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 Zn2+ drives NCp7 from an unfolded to a highly folded structure that is critical for its functions. Using the intrinsic fluorescence of Trp37, we investigated, by the stopped-flow technique, the folding of NCp7 distal finger through the pH dependence of its Zn2+ association and dissociation kinetics. Zn2+ 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 Zn2+-binding process have been determined. At neutral pH, the preferential pathway for the Zn2+-driven folding implies Zn2+ binding to the deprotonated Cys36 and His44 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 Zn2+ to optimize the binding geometry. This form then rapidly leads to the final native complex, through deprotonation of Cys39 and Cys49 residues and intramolecular substitution of coordinated water molecules. Zn2+ dissociation is also characterized by a multistep process and occurs fastest via the deprotonated Zn2+-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.