Recognition of an Essential Adenine at a Protein−RNA Interface: Comparison of the Contributions of Hydrogen Bonds and a Stacking Interaction

The energetic contribution of stacking interactions to the stabilization of protein-nucleic acid complexes is not well established.1 Stacking interactions between proteins and nucleic acid helices are uncommon because the nucleic acid bases are already involved in this interaction in the helix.2 However, bases in single-stranded regions of nucleic acids are able to stack with protein side chains without unfavorable conformational changes. Many RNA-binding proteins bind to single-stranded target sequences, and a number of structurally characterized RNAprotein complexes have displayed stacking interactions between RNA bases in single-stranded regions and amino acid side chains.3 We sought to compare the energetic contributions of stacking interactions and hydrogen bonds to the stability of the U1A-RNA complex by studying chemically modified systems. We show that (1) stacking interactions can rival hydrogen bonds in proteinRNA complexes and (2) the recognition of a single base by a combination of hydrogen bonds and stacking interactions is required for the stability of a high-affinity RNA-protein complex. We have studied the RNA complex of a 101 amino acid peptide comprised of the N-terminal RNP domain of the protein U1A, called U1A101.4 The RNP domain is one of the best-characterized RNA-binding domains and binds single-stranded RNA in loops or linear sequences.5 In the RNP domain there are three highly conserved aromatic residues that are thought to be essential for RNA binding through stacking interactions. The N-terminal RNP domain of U1A contains two of these conserved aromatic amino acids, Phe56 and Tyr13, and both are observed to participate in stacking interactions with RNA bases in structures obtained by X-ray crystallography and NMR spectroscopy.3a,f Phe56 and Tyr13 participate in stacking interactions with A6 and C5 respectively of stem loop 2 of U1 snRNA (Figure 1). The hydroxyl group of Tyr13 hydrogen bonds to Gln54, while Phe56 does not participate in other interactions with the RNA or peptide.3a,f Therefore, we have investigated the contribution of the stacking interaction between Phe56 and A6 to U1A101-RNA stability. To this end, we have mutated Phe56 to Ala, Leu, Tyr, Trp, and His. To probe for potential structural changes in the mutants, we examined their secondary structure by CD spectroscopy. U1A101 and the mutant peptides displayed similar CD spectra, characteristic of peptides containing both R-helix and â-sheet structure.6 We measured the stability of the mutant peptides to guanidinium chloride denaturation by CD at 223 nm.7 The Trp and Tyr mutants are as stable to denaturation as U1A101 within the error of our experiments, ∆G ) -8.9 kcal/mol, while the remaining mutants are less stable: Leu, ∆G ) -7.7 kcal/mol; His, ∆G ) -7.2 kcal/ mol; Ala, ∆G )-6.7 kcal/mol. The error in the measurements is approximately 1 kcal/mol due to the long extrapolation back to zero guanidinium concentration. The order of conformational stability of the mutant peptides correlates with the â-sheet-forming propensities of the mutated amino acids,8 rather than the stabilities of the RNA-peptide complexes presented below. The conformational destabilization of the Ala and Leu mutants, 1-2 kcal/ mol, is significantly less than the destabilization of their complexes with RNA. These results suggest that the low affinity of the Leu and Ala mutants for RNA is not due to a global change or destabilization of their conformations. Peptide-RNA equilibrium dissociation constants were measured by gel mobility shift assays.9 Mutation of Phe56 to Ala results in a 5.5 kcal/mol loss in binding energy (Table 1). A portion of this energy, 1.4 kcal/mol, is regained by mutation to Leu, suggesting that aliphatic hydrophobic interactions can compensate, in part, for the loss of the aromatic ring. The destabilization of the Ala and Leu mutant-RNA complexes probably results from both the disruption of the stacking interaction and the disruption of other interactions that are cooperative with the stacking interaction. The Trp and His mutants bind with only 0.6 kcal/mol lower affinity to RNA than the wild type peptide, suggesting that the binding site is tolerant of significant (1) Helene, C.; Maurizot, J.-C. CRC Crit. ReV. Biochem. 1981, 10, 213258. (2) Werner, M. H.; Clore, G. M.; Fisher, C. L.; Fisher, R. J.; Trinh, L.; Shiloach, J.; Gronenborn, A. M. Cell 1995, 83, 761-771. (3) (a) Oubridge, C.; Ito, N.; Evans, P. R.; Teo, C. H.; Nagai, K. Nature 1994, 372, 432-438. (b) Valegard, K.; Murray, J. B.; Stockley, P. G.; Stonehouse, N. J.; Liljas, L. Nature 1994, 371, 623-626. (c) Cavarelli, J.; Rees, B.; Ruff, M.; Thierry, J.-C.; Moras, D. Nature 1993, 362, 181-184. (d) Commans, S.; Plateau, P.; Blanquet, S.; Dardel, F. J. Mol. Biol. 1995, 253, 100-113. (e) Price, S. R.; Evans, P. R.; Nagai, K. Nature 1998, 394, 645-650. (f) Allain, F. H.; Howe, P. W. A.; Neuhaus, D.; Varani, G. EMBO J. 1997, 16, 5764-5774. (g) Convery, M. A.; Rowsell, S.; Stonehouse, N. J.; Ellington, A. D.; Hirao, I.; Murray, J. B.; Peabody, D. S.; Phillips, S. E. V.; Stockley, P. G. Nat. Struct. Biol. 1998, 5, 133-139. (h) Handa, N.; Nureki, O.; Kuimoto, K.; Kim, I.; Sakamoto, H.; Shimura, Y.; Muto, Y.; Yokoyama, S. Nature 1999, 398, 579-585. (4) Nagai, K.; Oubridge, C.; Jessen, T. H.; Li, J.; Evans, P. R. Nature 1990, 348, 515-520. (5) Varani, G. Acc. Chem. Res. 1997, 30, 189-195. (6) Venyaminov, S. Y.; Yang, J. T. Determination of Protein Secondary Structure; Fasman, G. D., Ed.; Plenum Press: New York, 1996; pp 69-107. (7) Santoro, M. M.; Bolen, D. W. Biochemistry 1988, 27, 8063-8068. (8) Smith, C. K.; Regan, L. Acc. Chem. Res. 1997, 30, 153-161. (9) Carey, J. Methods Enzymol. 1991, 208, 103-117. Figure 1. (A) Diagram of the complex formed between the N-terminal RNP domain of U1A and stem loop 2 from the X-ray cocrystal structure.3a Only a portion of stem loop 2, in black, is shown. The stacking interactions between Tyr13 and C5 and between Phe56, A6, and C7 are displayed. (B) Stem loop 2 of U1 snRNA. The adenine that stacks with Phe56 is in bold.