Scaffolding in the Spliceosome via Single α Helices

Abstract

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.

Human MFAP1 is a cryptic ortholog of the Saccharomyces cerevisiae Spp381 splicing factor

“…To exclude false positive hits that merely arise from co-occurrence of abundant, highly conserved domains, InParanoid uses a strict cut-off criterion of sequence coverage ≥ 50% and BLAST score ≥ 50. Taking into account the presumably low sequence conservation of MFAP1 due to the predicted structural disorder and the absence of folded protein domains [34], we also performed reciprocal best BLAST hit (RBH) searches of MFAP1 proteins against the same 273 sets of protein-coding genes with relaxed cut-off criteria (BLAST score ≥ 30, E-value ≤ 0.01). The RBH method has a relatively high specificity compared to other ortholog detection methods and its specificity is only marginally affected by changes in cut-off values [39].…”

“…3a) and of the structurally highly similar Prp38-MFAP1 complex from the thermophilic fungus Chaetomium thermophilum ( ct ; PDB ID: 5F5T, Fig. 3b) together with binding studies of arginine-to-alanine mutants of the first and second arginine (R282, R286), which were sufficient to disrupt Prp38-MFAP1, revealed a RxxxRxxR motif as a key Prp38-binding element of MFAP1 [34]. Strikingly, the C-terminal halves of K. lactis and S. cerevisiae Spp381 contain an identical or slightly modified motif, RxxxRxxR and RxxxRxxK, respectively (Fig.…”

“…Later, the protein was found in spliceosome preparations [31], was shown to interact with Prp38 in pull-down experiments and to be required for pre-mRNA processing [32]. Interactions between MFAP1 and other B-specific proteins were identified by yeast two-hybrid (Y2H) [21, 33] and in vitro binding studies [21, 34]. Due to its elongated, solvent exposed nature and predicted dense array of short protein binding motifs, MFAP1 was suggested to act as a scaffold or ruler that engages multiple binding partners [34].…”

“…Interactions between MFAP1 and other B-specific proteins were identified by yeast two-hybrid (Y2H) [21, 33] and in vitro binding studies [21, 34]. Due to its elongated, solvent exposed nature and predicted dense array of short protein binding motifs, MFAP1 was suggested to act as a scaffold or ruler that engages multiple binding partners [34]. Recently, the molecular details of the MFAP1-Prp38 interaction have been revealed by X-ray crystallography [34], representing one of the few structurally characterized interactions between B-specific proteins besides Snu23-Prp38 and Smu1-RED [35].…”

Characterization of long and stable de novo single alpha-helix domains provides novel insight into their stability

“…However, only a few of these have been characterized experimentally and in detail4569101112. Most recently, it has been shown that in some cases, a region of the SAH can mediate binding to other proteins1314. Thus it is important to understand the underlying stability of these intriguing domains, in order to understand their contribution to protein function.…”