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

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 α-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.