Three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (Vpu) from HIV-1.

The three-dimensional structure of the channel-forming trans-membrane domain of virus protein "u" (Vpu) of HIV-1 was determined by NMR spectroscopy in micelle and bilayer samples. Vpu(2-30+) is a 36-residue polypeptide that consists of residues 2-30 from the N terminus of Vpu and a six-residue "solubility tag" at its C terminus that facilitates the isolation, purification, and sample preparation of this highly hydrophobic minimal channel-forming domain. Nearly all of the resonances in the two-dimensional 1H/15N HSQC spectrum of uniformly 15N labeled Vpu(2-30+) in micelles are superimposable on those from the corresponding residues in the spectrum of full-length Vpu, which indicates that the structure of the trans-membrane domain is not strongly affected by the presence of the cytoplasmic domain at its C terminus. The two-dimensional 1H/15N PISEMA spectrum of Vpu(2-30+) in lipid bilayers aligned between glass plates has been fully resolved and assigned. The "wheel-like" pattern of resonances in the spectrum is characteristic of a slightly tilted membrane-spanning helix. Experiments were also performed on weakly aligned micelle samples to measure residual dipolar couplings and chemical shift anisotropies. The analysis of the PISA wheels and Dipolar Waves obtained from both weakly and completely aligned samples show that Vpu(2-30+) has a trans-membrane alpha-helix spanning residues 8-25 with an average tilt of 13 degrees. The helix is kinked slightly at Ile17, which results in tilts of 12 degrees for residues 8-16 and 15 degrees for residues 17-25. A structural fit to the experimental solid-state NMR data results in a three-dimensional structure with precision equivalent to an RMSD of 0.4 A. Vpu(2-30+) exists mainly as an oligomer on PFO-PAGE and forms ion-channels, a most frequent conductance of 96(+/- 6) pS in lipid bilayers. The structural features of the trans-membrane domain are determinants of the ion-channel activity that may be associated with the protein's role in facilitating the budding of new virus particles from infected cells.

[1]  C. Deber,et al.  Retention of native-like oligomerization states in transmembrane segment peptides: application to the Escherichia coli aspartate receptor. , 2001, Biochemistry.

[2]  S. Grzesiek,et al.  Solution NMR of proteins within polyacrylamide gels: Diffusional properties and residual alignment by mechanical stress or embedding of oriented purple membranes , 2000, Journal of biomolecular NMR.

[3]  S. Opella,et al.  Detection of 1H homonuclear NOE between amide sites in proteins with 1H15N heteronuclear correlation spectroscopy , 1989 .

[4]  S. Kan,et al.  A single‐coil triple resonance probe for NMR experiments , 1980 .

[5]  T. Klimkait,et al.  Molecular and biochemical analyses of human immunodeficiency virus type 1 vpu protein , 1989, Journal of virology.

[6]  T. Creighton.  The energetic ups and downs of protein folding , 1994, Nature Structural Biology.

[7]  A Nevzorov,et al.  Structure determination of membrane proteins by NMR spectroscopy. , 2002, Biochemistry and cell biology = Biochimie et biologie cellulaire.

[8]  M. Sansom,et al.  The pore domain of the nicotinic acetylcholine receptor: molecular modeling, pore dimensions, and electrostatics. , 1996, Biophysical journal.

[9]  A. Bax,et al.  Micelle-induced curvature in a water-insoluble HIV-1 Env peptide revealed by NMR dipolar coupling measurement in stretched polyacrylamide gel. , 2002, Journal of the American Chemical Society.

[10]  Che Ma,et al.  Expression, purification, and activities of full‐length and truncated versions of the integral membrane protein Vpu from HIV‐1 , 2002, Protein science : a publication of the Protein Society.

[11]  M. Martin,et al.  Human immunodeficiency virus type 1 Vpu protein regulates the formation of intracellular gp160-CD4 complexes , 1992, Journal of virology.

[12]  N. Unwin.  Acetylcholine receptor channel imaged in the open state , 1995, Nature.

[13]  A. Bax,et al.  Measurement of J and dipolar couplings from simplified two-dimensional NMR spectra. , 1998, Journal of magnetic resonance.

[14]  T. Klimkait,et al.  A novel gene of HIV-1, vpu, and its 16-kilodalton product. , 1988, Science.

[15]  A. Bax,et al.  High-resolution heteronuclear NMR of human ubiquitin in an aqueous liquid crystalline medium , 1997, Journal of biomolecular NMR.

[16]  T. Hope,et al.  Recent advances in the understanding of HIV accessory protein function , 2001, AIDS.

[17]  Roland L. Dunbrack,et al.  Prediction of protein side-chain rotamers from a backbone-dependent rotamer library: a new homology modeling tool. , 1997, Journal of molecular biology.

[18]  M. Billeter,et al.  MOLMOL: a program for display and analysis of macromolecular structures. , 1996, Journal of molecular graphics.

[19]  M. Summers,et al.  Structural biology of HIV. , 1999, Journal of molecular biology.

[20]  N Tjandra,et al.  Carbonyl CSA restraints from solution NMR for protein structure refinement. , 2001, Journal of the American Chemical Society.

[21]  S. Grzesiek,et al.  NMRPipe: A multidimensional spectral processing system based on UNIX pipes , 1995, Journal of biomolecular NMR.

[22]  Christopher Miller,et al.  Ion Channel Reconstitution , 1986, Springer US.

[23]  K. Strebel,et al.  HIV accessory proteins: multifunctional components of a complex system. , 2000, Advances in pharmacology.

[24]  N. Sarver,et al.  HIV accessory proteins as therapeutic targets , 1997, Nature Medicine.

[25]  S. Opella,et al.  Structural fitting of PISEMA spectra of aligned proteins. , 2003, Journal of magnetic resonance.

[26]  P. Gage,et al.  The Vpu protein of human immunodeficiency virus type 1 forms cation-selective ion channels , 1996, Journal of virology.

[27]  M. Peeters,et al.  Genetic characterization of accessory genes from human immunodeficiency virus type 1 group O strains. , 1998, AIDS research and human retroviruses.

[28]  A. Kukol,et al.  vpu transmembrane peptide structure obtained by site-specific fourier transform infrared dichroism and global molecular dynamics searching. , 1999, Biophysical journal.

[29]  Y. Ishii,et al.  Controlling residual dipolar couplings in high-resolution NMR of proteins by strain induced alignment in a gel , 2001, Journal of biomolecular NMR.

[30]  S. Oiki,et al.  Bundles of amphipathic transmembrane α‐helices as a structural motif for ion‐conducting channel proteins: Studies on sodium channels and acetylcholine receptors , 1990, Proteins.

[31]  J. Sodroski,et al.  Functional role of human immunodeficiency virus type 1 vpu. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

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

[33]  M. Klein,et al.  Simulation of the HIV‐1 Vpu transmembrane domain as a pentameric bundle , 1998, FEBS letters.

[34]  J. Sodroski,et al.  Identification of a protein encoded by the vpu gene of HIV-1 , 1988, Nature.

[35]  S. Opella,et al.  Simultaneous assignment and structure determination of a membrane protein from NMR orientational restraints , 2003, Protein science : a publication of the Protein Society.

[36]  J. Changeux,et al.  Mutations in the channel domain alter desensitization of a neuronal nicotinic receptor , 1991, Nature.

[37]  S. Opella,et al.  Experimental aspects of multidimensional solid-state NMR correlation spectroscopy. , 1999, Journal of magnetic resonance (San Diego, Calif. 1997 : Print).

[38]  M Montal,et al.  Correlation of the structural and functional domains in the membrane protein Vpu from HIV-1. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[39]  L. Huan,et al.  Novel method for evaluation of the oligomeric structure of membrane proteins. , 1999 .

[40]  U. Schubert,et al.  The two biological activities of human immunodeficiency virus type 1 Vpu protein involve two separable structural domains , 1996, Journal of virology.

[41]  Axel T. Brunger,et al.  X-PLOR Version 3.1: A System for X-ray Crystallography and NMR , 1992 .

[42]  R. Wittebort,et al.  Double and Triple Resonance Circuits for High-Frequency Probes , 1998 .

[43]  G. Veglia,et al.  Dipolar waves map the structure and topology of helices in membrane proteins. , 2003, Journal of the American Chemical Society.

[44]  P. Henklein,et al.  Identification of an ion channel activity of the Vpu transmembrane domain and its involvement in the regulation of virus release from HIV‐1‐infected cells , 1996, FEBS letters.

[45]  M. Montal,et al.  The Reconstituted Sodium Channel from Brain , 1986 .

[46]  J. Torres,et al.  Mapping the energy surface of transmembrane helix-helix interactions. , 2001, Biophysical journal.

[47]  R. Anholt,et al.  The Reconstituted Acetylcholine Receptor , 1986 .

[48]  S. Opella,et al.  Structure determination of membrane proteins by NMR spectroscopy. , 2004, Chemical reviews.

[49]  J H Prestegard,et al.  Partial alignment of biomolecules: an aid to NMR characterization. , 2001, Current opinion in chemical biology.

[50]  Carlos F. Lopez,et al.  Molecular dynamics investigation of membrane-bound bundles of the channel-forming transmembrane domain of viral protein U from the human immunodeficiency virus HIV-1. , 2002, Biophysical journal.

[51]  R. Lamb,et al.  Do Vpu and Vpr of human immunodeficiency virus type 1 and NB of influenza B virus have ion channel activities in the viral life cycles? , 1997, Virology.

[52]  F. D. Doty,et al.  A multinuclear double-tuned probe for applications with solids or liquids utilizing lumped tuning elements , 1981 .

[53]  P. Stewart,et al.  Protein structure by solid-state NMR spectroscopy , 1987, Quarterly Reviews of Biophysics.

[54]  Che Ma,et al.  Lanthanide ions bind specifically to an added "EF-hand" and orient a membrane protein in micelles for solution NMR spectroscopy. , 2000, Journal of magnetic resonance.

[55]  S. Opella,et al.  Structure of the coat protein in fd filamentous bacteriophage particles determined by solid-state NMR spectroscopy , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[56]  J. Waugh,et al.  Single coil probe with transmission‐line tuning for nuclear magnetic double resonance , 1976 .

[57]  J. Tomich,et al.  A molecular blueprint for the pore-forming structure of voltage-gated calcium channels. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[58]  J. Gesell,et al.  Structures of the M2 channel-lining segments from nicotinic acetylcholine and NMDA receptors by NMR spectroscopy , 1999, Nature Structural Biology.

[59]  Lawrence H. Pinto,et al.  Influenza virus M2 protein has ion channel activity , 1992, Cell.

[60]  S. Opella.  NMR and membrane proteins. , 1997, Nature structural biology.

[61]  S. Opella,et al.  Dipolar Waves as NMR maps of helices in proteins. , 2003, Journal of magnetic resonance.

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

[63]  A. Bax,et al.  Dipolar couplings in macromolecular structure determination. , 2001, Methods in enzymology.

[64]  T. Iwamoto,et al.  Synthetic peptides and four-helix bundle proteins as model systems for the pore-forming structure of channel proteins. I. Transmembrane segment M2 of the nicotinic cholinergic receptor channel is a key pore-lining structure. , 1993, The Journal of biological chemistry.

[65]  Randal R Ketchem,et al.  High-resolution conformation of gramicidin A in a lipid bilayer by solid-state NMR. , 1993, Science.

[66]  D. Trono,et al.  HIV accessory proteins: Leading roles for the supporting cast , 1995, Cell.

[67]  G. Veglia,et al.  Lanthanide Ion Binding to Adventitious Sites Aligns Membrane Proteins in Micelles for Solution NMR Spectroscopy , 2000 .

[68]  Bundles consisting of extended transmembrane segments of Vpu from HIV-1: computer simulations and conductance measurements. , 2002, Biochemistry.

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

[70]  G. Veglia,et al.  Dipolar waves as NMR maps of protein structure. , 2002, Journal of the American Chemical Society.

[71]  C. Walsh,et al.  Production, Purification, and Cleavage of Tandem Repeats of Recombinant Peptides , 1994 .

[72]  Structure and dynamics of a membrane protein in micelles from three solution NMR experiments , 2003, Journal of biomolecular NMR.

[73]  Y. Ishii,et al.  Alignment of Biopolymers in Strained Gels: A New Way To Create Detectable Dipole−Dipole Couplings in High-Resolution Biomolecular NMR , 2000 .

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