Molecular mechanism of the Zn2+-induced folding of the distal CCHC finger motif of the HIV-1 nucleocapsid protein.
HIV-1 nucleocapsid protein, NCp7, contains two highly conserved CCHC zinc fingers. Binding of Zn(2+) drives NCp7 from an unfolded to a highly folded structure that is critical for its functions. Using the intrinsic fluorescence of Trp(37), we investigated, by the stopped-flow technique, the folding of NCp7 distal finger through the pH dependence of its Zn(2+) association and dissociation kinetics. Zn(2+) binding was found to involve four different paths associated with the four deprotonated states of the finger. Each binding path involves the rapid formation of an intermediate complex that is subsequently rearranged and stabilized in a rate-limiting step. The equilibrium and kinetic rate constants of the full Zn(2+)-binding process have been determined. At neutral pH, the preferential pathway for the Zn(2+)-driven folding implies Zn(2+) binding to the deprotonated Cys(36) and His(44) residues, in the bidentate state of the finger. The resulting intermediate is then converted with a rate constant of 500 s(-1) into a more suitably folded form, probably through a rearrangement of the peptide backbone around Zn(2+) to optimize the binding geometry. This form then rapidly leads to the final native complex, through deprotonation of Cys(39) and Cys(49) residues and intramolecular substitution of coordinated water molecules. Zn(2+) dissociation is also characterized by a multistep process and occurs fastest via the deprotonated Zn(2+)-bound bidentate state with a rate constant of 3 s(-1). Due to their critical role in folding, the intermediates identified for the first time in this study may constitute potential targets for HIV therapy.
Bombarda
Elisa
E
UMR 7175, Centre National de la Recherche Scientifique, Université Louis Pasteur, Faculté de Pharmacie, Illkirch, France. elisa.bombarda@uni-bayreuth.de
Grell
Ernst
E
Roques
Bernard P
BP
Mély
Yves
Y
D020816:Amino Acid Motifs; D036022:Capsid Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D003198:Computer Simulation; D015683:Gene Products, gag / Q000737:chemistry* / Q000648:ultrastructure*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000648:ultrastructure*; D015032:Zinc / Q000737:chemistry*; D016335:Zinc Fingers; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; C080114:NC71 protein, Human immunodeficiency virus 1; D016601:RNA-Binding Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D015032:Zinc