High-resolution orientation and depth of insertion of the voltage-sensing S4 helix of a potassium channel in lipid bilayers.

The opening and closing of voltage-gated potassium (Kv) channels are controlled by several conserved Arg residues in the S4 helix of the voltage-sensing domain. The interaction of these positively charged Arg residues with the lipid membrane has been of intense interest for understanding how membrane proteins fold to allow charged residues to insert into lipid bilayers against free-energy barriers. Using solid-state NMR, we have now determined the orientation and insertion depth of the S4 peptide of the KvAP channel in lipid bilayers. Two-dimensional (15)N correlation experiments of macroscopically oriented S4 peptide in phospholipid bilayers revealed a tilt angle of 40 degrees and two possible rotation angles differing by 180 degrees around the helix axis. Remarkably, the tilt angle and one of the two rotation angles are identical to those of the S4 helix in the intact voltage-sensing domain, suggesting that interactions between the S4 segment and other helices of the voltage-sensing domain are not essential for the membrane topology of the S4 helix. (13)C-(31)P distances between the S4 backbone and the lipid (31)P indicate a approximately 9 A local thinning and 2 A average thinning of the DMPC (1,2-dimyristoyl-sn-glycero-3-phosphochloline)/DMPG (1,2-dimyristoyl-sn-glycero-3-phosphatidylglycerol) bilayer, consistent with neutron diffraction data. Moreover, a short distance of 4.6 A from the guanidinium C(zeta) of the second Arg to (31)P indicates the existence of guanidinium phosphate hydrogen bonding and salt bridges. These data suggest that the structure of the Kv gating helix is mainly determined by protein-lipid interactions instead of interhelical protein-protein interactions, and the S4 amino acid sequence encodes sufficient information for the membrane topology of this crucial gating helix.