Probing the Structure of the Dimeric KtrB Membrane Protein

Abstract

The KtrAB ion transporter is a complex of two proteins, KtrB and KtrA. The integral membrane protein KtrB is expected to adopt the structural architecture typified by the pore domain of potassium channels. Here we show that homo-dimerization of KtrB proteins is most likely a general property of this family of transporters. Using cysteine mutants and bifunctional cross-linkers we define regions of the Bacillus subtilis KtrB molecule that are close to the molecular 2-fold axis and to the dimer interface. Fitting of the cross-linking data to a potassium channel-like model suggests structural similarities between potassium channels and KtrB proteins in the extracellular half of the molecule and differences in the cytoplasmic regions.Regulation of intracellular concentrations of sodium and potassium ions is a characteristic of all living organisms and is achieved by a battery of macromolecules that include ion transporter membrane proteins. The KtrAB transporter family plays a role in these processes by mediating Na ϩ -dependent potassium ion uptake in many prokaryotes (1-3). At the genetic level this transporter is usually arranged as a two gene operon (3) encoding the KtrB membrane protein and the cytoplasmic KtrA protein. KtrB proteins are 400 -600 residues long and predicted to have 8 transmembrane helices (TMs) 2 (1-3). KtrA proteins are ϳ220 amino acids long and contain RCK domains that form octameric rings and bind adenine-ribose-phosphatecontaining compounds (4, 5). RCK domains are commonly found associated with membrane proteins, including potassium channels, and function as activity regulators.The KtrAB transporters are part of a large superfamily of ion transport membrane proteins that also includes the plant HKT, the yeast Trk1,2, and the prokaryotic TrK (1, 3). Some of the members of the superfamily, including KtrAB, are thought to be either Na , 3, 6). Sequence homology and hydrophobicity profiles have led to the proposal that all membrane proteins in this superfamily adopt the architecture of the ion pore of potassium channels (1, 2). One KtrB polypeptide is thought to contain four repeats of the TM-P loop -TM structural motif observed in potassium channels, such as KcsA (7, 8). The repeats, labeled A to D in Fig. 1A, do not have the same sequence, except for a few conserved positions (Fig. 1B), and are connected by relatively large cytoplasmic loops (15-36 residues), which show no similarity to one another. The experimentally determined transmembrane topology of HKT1 from Arabidopsis thaliana revealed 8 TMs that agree well with a KcsA-like architecture (9). Moreover, members of the superfamily tend to have one or more conserved glycine residues in the regions equivalent to the potassium channel selectivity filter sequence GYG (glycine-tyrosineglycine) (1, 2) (Fig. 1B). Mutation of these conserved glycines in either the KtrB or HKT proteins has been shown to cause changes in the transport properties as expected if these residues form part of the transport pathway (2, 10, 11). Although the evidence desc...

The structure of the KtrAB potassium transporter

“…KtrAB complex was assembled by mixing KtrB and KtrA. Preparation of proteoliposomes and 86 Rb + flux assay was performed as previously described with some variations 6,29 . For crystallization the complex was further purified and detergent exchanged to Cymal6 by size-exclusion chromatography.…”

“…Structure determination and representation made use of XDS 30 42 programs. Growth complementation assay followed protocols previously described 6,8 . Protein quality control was performed by mass spectrometry.…”

“…To assess the importance of positioning a carboxylate close to the intramembrane loop we generated truncations of the C-terminal residues and evaluated their impact in a growth complementation assay 3,6,8 of the TK2420 Escherichia coli strain. This strain has its major K + transport systems disabled and only grows in K + concentration ≥30 mM; however, when expressing wild-type KtrAB it can grow in K + concentrations of ~0.3 mM.…”

“…Presumably, the fourth residue, a conserved apolar residue which in the structure seems to function as a C terminus anchor, has a role in the positioning of the C terminus and therefore on the stability of the C-terminus-mediated lateral contact. Notably, we had previously shown that truncation of the last 10 or 15 residues of the C terminus abolishes strain complementation and destabilizes the KtrB dimer 6 . Although we cannot rule out more subtle functional roles for the C terminus, such as cooperativity between KtrB subunits, our results demonstrate that the C terminus has a role both in the stability of the KtrB homodimer and of the KtrAB complex.…”

“…3a) resembling other KtrA proteins20. The functional effect of these two ligands was then evaluated in a 86 Rb + uptake assay 6,9,23 by reconstituting KtrAB with ATP or ADP ( Supplementary Fig. 8a) in the presence of an excess of KtrA to minimize the formation of the KtrB-KtrA-KtrB complex.…”