Zinc binding to the HIV-1 nucleocapsid protein: a thermodynamic investigation by fluorescence spectroscopy.
The HIV-1 nucleocapsid protein, NCp7, is characterized by two CCHC zinc finger motifs which have been shown to stoichiometrically bind zinc in mature virions. Moreover, this binding of zinc proves to be critical in various NCp7 functions, especially in the encapsidation process. To further understand the central role of zinc binding to NCp7, we closely investigated the zinc binding properties of NCp7 and various deleted or substituted derivatives. To this end, the fluorescence of wither the naturally occurring Trp37 or the conservatively substituted Trp16 was used to monitor the binding of zinc to the N- and C-terminal finger motifs, respectively. At pH 7.5, the NCp7 proximal motif was found to bind zinc strongly with 2.8 x 10(14) M-1 binding constant about five times higher than the NCp7 distal motif. Moreover, the binding of zinc to one finger motif decreased the affinity of the second one, and this negative cooperativity was shown to be related to the spatial proximity of the zinc-saturated finger motifs. The binding seemed to be almost equally driven by entropy and enthalpy, and the binding information was essentially encoded by the finger motifs themselves whereas the other parts of the protein only played a marginal stabilization role. As expected, the Cys and His residues of the CCHC motifs were critical and competition between protons and zinc ions to these residues induced a steep pH-dependence of the zinc binding constants to both sites. Taken together, our data provide further evidence for the nonequivalence of the two NCp7 finger motifs.
Mély
Y
Y
Laboratoire de Biophysique, URA 491 du CNRS, Faculté de Pharmacie de Strasbourg I, B.P. 24, Illkirch, France.
De Rocquigny
H
H
Morellet
N
N
Roques
B P
BP
Gérad
D
D
D000595:Amino Acid Sequence; D002213:Capsid / Q000138:chemical synthesis / Q000378:metabolism*; D036022:Capsid Proteins; D003198:Computer Simulation; D015683:Gene Products, gag / Q000138:chemical synthesis / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D017384:Sequence Deletion; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014764:Viral Proteins; D015032:Zinc / Q000378:metabolism*; D016335:Zinc Fingers; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D015032:Zinc