Structural analysis of the intrinsically disordered splicing factor Spp2 and its binding to the DEAH-box ATPase Prp2

Abstract

The spliceosome consists of five small RNAs and more than 100 proteins. Almost 50% of the human spliceosomal proteins were predicted to be intrinsically disordered or to contain disordered regions, among them the G-patch protein Spp2. The G-patch region of Spp2 binds to the DEAH-box ATPase Prp2, and both proteins together are essential for promoting the transition from the Bact to the catalytically active B* spliceosome. Here we show by circular dichroism and nuclear magnetic resonance (NMR) spectroscopy that Spp2 is intrinsically disordered in solution. Crystal structures of a complex consisting of Prp2-ADP and the G-patch domain of Spp2 demonstrate that the G-patch gains a defined fold when bound to Prp2. While the N-terminal region of the G-patch always folds into an α-helix in five different crystal structures, the C-terminal part is able to adopt two alternative conformations. NMR studies further revealed that the N-terminal part of the Spp2 G-patch, which is the most conserved region in different G-patch proteins, transiently samples helical conformations, possibly facilitating a conformational selection binding mechanism. The structural analysis unveils the role of conserved residues of the G-patch in the dynamic interaction mode of Spp2 with Prp2, which is vital to maintain the binding during the Prp2 domain movements needed for RNA translocation.

Structural basis for DEAH-helicase activation by G-patch proteins

“…Third, XL-MS of the scB act complex was consistent with both scSpp2 brace-helix and brace-loop binding on WH and RecA2 (57). Consistently, in a parallel study, the Ficner group found an almost identical binding mode for ctSpp2 to ctPrp2 (59). Based on sequence comparison alone (SI Appendix, Fig.…”

ATP regulates RNA‐driven cold inducible RNA binding protein phase separation

“…Intrinsically disordered proteins (IDPs) and proteins harboring intrinsically disordered regions (IDRs) constitute 33% of the eukaryotic and 44% of the human proteome 1–3 . Their functional implications include essential biological processes like cell signaling, 4,5 gene transcription, 6 RNA splicing, 7,8 and cell cycle regulation 9,10 . Over the last decades, a number of studies have demonstrated the essential role of IDPs/IDRs in the formation of biomolecular condensates also referred as membraneless organelles 4,11,12 .…”

The G-patch activators Pfa1 and PINX1 exhibit different modes of interaction with the Prp43 RNA helicase

“…Our finding could not be explained by the presence of two functionally independent Prp43 binding sites within PINX1 as is the case for Pfa1 since the integrity of the G-patch domain is essential for full length PINX1 binding to Prp43 [29]. While this manuscript was in preparation, the crystal structures of helicase DHX15 bound to the G-patch of NKRF and of splicing helicase Prp2 bound to the G-patch of Spp2 were reported [39,51]. In both structures, the G-patch binds its partner helicase in a similar way, forming a flexible molecular brace across the helicase surface.…”

“…Unexpectedly, however, our results also show that the G-patch containing domains of Pfa1 and PINX1 have different requirements for binding to Prp43. We propose that PINX1 interacts with Prp43 as NKRF or Spp2 do with their partner helicase [39,51], while Pfa1 G-patch mode of binding to Prp43 appears significantly different.…”

“…In the cryo-EM structure of the yeast intron lariat spliceosome, the Ntr1 protein is present but the structure of its G-patch domain that mediates the interaction with Prp43 could not be resolved [48]. However, the crystal structures of helicase DHX15 bound to NKRF Gpatch and of helicase Prp2 bound to Spp2 G-patch have just been published, providing for the first time detailed molecular insights into DEAH protein/G-patch interactions [39,51] (see Fig. S1 for the structure of DHX15 bound to NKRF G-patch).…”