Structure of the HIV-1 nucleocapsid protein bound to the SL3 ψ-RNA recognition element

Science

Volumn 279, Issue 5349, 1998, Pages 384-388

  De Guzman, Roberto N ^a   Wu, Zheng Rong ^a   Stalling, Chelsea C ^a   Pappalardo, Lucia ^b   Borer, Philip N ^b   Summers, Michael F ^a  

^a UNIVERSITY OF MARYLAND BALTIMORE COUNTY   (United States)

^b SYRACUSE UNIVERSITY   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

VIRUS PROTEIN;

AMINO TERMINAL SEQUENCE; ARTICLE; BINDING AFFINITY; CARBOXY TERMINAL SEQUENCE; HUMAN IMMUNODEFICIENCY VIRUS 1; NONHUMAN; NUCLEAR MAGNETIC RESONANCE; PRIORITY JOURNAL; PROTEIN RNA BINDING; PROTEIN STRUCTURE; STRUCTURE ACTIVITY RELATION; STRUCTURE ANALYSIS; VIRUS NUCLEOCAPSID;

AMINO ACID SEQUENCE; BASE SEQUENCE; BINDING SITES; GENE PRODUCTS, GAG; GENOME, VIRAL; HIV-1; HYDROGEN BONDING; MAGNETIC RESONANCE SPECTROSCOPY; MODELS, MOLECULAR; MOLECULAR SEQUENCE DATA; NUCLEIC ACID CONFORMATION; NUCLEOCAPSID; PROTEIN CONFORMATION; PROTEIN FOLDING; PROTEIN STRUCTURE, SECONDARY; RNA, VIRAL; ZINC; ZINC FINGERS;

EID: 2242469712     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.279.5349.384     Document Type: Article

References (92)

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27. 13C]methanol and purified as described [R. T. Batey, J. L. Battiste, J. R. Williamson, Methods Enzymol. 261, 300 (1995)].
28. 13C]methanol and purified as described [R. T. Batey, J. L. Battiste, J. R. Williamson, Methods Enzymol. 261, 300 (1995)].
29. 13C]methanol and purified as described [R. T. Batey, J. L. Battiste, J. R. Williamson, Methods Enzymol. 261, 300 (1995)].
30. 13C]methanol and purified as described [R. T. Batey, J. L. Battiste, J. R. Williamson, Methods Enzymol. 261, 300 (1995)].
31. r of Zn-bound protein, observed = 6501 ± 2, calculated = 6500).
32. r of Zn-bound protein, observed = 6501 ± 2, calculated = 6500).
33. r of Zn-bound protein, observed = 6501 ± 2, calculated = 6500).
34. r = 6678), and NC-RNA complex elute with apparent molecular weights of 8.0, 7.6, and 13.7 KD, respectively.
35. d = [NC][RNA]/[NC -RNA] ∼ 100 nM) was estimated by native gel electrophoresis (15% acrylamide), performed using dilutions of an NMR-characterized NC-SL3 sample. Equilibrium concentrations of free RNA were determined by comparison of band intensities with those of known concentrations of SL3 RNA.
36. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
37. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
38. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
39. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
40. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
41. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
42. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
43. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
44. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
45. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
46. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
47. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
48. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
49. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
50. 15N-edited TOCSY (36) obtained with a 75-ms clean-MLEV-17 mixing period [C. Griesinger, G. Otting, K. Wüthrich, R. R. Ernst, J. Am. Chem. Soc. 110, 7870 (1988)] and sensitivity-improved gradient coherence selection [O. Zhang, L. E. Kay, J. P. Olivier, J. D. Forman-Kay, J. Biomol. NMR 4, 845 (1994)]; 2D rotating frame Overhauser effect (ROESY) [A. Bax and D. G. Davis, J. Magn. Reson. 63, 207 (1985)] (120 ms, 6.0-kHz continuous-wave spin-lock mixing pulse); and constant-time heteronuclear single-quantum coherence (HSQC) [G. W.
51. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
52. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
53. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
54. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
55. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
56. 12C-detected gradient-purged 3D HMQC-NOESY data [M. Ikura and A. Bax, J. Am. Chem. Soc. 114, 2433 (1992); W. Lee, M. J. Revington, C. Arrowsmith, L. E. Kay, FEBS Lett. 350, 87(1994)].
57. Structures were calculated with the program DY-ANA [P. Güntert, C. Mumenthaler, K. Wüthrich, J. Mol. Biol. 273, 283 (1997)]. Secondary structure elements were identified by analysis of NOE cross-peak patterns [K. Wuthrich, NMR of Proteins and Nucleic Acids (Wiley, New York, 1986)]. Upper-limit distance restraints of 2.7, 3.3, and 5.0 Å (with 0.5 Å added for NOEs involving methyl protons)-corresponding to strong-, medium-, and weak-in-tensity NOE cross-peaks, respectively-were used for both the NC protein and RNA. Loose torsion angle restraints were used for the RNA phosphodiester groups, which allowed sampling of both A- and B-helical conformations but prevented excessive fraying [D. S. Wutke, M. P. Foster, D. A. Case, J. M. Gottesfeld, P. E. Wright, J. Mol. Biol. 273, 183 (1997)]. Only functional restraints were included in the calculations (for example, in cases where a proton exhibited NOEs of different intensities to geminal methylene protons, restraints were used only for the stronger of the two cross-peaks). Convergence statistics were calculated with MOLMOL [R. Koradi, M. Billeter, K. Wuthrich, J. Mol. Graphics 14, 51 (1996)]. Color images were generated with MidasPlus [T. E. Ferrin, C. C. Huang, L. E. Javis, R. Langridge, ibid. 6, 13 (1988)], Molscript [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)], GRASP (A. Nicholls, GRASP: Graphical Representation and Analysis of Surface Properties, Columbia University), and Raster-30 [D. J. Bacon and W. F. Anderson, J. Mol. Graphics 6, 219 (1988)].
58. Structures were calculated with the program DY-ANA [P. Güntert, C. Mumenthaler, K. Wüthrich, J. Mol. Biol. 273, 283 (1997)]. Secondary structure elements were identified by analysis of NOE cross-peak patterns [K. Wuthrich, NMR of Proteins and Nucleic Acids (Wiley, New York, 1986)]. Upper-limit distance restraints of 2.7, 3.3, and 5.0 Å (with 0.5 Å added for NOEs involving methyl protons)-corresponding to strong-, medium-, and weak-in-tensity NOE cross-peaks, respectively-were used for both the NC protein and RNA. Loose torsion angle restraints were used for the RNA phosphodiester groups, which allowed sampling of both A- and B-helical conformations but prevented excessive fraying [D. S. Wutke, M. P. Foster, D. A. Case, J. M. Gottesfeld, P. E. Wright, J. Mol. Biol. 273, 183 (1997)]. Only functional restraints were included in the calculations (for example, in cases where a proton exhibited NOEs of different intensities to geminal methylene protons, restraints were used only for the stronger of the two cross-peaks). Convergence statistics were calculated with MOLMOL [R. Koradi, M. Billeter, K. Wuthrich, J. Mol. Graphics 14, 51 (1996)]. Color images were generated with MidasPlus [T. E. Ferrin, C. C. Huang, L. E. Javis, R. Langridge, ibid. 6, 13 (1988)], Molscript [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)], GRASP (A. Nicholls, GRASP: Graphical Representation and Analysis of Surface Properties, Columbia University), and Raster-30 [D. J. Bacon and W. F. Anderson, J. Mol. Graphics 6, 219 (1988)].
59. Structures were calculated with the program DY-ANA [P. Güntert, C. Mumenthaler, K. Wüthrich, J. Mol. Biol. 273, 283 (1997)]. Secondary structure elements were identified by analysis of NOE cross-peak patterns [K. Wuthrich, NMR of Proteins and Nucleic Acids (Wiley, New York, 1986)]. Upper-limit distance restraints of 2.7, 3.3, and 5.0 Å (with 0.5 Å added for NOEs involving methyl protons)-corresponding to strong-, medium-, and weak-in-tensity NOE cross-peaks, respectively-were used for both the NC protein and RNA. Loose torsion angle restraints were used for the RNA phosphodiester groups, which allowed sampling of both A- and B-helical conformations but prevented excessive fraying [D. S. Wutke, M. P. Foster, D. A. Case, J. M. Gottesfeld, P. E. Wright, J. Mol. Biol. 273, 183 (1997)]. Only functional restraints were included in the calculations (for example, in cases where a proton exhibited NOEs of different intensities to geminal methylene protons, restraints were used only for the stronger of the two cross-peaks). Convergence statistics were calculated with MOLMOL [R. Koradi, M. Billeter, K. Wuthrich, J. Mol. Graphics 14, 51 (1996)]. Color images were generated with MidasPlus [T. E. Ferrin, C. C. Huang, L. E. Javis, R. Langridge, ibid. 6, 13 (1988)], Molscript [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)], GRASP (A. Nicholls, GRASP: Graphical Representation and Analysis of Surface Properties, Columbia University), and Raster-30 [D. J. Bacon and W. F. Anderson, J. Mol. Graphics 6, 219 (1988)].
60. Structures were calculated with the program DY-ANA [P. Güntert, C. Mumenthaler, K. Wüthrich, J. Mol. Biol. 273, 283 (1997)]. Secondary structure elements were identified by analysis of NOE cross-peak patterns [K. Wuthrich, NMR of Proteins and Nucleic Acids (Wiley, New York, 1986)]. Upper-limit distance restraints of 2.7, 3.3, and 5.0 Å (with 0.5 Å added for NOEs involving methyl protons)-corresponding to strong-, medium-, and weak-in-tensity NOE cross-peaks, respectively-were used for both the NC protein and RNA. Loose torsion angle restraints were used for the RNA phosphodiester groups, which allowed sampling of both A- and B-helical conformations but prevented excessive fraying [D. S. Wutke, M. P. Foster, D. A. Case, J. M. Gottesfeld, P. E. Wright, J. Mol. Biol. 273, 183 (1997)]. Only functional restraints were included in the calculations (for example, in cases where a proton exhibited NOEs of different intensities to geminal methylene protons, restraints were used only for the stronger of the two cross-peaks). Convergence statistics were calculated with MOLMOL [R. Koradi, M. Billeter, K. Wuthrich, J. Mol. Graphics 14, 51 (1996)]. Color images were generated with MidasPlus [T. E. Ferrin, C. C. Huang, L. E. Javis, R. Langridge, ibid. 6, 13 (1988)], Molscript [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)], GRASP (A. Nicholls, GRASP: Graphical Representation and Analysis of Surface Properties, Columbia University), and Raster-30 [D. J. Bacon and W. F. Anderson, J. Mol. Graphics 6, 219 (1988)].
61. Structures were calculated with the program DY-ANA [P. Güntert, C. Mumenthaler, K. Wüthrich, J. Mol. Biol. 273, 283 (1997)]. Secondary structure elements were identified by analysis of NOE cross-peak patterns [K. Wuthrich, NMR of Proteins and Nucleic Acids (Wiley, New York, 1986)]. Upper-limit distance restraints of 2.7, 3.3, and 5.0 Å (with 0.5 Å added for NOEs involving methyl protons)-corresponding to strong-, medium-, and weak-in-tensity NOE cross-peaks, respectively-were used for both the NC protein and RNA. Loose torsion angle restraints were used for the RNA phosphodiester groups, which allowed sampling of both A- and B-helical conformations but prevented excessive fraying [D. S. Wutke, M. P. Foster, D. A. Case, J. M. Gottesfeld, P. E. Wright, J. Mol. Biol. 273, 183 (1997)]. Only functional restraints were included in the calculations (for example, in cases where a proton exhibited NOEs of different intensities to geminal methylene protons, restraints were used only for the stronger of the two cross-peaks). Convergence statistics were calculated with MOLMOL [R. Koradi, M. Billeter, K. Wuthrich, J. Mol. Graphics 14, 51 (1996)]. Color images were generated with MidasPlus [T. E. Ferrin, C. C. Huang, L. E. Javis, R. Langridge, ibid. 6, 13 (1988)], Molscript [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)], GRASP (A. Nicholls, GRASP: Graphical Representation and Analysis of Surface Properties, Columbia University), and Raster-30 [D. J. Bacon and W. F. Anderson, J. Mol. Graphics 6, 219 (1988)].
62. Structures were calculated with the program DY-ANA [P. Güntert, C. Mumenthaler, K. Wüthrich, J. Mol. Biol. 273, 283 (1997)]. Secondary structure elements were identified by analysis of NOE cross-peak patterns [K. Wuthrich, NMR of Proteins and Nucleic Acids (Wiley, New York, 1986)]. Upper-limit distance restraints of 2.7, 3.3, and 5.0 Å (with 0.5 Å added for NOEs involving methyl protons)-corresponding to strong-, medium-, and weak-in-tensity NOE cross-peaks, respectively-were used for both the NC protein and RNA. Loose torsion angle restraints were used for the RNA phosphodiester groups, which allowed sampling of both A- and B-helical conformations but prevented excessive fraying [D. S. Wutke, M. P. Foster, D. A. Case, J. M. Gottesfeld, P. E. Wright, J. Mol. Biol. 273, 183 (1997)]. Only functional restraints were included in the calculations (for example, in cases where a proton exhibited NOEs of different intensities to geminal methylene protons, restraints were used only for the stronger of the two cross-peaks). Convergence statistics were calculated with MOLMOL [R. Koradi, M. Billeter, K. Wuthrich, J. Mol. Graphics 14, 51 (1996)]. Color images were generated with MidasPlus [T. E. Ferrin, C. C. Huang, L. E. Javis, R. Langridge, ibid. 6, 13 (1988)], Molscript [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)], GRASP (A. Nicholls, GRASP: Graphical Representation and Analysis of Surface Properties, Columbia University), and Raster-30 [D. J. Bacon and W. F. Anderson, J. Mol. Graphics 6, 219 (1988)].
63. Structures were calculated with the program DY-ANA [P. Güntert, C. Mumenthaler, K. Wüthrich, J. Mol. Biol. 273, 283 (1997)]. Secondary structure elements were identified by analysis of NOE cross-peak patterns [K. Wuthrich, NMR of Proteins and Nucleic Acids (Wiley, New York, 1986)]. Upper-limit distance restraints of 2.7, 3.3, and 5.0 Å (with 0.5 Å added for NOEs involving methyl protons)-corresponding to strong-, medium-, and weak-in-tensity NOE cross-peaks, respectively-were used for both the NC protein and RNA. Loose torsion angle restraints were used for the RNA phosphodiester groups, which allowed sampling of both A- and B-helical conformations but prevented excessive fraying [D. S. Wutke, M. P. Foster, D. A. Case, J. M. Gottesfeld, P. E. Wright, J. Mol. Biol. 273, 183 (1997)]. Only functional restraints were included in the calculations (for example, in cases where a proton exhibited NOEs of different intensities to geminal methylene protons, restraints were used only for the stronger of the two cross-peaks). Convergence statistics were calculated with MOLMOL [R. Koradi, M. Billeter, K. Wuthrich, J. Mol. Graphics 14, 51 (1996)]. Color images were generated with MidasPlus [T. E. Ferrin, C. C. Huang, L. E. Javis, R. Langridge, ibid. 6, 13 (1988)], Molscript [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)], GRASP (A. Nicholls, GRASP: Graphical Representation and Analysis of Surface Properties, Columbia University), and Raster-30 [D. J. Bacon and W. F. Anderson, J. Mol. Graphics 6, 219 (1988)].
64. Structures were calculated with the program DY-ANA [P. Güntert, C. Mumenthaler, K. Wüthrich, J. Mol. Biol. 273, 283 (1997)]. Secondary structure elements were identified by analysis of NOE cross-peak patterns [K. Wuthrich, NMR of Proteins and Nucleic Acids (Wiley, New York, 1986)]. Upper-limit distance restraints of 2.7, 3.3, and 5.0 Å (with 0.5 Å added for NOEs involving methyl protons)-corresponding to strong-, medium-, and weak-in-tensity NOE cross-peaks, respectively-were used for both the NC protein and RNA. Loose torsion angle restraints were used for the RNA phosphodiester groups, which allowed sampling of both A- and B-helical conformations but prevented excessive fraying [D. S. Wutke, M. P. Foster, D. A. Case, J. M. Gottesfeld, P. E. Wright, J. Mol. Biol. 273, 183 (1997)]. Only functional restraints were included in the calculations (for example, in cases where a proton exhibited NOEs of different intensities to geminal methylene protons, restraints were used only for the stronger of the two cross-peaks). Convergence statistics were calculated with MOLMOL [R. Koradi, M. Billeter, K. Wuthrich, J. Mol. Graphics 14, 51 (1996)]. Color images were generated with MidasPlus [T. E. Ferrin, C. C. Huang, L. E. Javis, R. Langridge, ibid. 6, 13 (1988)], Molscript [P. J. Kraulis, J. Appl. Crystallogr. 24, 946 (1991)], GRASP (A. Nicholls, GRASP: Graphical Representation and Analysis of Surface Properties, Columbia University), and Raster-30 [D. J. Bacon and W. F. Anderson, J. Mol. Graphics 6, 219 (1988)].
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92. Supported by NIH grants GM42561 (M.F.S.) and GM32691 (P.N.B.). C.C.S. is an Undergraduate Meyerhoff Scholar at UMBC and L.P. is supported by a Syracuse University Graduate Scholarship. We thank B. M. Lee for collecting some of the NMR data, R. Wall for assistance in RNA purification, and R. Edwards and M. Zawrotny for technical support.