Zn(2+) 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 Zn(2+) dissociation.

The two highly conserved Zn(2+) finger motifs of the HIV-1 nucleocapsid protein, NCp7, strongly bind Zn(2+) through coordination of one His and three Cys residues. To further analyze the role of these residues, we investigated the Zn(2+) 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 Zn(2+)-coordinating residue is substituted with a noncoordinating one. Using the spectroscopic properties of Co(2+), 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 (1)H NMR to be similar to those of the native peptide, suggesting that the mutations do not affect the acid-base properties of the Zn(2+)-coordinating residues. The binding of Zn(2+) was monitored by using the fluorescence of Trp37 as an intrinsic probe. At pH 7.5, the apparent Zn(2+) 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 Zn(2+) binding proteins. As a consequence, the loss of viral infectivity following the mutation of one Zn(2+)-coordinating residue in vivo may not be related to the total loss of Zn(2+) binding. The pH dependence of Zn(2+) binding indicates that the coordinating residues bind Zn(2+) 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 Zn(2+). 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 Zn(2+)-coordinating residues. This implies that Cys49 may act as a switch for Zn(2+) dissociation in the distal finger motif of NCp7, a feature that may contribute to the high susceptibility of Cys49 to electrophilic attack.

Determination of the pK(a) of the four Zn2+-coordinating residues of the distal finger motif of the HIV-1 nucleocapsid protein: consequences on the binding of Zn2+.

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 is characterized by two highly conserved CCHC motifs that bind Zn2+ strongly. To elucidate the striking pH-dependence of the apparent Zn2+-binding constants of these motifs further, we investigated, using 1H NMR, potentiometry and fluorescence spectroscopy, the acid-base properties of the four Zn2+-coordinating residues of (35-50)NCp7, a peptide corresponding to the distal finger motif of NCp7. With the exception of the H(beta2) proton of Cys39, the pH-dependence of the H(beta) proton resonances of the three Cys residues and, the H(delta) and H(epsilon) resonances of His44 in the apopeptide could be fitted adequately with a single pK(a). This suggests that the protonating groups are non-interacting, a feature that was confirmed by a potentiometric titration. The pK(a) of His44, Cys36, Cys39, and Cys49 in the apopeptide were found to be 6.4, 8.0, 8.8 and 9.3, respectively. Accordingly, the deprotonation is almost sequential and may thus induce a sequential binding of Zn2+ to the four coordinating residues. The high pK(a) of Cys49 is probably related to the negative charge of the neighboring Asp48. Such a high pK(a) may be a general feature in nucleocapsid proteins (NCs), since an acidic residue generally occupies the (i-1) position of the C-terminal Cys residue of single-finger NCs and distal finger motifs in two-finger NCs. Molecular dynamics simulation suggested the formation of a hydrogen bonded network that weakly structured the Cys36-Cys39 segment in the apopeptide. This network depends on the protonation state of Cys36 and may thus explain the biphasic behavior of the pH-dependence of the Cys39 H(beta2) resonance. Finally, the pK(a) values were used to build up a model describing the coordination of Zn2+ to (35-50)NCp7 at equilibrium. It appears that each protonation step of the coordination complex decreases the Zn2+-binding constant by about four orders of magnitude and that a significant dissociation of Zn2+ from the holopeptide can be achieved in acidic cell compartments.