Membrane Region M-2C2 in Subunit KtrB of the K+ Uptake System KtrAB from Vibrio alginolyticus Forms a Flexible Gate Controlling K+ Flux AN ELECTRON PARAMAGNETIC RESONANCE STUDY

Autor(en): Haenelt, Inga
Wunnicke, Dorith
Mueller-Trimbusch, Meike
Vor der Brueggen, Marc
Kraus, Inga
Bakker, Evert P.
Steinhoff, Heinz-Juergen 
Stichwörter: AMINO-ACID SUBSTITUTIONS; Biochemistry & Molecular Biology; CRYSTAL-STRUCTURE; DISTANCE MEASUREMENTS; ESCHERICHIA-COLI K-12; FULL-LENGTH KCSA; GLYCINE RESIDUES; HIGH-AFFINITY; ION CHANNEL; KDPFABC COMPLEX; POTASSIUM-TRANSPORT
Erscheinungsdatum: 2010
Herausgeber: AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
Journal: JOURNAL OF BIOLOGICAL CHEMISTRY
Volumen: 285
Ausgabe: 36
Startseite: 28210
Seitenende: 28219
Zusammenfassung: 
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, M2C2 residue Thr-318R1 (R1 indicates the bound spin label) moved with respect to M-2B residue Asp-222R1 and M2C3 residue Val-331R1 but not with respect to M2C1 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.
DOI: 10.1074/jbc.M110.139311

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