title: Probing the RNA binding surface of the HIV-1 nucleocapsid protein by site-directed mutagenesis.
abstract: The highly conserved nucleocapsid protein domain in HIV-1 recognizes and binds SL3 in genomic RNA. In this work, we used the structure of the NCp7-SL3 RNA complex to guide the construction of 16 NCp7 mutants to probe the RNA binding surface of the protein [De Guzman, R. N., et al. (1998) Science 279, 384-388]. Thirteen residues with functional or structural significance were mutated individually to Ala (Asn(5), Phe(6), Val(13), Phe(16), Asn(17), Gly(19), Glu(21), Ile(24), Gln(45), Met(46), Gly(22), Pro(31), and Gly(40)), and three salt bridge switch mutants exchanged Lys and Glu (Lys(14)-Glu(21), Lys(33)-Glu(42), and Lys(38)-Glu(51)). Dissociation constants (Kd) determined by fluorescence titration and isothermal titration calorimetry were used to compare affinities of SL3 for the variant proteins to that for the wild type. The F16A (Phe(16) to Ala) variant showed a 25-fold reduction in affinity, consistent with a loss of organized structure in f1, the protein's first zinc finger. I24A, Q45A, and M46A reduced affinity by 2-5-fold; these residues occupy nearly equivalent positions in f1 and f2. E21A increased affinity by 3-fold, perhaps because of the mutant's increased net positive charge. Among the salt bridge switch mutants, only K14E/E21K in f1 caused a substantial change in affinity (5-fold reduction), binding SL3 with a biphasic binding isotherm. Aside from these six variants, most of the mutations studied have relatively minor effects on the stability of the complex. We conclude that many side chain interactions in the wild-type complex contribute little to stability or can be compensated by new contacts in the mutants.
authors.affiliation: Graduate Program in Structural Biology, Biochemistry, and Biophysics and ‡Department of Chemistry, Syracuse University , Syracuse, New York 13244-4100, United States.
authors.forename: Wei
authors.initials: W
authors.lastname: Ouyang
authors.forename: Stephen
authors.initials: S
authors.lastname: Okaine
authors.forename: Mark P
authors.initials: MP
authors.lastname: McPike
authors.forename: Yong
authors.initials: Y
authors.lastname: Lin
authors.forename: Philip N
authors.initials: PN
authors.lastname: Borer
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D016679:Genome, Viral; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D020038:RNA, Spliced Leader / Q000235:genetics / Q000378:metabolism*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
chemical_list: C071884:NCP7 protein, Human immunodeficiency virus 1; D020038:RNA, Spliced Leader; D012367:RNA, Viral; D054301:gag Gene Products, Human Immunodeficiency Virus