Abstract
KtrB, the K(+)-translocating subunit of the Na(+)-dependent bacterial K(+) uptake system KtrAB, consists of four M(1)PM(2) 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 V(max) values for K(+) uptake via KtrB. However, several deletion variants also exhibited higher K(m) values for K(+) uptake and at least one deletion variant, KtrB(Delta326-328), 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.
Highlights
Forschungsgemeinschaft. □S The on-line version of this article contains a supplemental table, Figs. 1–3, and additional explanations and references. 1 To whom correspondence should be addressed: Abteilung Mikrobiologie, type, like KcsA [10, 11] or Kir [12], by multiple gene duplications and gene fusions [7]
Durell and Guy [13] have proposed that M2C2 forms either a loop within the membrane connecting the apolar membrane spanning stretches M2C1 and M2C3 with each other, or that M2C1 and M2C2 span the membrane together and that helix M2C3 is located within the inner surface of the membrane
We assume that a flexible M2C2 loop fills the cavity just below the p-loop and that VaKtrB M2C2-residue Lys325 forms a salt bridge with VaKtrB residue Asp222 from membrane span M2B [13]
Summary
Forschungsgemeinschaft. □S The on-line version of this article (available at http://www.jbc.org) contains a supplemental table, Figs. 1–3, and additional explanations and references. 1 To whom correspondence should be addressed: Abteilung Mikrobiologie, type, like KcsA [10, 11] or Kir [12], by multiple gene duplications and gene fusions [7]. With single conserved glycine residues in SKT proteins, this filter appears to have a simpler structure than in Kϩ channels, in which the filter is formed by the well conserved p-loop sequence TVGYG from each subunit [18]. In particular, the C termini from the membrane spans M2C and M2D deviated from that of KcsA-M2 This may reflect the difference in function between the channel KcsA and the transporter KtrB [13]. KtrB may form dimers [26] In their modeling studies, Durell and Guy [13] focused on membrane span M2C. Guy model [13], M2C1 and M2C3 span the membrane, and M2C2 forms a loop that fills the cavity just beneath the p-loops. The conserved lysine residue ( Lys325 in VaKtrB from Vibrio alginolyticus; see vertical arrow in Fig. 1B) may form a salt bridge with
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