High-Resolution Orientation and Depth of Insertion of the Voltage-Sensing S4 Helix of a Potassium Channel in Lipid Bilayers

摘要

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’s 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. We have now determined the orientation and insertion depth of the S4 peptide of the KvAP channel in lipid bilayers using solid-state NMR. 2D ¹⁵N correlation experiments of macroscopically oriented S4 peptide in phospholipid bilayers revealed a tilt angle of 40°, and two possible rotation angles differing by 180° 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 is not essential for the membrane topology of the S4 helix. ¹³C-³¹P distances between the S4 backbone and the lipid ³¹P indicate a ~9 Å local thinning and 2 Å average thinning of the DMPC/DMPG bilayer, consistent with neutron diffraction data. Moreover, a short distance of 4.6 Å from the guanidinium Cζ of the second Arg to ³¹P indicates the existence of guanidinium-phosphate hydrogen bonding and salt bridge. These data suggest that the structure of the Kv gating helix is mainly determined by protein-lipid interactions instead of inter-helical protein-protein interactions, and the S4 amino acid sequence encodes sufficient information for the membrane topology of this crucial gating helix.