Multiple U2AF65 binding sites within SF3b155:: Thermodynamic and spectroscopic characterization of protein-protein interactions among pre-mRNA splicing factors

Essential, protein-protein complexes between the large subunit of the U2 small nuclear RNA auxiliary factor (U2AF(65)) with the splicing factor I (SF1) or the spliceosomal component SF3b155 are exchanged during a critical, ATP-dependent step of pre-mRNA splicing. Both SF1 and the N-terminal domain of SF3b155 interact with a U2AF(65) homology motif (UHM) of U2AF(65). SF3b155 contains seven tryptophan-containing sites with sequence similarity to the previously characterized U2AF(65)-binding domain of SF1. We show that the SF3b155 domain lacks detectable secondary structure using circular dichroism spectroscopy, and demonstrate that five of the tryptophan-containing SF3b155 sites are recognized by the U2AF(65)-UHM using intrinsic tryptophan fluorescence experiments with SF3b155 variants. When compared with SF1, similar spectral shifts and sequence requirements indicate that U2AF interactions with each of the SF3b155 sites are similar to the minimal SF1 site. However, thermodynamic comparison of SF1 or SF3b155 proteins with minimal peptides demonstrates that formation the SF1/U2AF(65) complex is likely to affect regions of SF1 beyond the previously identified, linear interaction site, in a remarkably distinct manner from the local U2AF(65) binding mode of SF3b155. Furthermore, the complex of the SF1/U2AF(65) interacting domains is stabilized by 3.3 kcal mol(-1) relative to the complex of the SF3b155/U2AF(65) interacting domains, consistent with the need for ATP hydrolysis to drive exchange of these partners during pre-mRNA splicing. We propose that the multiple U2AF(65) binding sites within SF3b155 regulate conformational rearrangements during spliceosome assembly. Comparison of the SF3b155 sites defines an (R/K)(n)XRW(DE) consensus sequence for predicting U2AF(65)-UHM ligands from genomic sequences, where parentheses denote residues that contribute to, but are not required for binding. (c) 2005 Elsevier Ltd. All rights reserved.