Investigation of zinc-binding affinities of Moloney murine leukemia virus nucleocapsid protein and its related zinc finger and modified peptides.
Nucleocapsid proteins of retroviruses are small basic, nucleic acid-binding proteins with either one or two "Cys-His" boxes, which have been shown to be involved in genomic RNA dimerization, encapsidation, and replication primer tRNA annealing to the initiation site for reverse transcription. The nucleocapsid (NC) protein of Moloney murine leukemia virus (MoMuLV NCp10) is made up of 56 residues with one Cys-His motif. The Zn(2+)-binding affinities and induced conformational changes of NCp10 were investigated by following the fluorescence of Trp 35 located in the Cys-His domain. At pH 7.5, NCp10 was shown to bind Zn2+ at a 1 : 1 ratio with a very high apparent binding constant of 1.2 (+/- 0.3).10(13)M-1. A similar apparent binding constant was obtained for a 19-residue peptide encompassing the Cys-His box, designated the "zinc finger motif," indicating that it contains most if not all the information to bind Zn2+ tightly. Changing Trp 35 to Phe in the peptide did not affect the Zn2+ affinity, indicating that Trp 35 is not crucial for Zn2+ binding. On the contrary, replacing Cys 29 by Ser, the chemical modification or oxidation of the three Cys sharply reduced Zn2+ affinity, confirming the essential role of Cys in Zn2+ binding. In addition, fluorescence and energy transfer data suggested that Zn2+ binding modifies the Trp 35 environment but not its solvent exposure, and increases the average distance between Tyr 28 and Trp 35 by about 2 A. These data suggest that Zn2+ binding to retroviral NC protein is biologically relevant.
Mely
Y
Y
Laboratoire de Biophysique, CNRS UA 491, Université Louis Pasteur, Faculté de Pharmacie de Strasbourg, Illkirch, France.
Cornille
F
F
Fournié-Zaluski
M C
MC
Darlix
J L
JL
Roques
B P
BP
Gérard
D
D
D000595:Amino Acid Sequence; D002213:Capsid / Q000378:metabolism*; D008969:Molecular Sequence Data; D008979:Moloney murine leukemia virus / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism*; D014758:Viral Core Proteins / Q000378:metabolism*; D015032:Zinc / Q000378:metabolism*; D016335:Zinc Fingers / Q000502:physiology*
D010455:Peptides; D014758:Viral Core Proteins; D015032:Zinc