' s personal copy High-Resolution Orientation and Depth of Insertion of the Voltage-Sensing S 4 Helix of a Potassium Channel in Lipid Bilayers

Received 15 February 2010; received in revised form 17 June 2010; accepted 23 June 2010 Available online 30 June 2010 The opening and closing of voltage-gated potassium (Kv) channels are controlled by several conserved Arg residues in the S4 helix of the voltagesensing 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 N 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 are not essential for the membrane topology of the S4 helix. C–P distances between the S4 backbone and the lipid P indicate a ∼9 Å local thinning and 2 Å 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 Å from the guanidinium C of the second Arg to 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. © 2010 Elsevier Ltd. All rights reserved.

[1]  J. Nagle,et al.  Structure of fully hydrated fluid phase DMPC and DLPC lipid bilayers using X-ray scattering from oriented multilamellar arrays and from unilamellar vesicles. , 2005, Biophysical journal.

[2]  Wenbin Luo,et al.  Structure and function of the influenza A M2 proton channel. , 2009, Biochemistry.

[3]  M. E. Rose,et al.  Elementary Theory of Angular Momentum , 1957 .

[4]  Fredrik Elinder,et al.  Structure, Function, and Modification of the Voltage Sensor in Voltage-Gated Ion Channels , 2008, Cell Biochemistry and Biophysics.

[5]  E. Perozo,et al.  Molecular Architecture of the KvAP Voltage-Dependent K+ Channel in a Lipid Bilayer , 2004, Science.

[6]  M. Hong,et al.  Asymmetric insertion of membrane proteins in lipid bilayers by solid-state NMR paramagnetic relaxation enhancement: a cell-penetrating Peptide example. , 2008, Journal of the American Chemical Society.

[7]  M. Cadene,et al.  X-ray structure of a voltage-dependent K+ channel , 2003, Nature.

[8]  S. Opella,et al.  A solid-state NMR index of helical membrane protein structure and topology. , 2000, Journal of magnetic resonance.

[9]  Wenbin Luo,et al.  Side-chain conformation of the M2 transmembrane peptide proton channel of influenza a virus from 19F solid-state NMR. , 2007, The journal of physical chemistry. B.

[10]  A. Waring,et al.  Orientation and dynamics of an antimicrobial peptide in the lipid bilayer by solid-state NMR spectroscopy. , 2001, Biophysical journal.

[11]  S. Opella,et al.  Bicelle samples for solid-state NMR of membrane proteins , 2007, Nature Protocols.

[12]  Huey W. Huang.  Molecular mechanism of antimicrobial peptides: the origin of cooperativity. , 2006, Biochimica et biophysica acta.

[13]  Mauricio Montal,et al.  Three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (Vpu) from HIV-1. , 2003, Journal of molecular biology.

[14]  Mei Hong,et al.  Effects of guanidinium-phosphate hydrogen bonding on the membrane-bound structure and activity of an arginine-rich membrane peptide from solid-state NMR spectroscopy. , 2008, Angewandte Chemie.

[15]  G. von Heijne,et al.  Interface connections of a transmembrane voltage sensor. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[16]  S. Opella,et al.  Three-dimensional structure of the transmembrane domain of Vpu from HIV-1 in aligned phospholipid bicelles. , 2006, Biophysical journal.

[17]  A. Waring,et al.  Solid-state NMR investigation of the depth of insertion of protegrin-1 in lipid bilayers using paramagnetic Mn2+. , 2003, Biophysical journal.

[18]  R. MacKinnon,et al.  Phospholipids and the origin of cationic gating charges in voltage sensors , 2006, Nature.

[19]  A. Waring,et al.  Membrane thinning effect of the beta-sheet antimicrobial protegrin. , 2000, Biochemistry.

[20]  David L. Worcester,et al.  Structure and hydration of membranes embedded with voltage-sensing domains , 2009, Nature.

[21]  E. Campbell,et al.  Atomic structure of a voltage-dependent K+ channel in a lipid membrane-like environment , 2007, Nature.

[22]  Zhe Lu,et al.  Removal of phospho-head groups of membrane lipids immobilizes voltage sensors of K+ channels , 2008, Nature.

[23]  G. Heijne,et al.  Recognition of transmembrane helices by the endoplasmic reticulum translocon , 2005, Nature.

[24]  Timothy A Cross,et al.  Transmembrane helix uniformity examined by spectral mapping of torsion angles. , 2008, Structure.

[25]  Roderick MacKinnon,et al.  Calibrated Measurement of Gating-Charge Arginine Displacement in the KvAP Voltage-Dependent K+ Channel , 2005, Cell.

[26]  R. Prosser,et al.  Current applications of bicelles in NMR studies of membrane-associated amphiphiles and proteins. , 2006, Biochemistry.

[27]  K. Swartz,et al.  Sensing voltage across lipid membranes , 2008, Nature.

[28]  E. Dufourc,et al.  Toward bicelle stability with ether-linked phospholipids: temperature, composition, and hydration diagrams by 2H and 31P solid-state NMR. , 2005, Langmuir : the ACS journal of surfaces and colloids.

[29]  C. Rienstra,et al.  Measurement of 13C−15N Distances in Uniformly 13C Labeled Biomolecules: J-Decoupled REDOR , 1999 .

[30]  Oleg Jardetzky,et al.  Probability‐based protein secondary structure identification using combined NMR chemical‐shift data , 2002, Protein science : a publication of the Protein Society.

[31]  A. Waring,et al.  Orientation of a beta-hairpin antimicrobial peptide in lipid bilayers from two-dimensional dipolar chemical-shift correlation NMR. , 2006, Biophysical journal.

[32]  R. Griffin,et al.  Frequency-Switched Lee—Goldburg Sequences in Solids , 1990 .

[33]  M. Sansom,et al.  How Does a Voltage Sensor Interact with a Lipid Bilayer? Simulations of a Potassium Channel Domain , 2007, Structure.

[34]  Youxing Jiang,et al.  The principle of gating charge movement in a voltage-dependent K+ channel , 2003, Nature.

[35]  J Wang,et al.  Imaging membrane protein helical wheels. , 2000, Journal of magnetic resonance.

[36]  M. Hohwy,et al.  Broadband dipolar recoupling in the nuclear magnetic resonance of rotating solids: A compensated C7 pulse sequence , 1998 .

[37]  Benoît Roux,et al.  Dynamics of the Kv1.2 voltage-gated K+ channel in a membrane environment. , 2007, Biophysical journal.

[38]  Sanguk Kim,et al.  2D solid state NMR spectral simulation of 310, α, and π-helices , 2004 .

[39]  M. B. Banaszak Holl,et al.  Membrane thinning due to antimicrobial peptide binding: an atomic force microscopy study of MSI-78 in lipid bilayers. , 2005, Biophysical journal.

[40]  M. Auger,et al.  Bicelles as model membranes for solid‐ and solution‐state NMR studies of membrane peptides and proteins , 2005 .

[41]  J Wang,et al.  Structure of the transmembrane region of the M2 protein H+ channel , 2001, Protein science : a publication of the Protein Society.

[42]  H De Loof,et al.  Amphipathic helix motif: Classes and properties , 1990, Proteins.

[43]  Kurt W. Zilm.  Multidimensional solid-state NMR and polymers , 1997 .

[44]  A. Waring,et al.  Phosphate-mediated arginine insertion into lipid membranes and pore formation by a cationic membrane peptide from solid-state NMR. , 2007, Journal of the American Chemical Society.

[45]  T. Gullion,et al.  Rotational-Echo, Double-Resonance NMR , 1989 .

[46]  J. Killian,et al.  Lipid dependence of membrane anchoring properties and snorkeling behavior of aromatic and charged residues in transmembrane peptides. , 2002, Biochemistry.

[47]  Kiyonori Takegoshi,et al.  13C–1H dipolar-assisted rotational resonance in magic-angle spinning NMR , 2001 .

[48]  R. Bertram,et al.  Backbone structure of the amantadine-blocked trans-membrane domain M2 proton channel from Influenza A virus. , 2007, Biophysical journal.

[49]  Francisco Bezanilla,et al.  Voltage-Sensing Residues in the S2 and S4 Segments of the Shaker K+ Channel , 1996, Neuron.

[50]  Ayyalusamy Ramamoorthy,et al.  High-Resolution Heteronuclear Dipolar Solid-State NMR Spectroscopy , 1994 .

[51]  Wenbin Luo,et al.  Structure and Function of the Influenza A M 2 Proton Channel , 2009 .

[52]  A. Waring,et al.  Solid-State NMR Investigation of the Depth of Insertion of Protegrin-1 in Lipid Bilayers Using Paramagnetic Mn 2 1 , 2003 .

[53]  Conggang Li,et al.  Solid-state NMR characterization of conformational plasticity within the transmembrane domain of the influenza A M2 proton channel. , 2007, Biochimica et biophysica acta.

[54]  S. Opella,et al.  Structure of the coat protein in Pf1 bacteriophage determined by solid-state NMR spectroscopy. , 2004, Journal of molecular biology.

[55]  A. A. Alabi,et al.  Portability of paddle motif function and pharmacology in voltage sensors , 2007, Nature.

[56]  A. Waring,et al.  Roles of arginine and lysine residues in the translocation of a cell-penetrating peptide from (13)C, (31)P, and (19)F solid-state NMR. , 2009, Biochemistry.