Gain of Function Mutations in Membrane Region M-2C2 of KtrB Open a Gate Controlling K+ Transport by the KtrAB System from Vibrio alginolyticus
Vor der Brueggen, Marc
Bakker, Evert P.
|ADAPTATION; AMINO-ACID SUBSTITUTIONS; Biochemistry & Molecular Biology; CHANNEL; ESCHERICHIA-COLI K-12; GLYCINE RESIDUES; HIGH-AFFINITY; KDPFABC COMPLEX; POTASSIUM-TRANSPORT; PROTEIN; SELECTIVITY
|AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
|JOURNAL OF BIOLOGICAL CHEMISTRY
KtrB, the K+-translocating subunit of the Na+-dependent bacterial K+ uptake system KtrAB, consists of four M1PM2 domains, in which M-1 and M-2 are transmembrane helices and P indicates a p-loop that folds back from the external medium into the cell membrane. The transmembrane stretch M-2C is, with its 40 residues, unusually long. It consists of three parts, the hydrophobic helices M-2C1 and M-2C3, which are connected by a nonhelical M-2C2 region, containing conserved glycine, alanine, serine, threonine, and lysine residues. Several point mutations in M-2C2 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 V-max for K+ transport. Na+ translocation by KtrB was not affected. Partial to complete deletions of M-2C2 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, KtrB(Delta 326-328), 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 M-2C2 forms a flexible structure within the membrane allowing M-2C3 to be directed either to the cytoplasm or (artificially) to the periplasm. These data are interpreted to mean (i) that region M-2C2 forms a flexible gate controlling K+ translocation at the cytoplasmic side of KtrB, and (ii) that M-2C2 is required for the interaction between KtrA and KtrB.
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