Gain of Function Mutations in Membrane Region M2C2 of KtrB Open a Gate Controlling K+ Transport by the KtrAB System from Vibrio alginolyticus

• Published in: The Journal of biological chemistry Vol. 285; no. 14; pp. 10318 - 10327
• Main Authors: Hänelt, Inga, Löchte, Sara, Sundermann, Lea, Elbers, Katharina, Vor der Brüggen, Marc, Bakker, Evert P.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02-04-2010; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Bacteria; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Bacterial Proteins - metabolism; Biological Transport; Cation Transport Proteins - chemistry; Cation Transport Proteins - genetics; Cation Transport Proteins - metabolism; Cell Membrane - metabolism; Enzyme Kinetics; Escherichia coli - genetics; Escherichia coli - metabolism; Membrane Biology; Membrane Proteins; Molecular Sequence Data; Mutation - genetics; Potassium - metabolism; Potassium Transport; Sequence Homology, Amino Acid; Site-directed Mutagenesis; Sodium - metabolism; Vibrio alginolyticus; Vibrio alginolyticus - genetics; Vibrio alginolyticus - metabolism; Membrane Proteins; Site-directed Mutagenesis; Bacteria; Enzyme Kinetics; Potassium Transport
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M109.089870

KtrB, the K+-translocating subunit of the Na+-dependent bacterial K+ uptake system KtrAB, consists of four M1PM2 domains, in which M1 and M2 are transmembrane helices and P indicates a p-loop that folds back from the external medium into the cell membrane. The transmembrane stretch M2C is, with its 40 residues, unusually long. It consists of three parts, the hydrophobic helices M2C1 and M2C3, which are connected by a nonhelical M2C2 region, containing conserved glycine, alanine, serine, threonine, and lysine residues. Several point mutations in M2C2 led to a huge gain of function of K+ uptake by KtrB from the bacterium Vibrio alginolyticus. This effect was exclusively due to an increase in Vmax for K+ transport. Na+ translocation by KtrB was not affected. Partial to complete deletions of M2C2 also led to enhanced Vmax values for K+ uptake via KtrB. However, several deletion variants also exhibited higher Km values for K+ uptake and at least one deletion variant, KtrBΔ326–328, also transported Na+ faster. The presence of KtrA did not suppress any of these effects. For the deletion variants, this was due to a diminished binding of KtrA to KtrB. PhoA studies indicated that M2C2 forms a flexible structure within the membrane allowing M2C3 to be directed either to the cytoplasm or (artificially) to the periplasm. These data are interpreted to mean (i) that region M2C2 forms a flexible gate controlling K+ translocation at the cytoplasmic side of KtrB, and (ii) that M2C2 is required for the interaction between KtrA and KtrB.