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

摘要

Essential, protein-protein complexes between the large subunit of the U2 small nuclear RNA Auxiliary Factor (U2AF⁶⁵) with the Splicing Factor 1 (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 homology motif (UHM) of U2AF⁶⁵. SF3b155 contains seven tryptophan-containing sites with sequence similarity to the previously characterized U2AF⁶⁵-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 U2AF⁶⁵-UHM using intrinsic tryptophan fluorescence experiments with SF3b155 variants. When compared with SF1, similar spectral shifts and sequence requirements indicate that U2AF⁶⁵ 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/U2AF⁶⁵ complex is likely to affect regions of SF1 beyond the previously identified, linear interaction site, in a remarkably distinct manner from the local U2AF⁶⁵ binding mode of SF3b155. Furthermore, the complex of the SF1/U2AF⁶⁵ interacting domains is stabilized by 3.3 kcal mol⁻¹ relative to the complex of the SF3b155/U2AF⁶⁵ interacting domains, consistent with the need for ATP hydrolysis to drive exchange of these partners during pre-mRNA splicing. We propose that the multiple U2AF⁶⁵ binding sites within SF3b155 regulate conformational rearrangements during spliceosome assembly. Comparison of the SF3b155 sites defines an (R/K)nXRW(DE) consensus sequence for predicting U2AF⁶⁵-UHM ligands from genomic sequences, where parentheses denote residues that contribute to, but are not required for binding.