Substitution of an essential adenine in the U1A-RNA complex with a non-polar isostere.
The RNA recognition motif (RRM) binds to single-stranded RNA target sites of diverse sequences and structures. A conserved mode of base recognition by the RRM involves the simultaneous formation of a network of hydrogen bonds with the base functional groups and a stacking interaction between the base and a highly conserved aromatic amino acid. We have investigated the energetic contribution of the functional groups involved in the recognition of an essential adenine, A6, in stem-loop 2 of U1 snRNA by the N-terminal RRM of the U1A protein. Previously, we found that elimination of individual hydrogen bond donors and acceptors on A6 destabilized the complex by 0.8-1.9 kcal/mol, while mutation of the aromatic amino acid (Phe56) that stacks with A6 to Ala destabilized the complex by 5.5 kcal/mol. Here we continue to probe the contribution of A6 to complex stability through mutation of both the RNA and protein. We have removed two hydrogen-bonding functional groups by introducing a U1A mutation, Ser91Ala, and replacing A6 with tubercidin, purine, or 1-deazaadenine. We find that the complex is destabilized an additional 1.2-2.6 kcal/mol by the elimination of the second hydrogen bond donor or acceptor. Surprisingly, deletion of all of the functional groups involved in hydrogen bonds with the U1A protein by substituting adenine with 4-methylindole reduced the binding free energy by only 2.0 kcal/mol. Experiments with U1A proteins containing mutations of Phe56 suggested that improved stacking interactions due to the greater hydrophobicity of 4-methylindole than adenine may be partly responsible for the small destabilization of the complex upon substitution of 4-methylindole for A6. The data imply that hydrophobic interactions can compensate energetically for the disruption of the complex hydrogen-bonding network between nucleotide and protein.
Tuite
Jacob B
JB
Department of Chemistry, Wesleyan University, Middletown, CT 06459, USA.
Shiels
Jerome C
JC
Baranger
Anne M
AM
D000225:Adenine / Q000737:chemistry*; D020816:Amino Acid Motifs; D001483:Base Sequence; D001665:Binding Sites; D006860:Hydrogen Bonding; D007211:Indoles / Q000737:chemistry; D046911:Macromolecular Substances; D008958:Models, Molecular; D009154:Mutation; D011485:Protein Binding; D012342:RNA, Small Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D007211:Indoles; D046911:Macromolecular Substances; D012342:RNA, Small Nuclear; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C405483:U1 small nuclear RNA; C075339:U1A protein; D000225:Adenine