Mechanism of zinc coordination by point-mutated structures of the distal CCHC binding motif of the HIV-1 NCp7 protein.
The kinetics of Zn(2+) 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 Zn(2+) and the two model peptides depend on pH. The results are interpreted on the basis of a multistep reaction model involving three Zn(2+) 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 x 10(6) and 3.9 x 10(7) M(-)(1) s(-)(1), respectively. For Ala44(35-50)NCp7, intermolecular Zn(2+) binding predominantly occurs via the deprotonated Cys36 in the monodentate state with a rate constant of 3.6 x 10(7) M(-)(1) s(-)(1). In both mutants, the final state of the Zn(2+) 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 Zn(2+) from both peptides is again characterized by a multistep process and takes place fastest via the protonated Zn(2+)-bound bidentate and monodentate states, with rate constants of approximately 0.3 and approximately 10(3) s(-)(1), respectively, for Ser49(35-50)NCp7 and approximately 4 x 10(-)(3) and approximately 500 s(-)(1), respectively, for Ala44(35-50)NCp7.
Bombarda
Elisa
E
UMR 7034 du CNRS, Laboratoire de Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, Faculté de Pharmacie, Université Louis Pasteur, 74, Route du Rhin, F-67401 Illkirch Cedex, France.
Roques
Bernard P
BP
Mély
Yves
Y
Grell
Ernst
E
D000409:Alanine / Q000235:genetics; D020816:Amino Acid Motifs / Q000235:genetics; D000595:Amino Acid Sequence; D036022:Capsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003545:Cysteine / Q000235:genetics*; D015683:Gene Products, gag / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000737:chemistry* / Q000235:genetics; D006639:Histidine / Q000235:genetics*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding / Q000235:genetics; D012694:Serine / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000737:chemistry*; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D010446:Peptide Fragments; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D012694:Serine; D006639:Histidine; D015032:Zinc; D003545:Cysteine; D000409:Alanine