characteristic of secretion NTPases were mutated, and complementation of twitching motility was tested. Mutation of conserved WA or WB residues in any of the three ATPases abrogated twitching motility, and for the WA mutant of PilT caused loss of polar localization. The requirement for three invariant acidic residues in the Asp Box motif, and for two invariant His residues in the His Box motif varied, with PilB being the least tolerant of changes. In all three proteins, the third acidic residue in the Asp Box and the second His of the His Box were crucial for function; mutation of these residues caused loss of PilT ATPase activity in vitro. Modelling of the effects of these mutations on the crystal structures of Aquifex aeolicus PilT and Vibrio cholerae EpsE (a PilB homologue) showed that the critical Asp Box and His Box residues contribute to a catalytic pocket that surrounds the ligand. These results provide experimental evidence differentiating widely conserved Asp and His Box residues that are essential for function from those whose roles are modulated by specific local environments.
authors.affiliation: Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada.
authors.forename: Poney
authors.initials: P
authors.lastname: Chiang
authors.affiliation: Molecular Structure and Function Program, Hospital for Sick Children Research Institute, Toronto, ON, Canada.
authors.forename: Liliana M
authors.initials: LM
authors.lastname: Sampaleanu
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada.
authors.forename: Melissa
authors.initials: M
authors.lastname: Ayers
authors.affiliation: Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada.
authors.forename: Markian
authors.initials: M
authors.lastname: Pahuta
authors.affiliation: Department of Biochemistry, University of Toronto, Toronto, ON, Canada.|Molecular Structure and Function Program, Hospital for Sick Children Research Institute, Toronto, ON, Canada.
authors.forename: P Lynne
authors.initials: PL
authors.lastname: Howell
authors.affiliation: Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, ON, Canada.|Molecular Structure and Function Program, Hospital for Sick Children Research Institute, Toronto, ON, Canada.|Institute of Medical Sciences, University of Toronto, Toronto, ON, Canada.
authors.forename: Lori L
authors.initials: LL
authors.lastname: Burrows
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D036781:Fimbriae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005816:Genetic Complementation Test; D008124:Locomotion / Q000235:genetics; D008958:Models, Molecular; D020409:Molecular Motor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D043583:Nucleoside-Triphosphatase / Q000235:genetics / Q000378:metabolism*; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D020409:Molecular Motor Proteins; C091957:PilU protein, Pseudomonas aeruginosa; D036781:Fimbriae Proteins; D010088:Oxidoreductases; C068255:pilB protein, Bacteria; D000251:Adenosine Triphosphatases; D043583:Nucleoside-Triphosphatase