Zn2+ binding properties of single-point mutants of the C-terminal zinc finger of the HIV-1 nucleocapsid protein:: Evidence of a critical role of cysteine 49 in Zn2+ dissociation

The two highly conserved Zn2+ finger motifs of the HIV-1 nucleocapsid protein, NCp7, strongly bind Zn2+ through coordination of one His and three Cys residues. To further analyze the role of these residues, we investigated the Zn2+ binding and acid-base properties of four single-point mutants of a short peptide corresponding to the distal finger motif of NCp7. In each mutant, one Zn2+-coordinating residue is substituted with a noncoordinating one. Using the spectroscopic properties of Co2+, we first establish that the four mutants retain their ability to bind a metal cation through a four- or five-coordinate geometry with the vacant ligand position(s) presumably occupied by water molecule(s). Moreover, the pK(a) values of the three Cys residues of the mutant apopeptide where His44 is substituted with Ala are found by H-1 NMR to be similar to those of the native peptide, suggesting that the mutations do not affect the acid-base properties of the Zn2(+-)coordinating residues. The binding of Zn2+ was monitored by using the fluorescence of Trp37 as an intrinsic probe. At pH 7.5, the apparent Zn2+ binding constants (between 1.6 x 10(8) and 1.3 x 10(10) M-1) of the four mutants are strongly reduced compared to those of the native peptide but are similar to those of various host Zn2+ binding proteins. As a consequence, the loss of viral infectivity following the mutation of one Zn2+-coordinating residue in vivo may not be related to the total loss of Zn2+ binding. The pH dependence of Zn2+ binding indicates that the coordinating residues bind Zn2+ stepwise and that the free energy provided by the binding of a given residue may be modulated by the entropic contribution of the residues already bound to Zn2+. Finally, the pK(a) of Cys49 in the holopeptide is found to be 5.0, a value that is at least 0.7 unit higher than those for the other Zn2+-coordinating residues. This implies that Cys49 may act as a switch for Zn2+ dissociation in the distal finger motif of NCp7, a feature that may contribute to the high susceptibility of Cys49 to electrophilic attack.