Affinities of packaging domain loops in HIV-1 RNA for the nucleocapsid protein.
To design anti-nucleocapsid drugs, it is useful to know the affinities the protein has for its natural substrates under physiological conditions. Dissociation equilibrium constants are reported for seven RNA stem-loops bound to the mature HIV-1 nucleocapsid protein, NCp7. The loops include SL1, SL2, SL3, and SL4 from the major packaging domain of genomic RNA. The binding assay is based on quenching the fluorescence of tryptophan-37 in the protein by G residues in the single-stranded loops. Tightly bound RNA molecules quench nearly all the fluorescence of freshly purified NCp7 in 0.2 M NaCl. In contrast, when the GGAG-tetraloop of tight-binding SL3 is replaced with UUCG or GAUA, quenching is almost nil, indicating very low affinity. Interpreting fluorescence titrations in terms of a rapidly equilibrating 1:1 complex explains nearly all of the experimental variance for the loops. Analyzed in this way, the highest affinities are for 20mer SL3 and 19mer SL2 hairpin constructs (K(d) = 28 +/- 3 and 23 +/- 2 nM, respectively). The 20mer stem-UUCG-loop and GAUA-loop constructs have <0.5% of the affinity for NCp7 relative to SL3. Affinities relative to SL3 for the other stem-loops are the following: 10% for a 16mer construct to model SL4, 30% for a 27mer model of the 9-residue apical loop of SL1, and 20% for a 23mer model of a 1 x 3 asymmetric internal loop in SL1. A 154mer construct that includes all four stem-loops binds tightly to NCp7, with the equivalent of three NCp7 molecules bound with high affinity per RNA; it is also possible that two strong sites and several weaker ones combine to give the appearance of three strong sites.
Shubsda
Michael F
MF
Department of Chemistry, Syracuse University, Syracuse, New York 13244-4100, USA.
Paoletti
Andrew C
AC
Hudson
Bruce S
BS
Borer
Philip N
PN
D001483:Base Sequence; D001665:Binding Sites; D002213:Capsid / Q000737:chemistry / Q000378:metabolism*; D036022:Capsid Proteins; D015683:Gene Products, gag / Q000737:chemistry / Q000378:metabolism*; D015497:HIV-1 / Q000502:physiology*; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D012965:Sodium Chloride / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000378:metabolism; D014764:Viral Proteins; D019065:Virus Assembly; D054301:gag Gene Products, Human Immunodeficiency Virus
D036022:Capsid Proteins; D015683:Gene Products, gag; C071884:NCP7 protein, Human immunodeficiency virus 1; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D014764:Viral Proteins; D054301:gag Gene Products, Human Immunodeficiency Virus; D012965:Sodium Chloride; D014364:Tryptophan