Structure/function mapping of amino acids in the N-terminal zinc finger of the human immunodeficiency virus type 1 nucleocapsid protein: residues responsible for nucleic acid helix destabilizing activity.
The nucleocapsid protein (NC) of HIV-1 is 55 amino acids in length and possesses two CCHC-type zinc fingers. Finger one (N-terminal) contributes significantly more to helix destabilizing activity than finger two (C-terminal). Five amino acids differ between the two zinc fingers. To determine at the amino acid level the reason for the apparent distinction between the fingers, each different residue in finger one was incrementally replaced by the one at the corresponding location in finger two. Mutants were analyzed in annealing assays with unstructured and structured substrates. Three groupings emerged: (1) those similar to wild-type levels (N17K, A25M), (2) those with diminished activity (I24Q, N27D), and (3) mutant F16W, which had substantially greater helix destabilizing activity than that of the wild type. Unlike I24Q and the other mutants, N27D was defective in DNA binding. Only I24Q and N27D showed reduced strand transfer in in vitro assays. Double and triple mutants F16W/I24Q, F16W/N27D, and F16W/I24Q/N27D all showed defects in DNA binding, strand transfer, and helix destabilization, suggesting that the I24Q and N27D mutations have a dominant negative effect and abolish the positive influence of F16W. Results show that amino acid differences at positions 24 and 27 contribute significantly to finger one's helix destabilizing activity.
Narayanan
Nirupama
N
Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, Maryland 20742, USA.
Gorelick
Robert J
RJ
DeStefano
Jeffrey J
JJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D009696:Nucleic Acids / Q000737:chemistry / Q000378:metabolism; D019590:Nucleocapsid Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010449:Peptide Mapping; D016335:Zinc Fingers / Q000235:genetics
D009696:Nucleic Acids; D019590:Nucleocapsid Proteins; D004247:DNA