Membrane region M2C2 in subunit KtrB of the K+ uptake system KtrAB from Vibrio alginolyticus forms a flexible gate controlling K+ flux: an electron paramagnetic resonance study.
Transmembrane stretch M(2C) from the bacterial K(+)-translocating protein KtrB is unusually long. In its middle part, termed M(2C2), it contains several small and polar amino acids. This region is flanked by the two alpha-helices M(2C1) and M(2C3) and may form a flexible gate at the cytoplasmic side of the membrane controlling K(+) translocation. In this study, we provide experimental evidence for this notion by using continuous wave and pulse EPR measurements of single and double spin-labeled cysteine variants of KtrB. Most of the spin-labeled residues in M(2C2) were shown to be immobile, pointing to a compact structure. However, the high polarity revealed for the microenvironment of residue positions 317, 318, and 327 indicated the existence of a water-accessible cavity. Upon the addition of K(+) ions, M(2C2) residue Thr-318R1 (R1 indicates the bound spin label) moved with respect to M(2B) residue Asp-222R1 and M(2C3) residue Val-331R1 but not with respect to M(2C1) residue Met-311R1. Based on distances determined between spin-labeled residues of double-labeled variants of KtrB in the presence and absence of K(+) ions, structural models of the open and closed conformations were developed.
Hänelt
Inga
I
Department of Microbiology, University of Osnabrück, D-49076 Osnabrück, Germany. i.haenelt@rug.nl
Wunnicke
Dorith
D
Müller-Trimbusch
Meike
M
Vor der Brüggen
Marc
M
Kraus
Inga
I
Bakker
Evert P
EP
Steinhoff
Heinz-Jürgen
HJ
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001692:Biological Transport; D027682:Cation Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism*; D016764:Cell Polarity; D003545:Cysteine; D004578:Electron Spin Resonance Spectroscopy; D014644:Genetic Variation; D008958:Models, Molecular; D009068:Movement; D011188:Potassium / Q000378:metabolism*; D011487:Protein Conformation; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013113:Spin Labels
D001426:Bacterial Proteins; D027682:Cation Transport Proteins; C113206:KtrB protein, Bacteria; D021122:Protein Subunits; D013113:Spin Labels; D003545:Cysteine; D011188:Potassium