Biological Crystallography Structures of the Hiv-1 Capsid Protein Dimerization Domain at 2.6 a Ê Resolution

The human immunodeficiency virus type I (HIV-1) capsid protein is initially synthesized as the central domain of the Gag polyprotein, and is subsequently proteolytically processed into a discrete 231-amino-acid protein that forms the distinctive conical core of the mature virus. The crystal structures of two proteins that span the C-terminal domain of the capsid are reported here: one encompassing residues 146-231 (CA146-231) and the other extending to include the 14-residue p2 domain of Gag (CA146-p2). The isomorphous CA146-231 and CA146-p2 structures were determined by molecular replacement and have been refined at 2.6 A resolution to R factors of 22.3 and 20.7% (Rfree = 28.1 and 27.5%), respectively. The ordered domains comprise residues 148-219 for CA146-231 and 148-218 for CA146-p2, and their refined structures are essentially identical. The proteins are composed of a 310 helix followed by an extended strand and four alpha-helices. A crystallographic twofold generates a dimer that is stabilized by parallel packing of an alpha-helix 2 across the dimer interface and by packing of the 310 helix into a groove created by alpha-helices 2 and 3 of the partner molecule. CA146-231 and CA146-p2 dimerize with the full affinity of the intact capsid protein, and their structures therefore reveal the essential dimer interface of the HIV-1 capsid.

[1]  I. Jones,et al.  Gag-Gag interactions in the C-terminal domain of human immunodeficiency virus type 1 p24 capsid antigen are essential for Gag particle assembly. , 1996, The Journal of general virology.

[2]  A. Brunger.  Free R value: a novel statistical quantity for assessing the accuracy of crystal structures. , 1992 .

[3]  V. Georgiev.  Virology , 1955, Nature.

[4]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[5]  Z. Otwinowski,et al.  [20] Processing of X-ray diffraction data collected in oscillation mode. , 1997, Methods in enzymology.

[6]  S. Höglund,et al.  Functional domains of the capsid protein of human immunodeficiency virus type 1 , 1994, Journal of virology.

[7]  G. Ellman,et al.  Tissue sulfhydryl groups. , 1959, Archives of biochemistry and biophysics.

[8]  A. Kaplan,et al.  The p2 domain of human immunodeficiency virus type 1 Gag regulates sequential proteolytic processing and is required to produce fully infectious virions , 1994, Journal of virology.

[9]  D. G. Watts,et al.  Determining confidence intervals for parameters derived from analysis of equilibrium analytical ultracentrifugation data. , 1994, Methods in enzymology.

[10]  E. Barklis,et al.  Structural analysis of human immunodeficiency virus type 1 Gag protein interactions, using cysteine-specific reagents , 1996, Journal of virology.

[11]  Y. Zhoa,et al.  Complementation of human immunodeficiency virus (HIV-1) gag particle formation. , 1994, Virology.

[12]  B. Gay,et al.  Phenotypic characterization of insertion mutants of the human immunodeficiency virus type 1 Gag precursor expressed in recombinant baculovirus-infected cells , 1994, Journal of virology.

[13]  J. Luban,et al.  Specific incorporation of cyclophilin A into HIV-1 virions , 1994, Nature.

[14]  I. Jones,et al.  Distinct signals in human immunodeficiency virus type 1 Pr55 necessary for RNA binding and particle formation. , 1992, The Journal of general virology.

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

[16]  S. Höglund,et al.  Role of the major homology region of human immunodeficiency virus type 1 in virion morphogenesis , 1994, Journal of virology.

[17]  S. Sandmeyer,et al.  Mutations in the Ty3 major homology region affect multiple steps in Ty3 retrotransposition , 1996, Journal of virology.

[18]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[19]  W. Sundquist,et al.  Crystal Structure of Human Cyclophilin A Bound to the Amino-Terminal Domain of HIV-1 Capsid , 1996, Cell.

[20]  良二 上田.  J. Appl. Cryst.の発刊に際して , 1970 .

[21]  J. Luban,et al.  Linker insertion mutations in the human immunodeficiency virus type 1 gag gene: effects on virion particle assembly, release, and infectivity , 1995, Journal of virology.

[22]  B. Matthews.  Solvent content of protein crystals. , 1968, Journal of molecular biology.

[23]  W. Sundquist,et al.  Proteolytic refolding of the HIV‐1 capsid protein amino‐terminus facilitates viral core assembly , 1998, The EMBO journal.

[24]  F. Studier,et al.  Use of T7 RNA polymerase to direct expression of cloned genes. , 1990, Methods in enzymology.

[25]  J. Navaza,et al.  AMoRe: an automated package for molecular replacement , 1994 .

[26]  W. Hendrickson.  Determination of macromolecular structures from anomalous diffraction of synchrotron radiation. , 1991, Science.

[27]  R. Gilcher.  Human retroviruses and AIDS. , 1988, The Journal of the Oklahoma State Medical Association.

[28]  H. Kräusslich.  Morphogenesis and Maturation of Retroviruses , 1996, Current Topics in Microbiology and Immunology.

[29]  W. Sundquist,et al.  Structure of the carboxyl-terminal dimerization domain of the HIV-1 capsid protein. , 1997, Science.

[30]  E. Engleman,et al.  Identification of protein intermediates in the processing of the p55 HIV-1 gag precursor in cells infected with recombinant vaccinia virus. , 1989, The Journal of biological chemistry.

[31]  J. Sodroski,et al.  Functional association of cyclophilin A with HIV-1 virions , 1994, Nature.

[32]  C. Carter,et al.  Assembly of recombinant human immunodeficiency virus type 1 capsid protein in vitro , 1992, Journal of virology.

[33]  V. Biou,et al.  [31] Treatment of multiwavelength anomalous diffraction data as a special case of multiple isomorphous replacement. , 1997, Methods in enzymology.

[34]  E. Lillehoj,et al.  The gag gene products of human immunodeficiency virus type 1: alignment within the gag open reading frame, identification of posttranslational modifications, and evidence for alternative gag precursors , 1988, Journal of virology.

[35]  R. Wagner,et al.  Identification of a region in the Pr55gag-polyprotein essential for HIV-1 particle formation. , 1993, Virology.

[36]  P. V. von Hippel,et al.  Calculation of protein extinction coefficients from amino acid sequence data. , 1989, Analytical biochemistry.

[37]  C. Gelfand,et al.  Spectral analysis and tryptic susceptibility as probes of HIV-1 capsid protein structure. , 1994, Virology.

[38]  S. S. Hong,et al.  Assembly-defective point mutants of the human immunodeficiency virus type 1 Gag precursor phenotypically expressed in recombinant baculovirus-infected cells , 1993, Journal of virology.

[39]  H. Schägger,et al.  Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. , 1987, Analytical biochemistry.

[40]  W. Haseltine,et al.  A major retroviral core protein related to EPA and TIMP , 1985, Nature.

[41]  B. Gay,et al.  Sequence requirements for encapsidation of deletion mutants and chimeras of human immunodeficiency virus type 1 Gag precursor into retrovirus-like particles , 1995, Journal of virology.

[42]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[43]  J. Culp,et al.  Characterization of HIV‐1 p24 self‐association using analytical affinity chromatography , 1992, Proteins.

[44]  J. Wills,et al.  Genetic analysis of the major homology region of the Rous sarcoma virus Gag protein , 1995, Journal of virology.

[45]  Gilcher Ro.  Human retroviruses and AIDS. , 1988 .

[46]  H. Gendelman,et al.  Production of acquired immunodeficiency syndrome-associated retrovirus in human and nonhuman cells transfected with an infectious molecular clone , 1986, Journal of virology.

[47]  M. Hammarskjöld,et al.  Characterization of deletion mutations in the capsid region of human immunodeficiency virus type 1 that affect particle formation and Gag-Pol precursor incorporation , 1995, Journal of virology.

[48]  Wesley I. Sundquist,et al.  Structure of the Amino-Terminal Core Domain of the HIV-1 Capsid Protein , 1996, Science.

[49]  E. Barklis,et al.  Assembly, processing, and infectivity of human immunodeficiency virus type 1 gag mutants , 1993, Journal of virology.

[50]  Isma'il ibn Ali al-Sadiq.  AIDS , 1986, The Lancet.

[51]  S. Goff,et al.  The role of Gag in human immunodeficiency virus type 1 virion morphogenesis and early steps of the viral life cycle , 1996, Journal of virology.

[52]  W. Sundquist,et al.  Molecular recognition in the HIV-1 capsid/cyclophilin A complex. , 1997, Journal of molecular biology.

[53]  J. Luban,et al.  Specificity and sequence requirements for interactions between various retroviral Gag proteins , 1994, Journal of virology.