The spliceosomal B complex-specific protein Prp38 forms a complex with the intrinsically unstructured proteins MFAP1 and Snu23. Our binding and crystal structure analyses show that MFAP1 and Snu23 contact Prp38 via ER/K motif-stabilized single α helices, which have previously been recognized only as rigid connectors or force springs between protein domains. A variant of the Prp38-binding single α helix of MFAP1, in which ER/K motifs not involved in Prp38 binding were mutated, was less α-helical in isolation and showed a reduced Prp38 affinity, with opposing tendencies in interaction enthalpy and entropy. Our results indicate that the strengths of single α helix-based interactions can be tuned by the degree of helix stabilization in the unbound state. MFAP1, Snu23, and several other spliceosomal proteins contain multiple regions that likely form single α helices via which they might tether several binding partners and act as intermittent scaffolds that facilitate remodeling steps during assembly of an active spliceosome.
Spliceosomal Prp38 proteins contain a conserved amino-terminal domain, but only higher eukaryotic orthologs also harbor a carboxy-terminal RS domain, a hallmark of splicing regulatory SR proteins. We show by crystal structure analysis that the amino-terminal domain of human Prp38 is organized around three pairs of antiparallel α-helices and lacks similarities to RNAbinding domains found in canonical SR proteins. Instead, yeast two-hybrid analyses suggest that the amino-terminal domain is a versatile protein-protein interaction hub that possibly binds 12 other spliceosomal proteins, most of which are recruited at the same stage as Prp38. By quantitative, alanine surface-scanning two-hybrid screens and biochemical analyses we delineated four distinct interfaces on the Prp38 amino-terminal domain. In vitro interaction assays using recombinant proteins showed that Prp38 can bind at least two proteins simultaneously via two different interfaces. Addition of excess Prp38 amino-terminal domain to in vitro splicing assays, but not of an interaction-deficient mutant, stalled splicing at a precatalytic stage. Our results show that human Prp38 is an unusual SR protein, whose amino-terminal domain is a multi-interface protein-protein interaction platform that might organize the relative positioning of other proteins during splicing.
The proteins Smu1 and RED have been jointly implicated in the regulation of alternative splicing, mitosis, and influenza virus infection, but how they interact and whether their diverse cellular functions are coupled is unknown. We identified an N-terminal region of Smu1 and a central region of RED that stably interact. Structural analyses revealed that the RED-binding region of Smu1 contains an N-terminal LisH motif linked to a core domain and a C-terminal α helix that folds back onto the LisH motif. Smu1 dimerizes via its LisH motif and C-terminal α helix and undergoes global conformational changes upon RED binding. In the ensuing hetero-tetrameric Smu1-RED complex, two molecules of RED use short α helices to bind hydrophobic grooves of two Smu1 core domains. Our results show how Smu1 and RED form a functional module that exhibits intriguing similarities to transcriptional co-repressor complexes, arranging multiple additional protein-protein interaction sites for contacting splicing and/or chromatin factors.
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