Residues of the cytoplasmic domain of MotA essential for torque generation in the bacterial flagellar motor.
The MotA protein of Escherichia coli is a component of the flagellum that functions, together with MotB, in transmembrane proton conduction. MotA and MotB are believed to form the stator of the flagellar motor. They are integral membrane proteins; MotA has a large (ca 22 kDa) domain in the cytoplasm, and MotB a much smaller one (ca 3 kDa). Recent work suggests that cytoplasmically located parts of MotA and/or MotB might be present at the active site for torque generation in the motor. To test the proposal that the cytoplasmic domain of MotA functions in torque generation, and to identify the amino acid residues most important for function, we have carried out a mutational analysis of this domain. Using random mutagenesis, many mutations of cytoplasmic residues of MotA were isolated, which either abolish or impair torque generation. In most cases the residues affected are not conserved, and many of the replacements involve loss or gain of a proline residue, which suggests that these mutations disrupt function by altering the protein conformation rather than by directly affecting residues of an active site. Using site-directed mutagenesis, the conserved residues in the cytoplasmic domain of MotA were replaced, either singly or, in the case of charged residues, in various combinations. The results identify four residues of MotA that are important for motor function. These are Arg90 and Glu98, located in the cytoplasmic domain, and Pro173 and Pro222, located at the interface between the cytoplasmic domain and the membrane-spanning domain. Possible roles for these residues in torque generation are discussed.
Zhou
J
J
Department of Biology, University of Utah, Salt Lake City, UT 84112, USA.
Blair
D F
DF
D000595:Amino Acid Sequence; D001120:Arginine / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D016764:Cell Polarity; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004734:Energy Metabolism; D004926:Escherichia coli / Q000502:physiology*; D005407:Flagella / Q000502:physiology*; D018698:Glutamic Acid / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008961:Models, Structural; D016296:Mutagenesis; D011392:Proline / Q000235:genetics; D013329:Structure-Activity Relationship; D019415:Torque
D001426:Bacterial Proteins; D008565:Membrane Proteins; C063010:MotA protein, Bacteria; D018698:Glutamic Acid; D001120:Arginine; D011392:Proline