Nucleobase amino acids incorporated into the HIV-1 nucleocapsid protein increased the binding affinity and specificity for a hairpin RNA.
L-alpha-amino acids with a nucleobase in the side chain (nucleobase amino acids; NBAs) were used to enhance the function of RNA-binding proteins that recognize structured RNA. These NBAs were utilized in the three-dimensional structure of the protein to enhance RNA binding affinity and specificity as a result of selective recognition of NBAs by RNA bases. NBA units were incorporated at various positions into the HIV-1 nucleocapsid protein NCp7 (residues 1-55), which contains two CCHC-type (Cys-X(2)-Cys-X(4)-His-X(4)-Cys-type; X=an amino acid residue) zinc knuckle domains. The binding ability was evaluated by using the stem-loop (SL)3 region of HIV-1 Psi-RNA. Visible light absorption measurements revealed that two zinc ions bound strongly and quantitatively to the NBA-NCp7 molecule and to the wild-type NCp7 protein. This result indicates that the incorporation of NBA units composed of L-alpha-amino acids did not influence the formation of the specific structure of NCp7. Binding analysis with fluorescein-labeled SL3 RNA revealed that incorporation of NBA units into the NCp7 protein at appropriate positions increased its RNA binding affinity and specificity. An NBA-NCp7 protein that possessed cytosine and guanine NBA units at positions 13 and 46, respectively, showed a binding affinity for SL3 RNA ninefold higher than that of wild-type NCp7 as a result of the specific and cooperative interaction of the NBA units with RNA bases. These results clearly demonstrate that inclusion of NBA units in the three-dimensional structure of an RNA-binding protein is a useful strategy for enhancing the function of the protein.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry*; D001483:Base Sequence; D005452:Fluoresceins; D005454:Fluorescence Polarization; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009690:Nucleic Acid Conformation; D019590:Nucleocapsid Proteins / Q000138:chemical synthesis* / Q000378:metabolism*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D013053:Spectrophotometry / Q000379:methods; D013379:Substrate Specificity; D015032:Zinc / Q000737:chemistry / Q000378:metabolism
D000596:Amino Acids; D005452:Fluoresceins; D019590:Nucleocapsid Proteins; D012367:RNA, Viral; D015032:Zinc