Bombarda, E.

Grell, E.

Mély, Y.

Roques, B.

The kinetics of Zn2+ binding by two point-mutated forms of the HIV-1 NCp7 C-terminal zinc finger, each containing tridentate binding motif HCC [Ser49(35−50)NCp7] or CCC [Ala44(35−50)NCp7], has been studied by stopped-flow spectrofluorimetry. Both the formation and dissociation rate constants of the complexes between Zn2+ and the two model peptides depend on pH. The results are interpreted on the basis of a multistep reaction model involving three Zn2+ binding paths due to three deprotonated states of the coordinating motif, acting as monodentate, bidentate, and tridentate ligands. For Ser49(35−50)NCp7 around neutral pH, binding preferentially occurs via the deprotonated Cys36 in the bidentate state also involving His44. The binding rate constants for the monodentate and bidentate states are 1 × 106 and 3.9 × 107 M-1 s-1, respectively. For Ala44(35−50)NCp7, intermolecular Zn2+ binding predominantly occurs via the deprotonated Cys36 in the monodentate state with a rate constant of 3.6 × 107 M-1 s-1. In both mutants, the final state of the Zn2+ complex is reached by subsequent stepwise ligand deprotonation and intramolecular substitution of coordinated water molecules. The rate constants for the intermolecular binding paths of the bidentate and tridentate states of Ala44(35−50)NCp7 and of the tridentate state of Ser49(35−50)NCp7 are much smaller than expected according to electrostatic considerations. This is attributed to conformational constraints required to achieve proper metal coordination during folding. The dissociation of Zn2+ from both peptides is again characterized by a multistep process and takes place fastest via the protonated Zn2+-bound bidentate and monodentate states, with rate constants of ∼0.3 and ∼103 s-1, respectively, for Ser49(35−50)NCp7 and ∼4 × 10-3 and ∼500 s-1, respectively, for Ala44(35−50)NCp7

Mechanism of Zinc Coordination by Point-Mutated Structures of the Distal CCHC Binding Mofif of the HIV-1 NCp<sub>7</sub> Protein

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