CONFORMATIONAL BEHAVIOR OF THE ACTIVE AND INACTIVE FORMS OF THE NUCLEOCAPSID NCP7 OF HIV-1 STUDIED BY H-1-NMR

被引:137
作者
MORELLET, N
DEROCQUIGNY, H
MELY, Y
JULLIAN, N
DEMENE, H
OTTMANN, M
GERARD, D
DARLIX, JL
FOURNIEZALUSKI, MC
ROQUES, BP
机构
[1] FAC PHARM ILLKIRCH,BIOPHYS LAB,F-67401 ILLKIRCH GRAFFENS,FRANCE
[2] ENS LYON,BIOL MOLEC & CELLULAIRE LAB,F-69634 LYON 07,FRANCE
关键词
HIV-1; NUCLEOCAPSID NCP7; NMR; PROTEIN STRUCTURE; ZINC FINGER;
D O I
10.1016/S0022-2836(05)80033-6
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The nucleocapsid protein NCp7 of the human immunodeficiency virus type I (HIV-1) is a 72 amino acid peptide containing two zinc fingers of the type CX2CX4HX4C linked by a short basic sequence 29RAPRKKG35. NCp7 was shown to activate in vitro both viral RNA dimerization and replication primer tRNALys,3 annealing to the initiation site of reverse transcription. In order to clarify the possible structural role of the zinc fingers in the various functions of NCp7, complete sequence specific 1H NMR assignment of the entire protein was achieved by two-dimensional NMR experiments. Moreover, to characterize the role of the peptide linker in NCp7 folding, a synthetic analogue with an inversion of Pro31 configuration was studied by NMR and fluorescence techniques. Several long range NOEs implying amino acid protons from the folded zinc fingers and the spacer, such as Ala25 and Trp37, Phe16 and Trp37, Arg32 and Trp37, Ly3 and Trp37, Cyauthor8 and Ly3 disappeared in the d-Pro31 (12-53)NCp7, confirming the spatial proximity of the two CCHC boxes observed in the (13-51)NCp7. This was also confirmed by iodide fluorescence quenching experiments. The N and C-terminal parts of NCp7 displayed a large flexibility except for two short sequences Tyr56 to Gly58 and Tyr64 to Gly66, which seemed to oscillate between random-coil and helical conformations. The biological relevance of the structural characteristics of NCp7 was studied in vitro and in vivo. Substitution of Pro31 by d-Pro31 in the active (13-64)NCp7 peptide led to a severe reduction of dimerization in vitro. Moreover, site-directed mutagenesis subtituting Leu for Pro31 resulted in the formation of non-infectious and immature viral particles. These results suggest that the spatial proximity of the zinc fingers induced by the peptide linker, plays a critical role in encapsidation of genomic RNA and morphogenesis of HIV-1 infectious particles. © 1994 Academic Press Limited.
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页码:287 / 301
页数:15
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