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 cha racterized 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 H-1 NMR, potentiometry and fluorescence spectroscopy, the acid-base properties of the four Zn2+-coord inating residues of (35-50)NCp7, a peptide corresponding to the distal fing er 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 fi tted adequately with a single pK(a). This suggests that the protonating gro ups are non-interacting, a feature that was confirmed by a potentiometric t itration. The pK(a) of His44, Cys36, Cys39, and Cys49 in the apopeptide wer e found to be 6.4, 8.0, 8.8 and 9.3, respectively. Accordingly, the deproto nation 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 re lated to the negative charge of the neighboring Asp48. Such a high pK(a) ma y be a general feature in nucleocapsid proteins (NCs), since an acidic resi due generally occupies the (i - 1) position of the C-terminal Cys residue o f single-finger NCs and distal finger motifs in two-finger NCs. Molecular d ynamics simulation suggested the formation of a hydrogen bonded network tha t weakly structured the Cys36-Cys39 segment in the apopeptide. This network depends on the protonation state of Cys36 and may thus explain the biphasi c behavior of the pH-dependence of the Cys39 H-beta2 resonance. Finally, th e 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 fo ur orders of magnitude and that a significant dissociation of Zn2+ from the holopeptide can be achieved in acidic cell compartments. (C) 2001 Academic Press.