Probing the RNA binding surface of the HIV-1 nucleocapsid protein by site-directed mutagenesis

Wei Ouyang, Stephen Okaine, Mark P. McPike, Yong Lin, Philip N. Borer

Research output: Contribution to journalArticle

5 Scopus citations

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 (Asn5, Phe6, Val13, Phe16, Asn17, Gly19, Glu21, Ile24, Gln45, Met46, Gly22, Pro 31, and Gly40), and three salt bridge switch mutants exchanged Lys and Glu (Lys14-Glu21, Lys 33-Glu42, and Lys38-Glu51). 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 (Phe16 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.

Original languageEnglish (US)
Pages (from-to)3358-3368
Number of pages11
JournalBiochemistry
Volume52
Issue number19
DOIs
StatePublished - May 14 2013

ASJC Scopus subject areas

  • Biochemistry

Fingerprint Dive into the research topics of 'Probing the RNA binding surface of the HIV-1 nucleocapsid protein by site-directed mutagenesis'. Together they form a unique fingerprint.

  • Cite this

    Ouyang, W., Okaine, S., McPike, M. P., Lin, Y., & Borer, P. N. (2013). Probing the RNA binding surface of the HIV-1 nucleocapsid protein by site-directed mutagenesis. Biochemistry, 52(19), 3358-3368. https://doi.org/10.1021/bi400125z