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 Hβ2 proton of Cys39, the pH-dependence of the Hβ proton resonances of the three Cys residues and, the Hδ and H>ε resonances of His44 in the apopeptide could be fitted adequately with a single pKa. This suggests that the protonating groups are non-interacting, a feature that was confirmed by a potentiometric titration. The pKa 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 pKa of Cys49 is probably related to the negative charge of the neighboring Asp48. Such a high pKa 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 Hβ2 resonance. Finally, the pKa 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