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Edited by Michael Sattler, Helmholtz Zentrum München, Neuherberg, Germany, and accepted by Editorial Board Member Axel T. Brunger December 30, 2019 (received for review May 9, 2019)
Article Figures & SI
Figures
- Fig. 1.
Disorder and secondary structure analysis of yeast Spp2 in solution. (A) Analysis of the scSpp2 amino acid sequence by the Genesilico MetaDisorder prediction server. The probability to form disordered regions (y-axis) is shown with respect to the residue number (x-axis). (B) CD spectra of scSpp210–185 (blue line) and scSpp2100–150 (red line). The CD in mdeg (y-axis) is shown with respect to the wavelength (x-axis). (C) Two-dimensional 1H-15N HSQC spectrum of scSpp210–185 at 288 K and pH 6.5.
- Fig. 2.
Structural overview of the ctSpp2 G-patch bound to ctPrp2. (A) The model of the ctPrp2-ctSpp2211–254 complex depicted as a cartoon model, with ctPrp2 displayed semitransparently. N-terminal residues (270 to 296) of ctPrp2 are shown in black, the RecA1 domain (297 to 476) is shown in orange, the RecA2 domain (477 to 653) is shown in blue, the WH domain (654 to 721) is shown in gray, the HB domain (722 to 840) is shown in wheat, and the OB domain (841 to 921) is shown in green. Two alternative conformations of the ctSpp2 G-patch were found in complex structures obtained from five crystal forms (CFs). ctSpp2211–254 molecules exhibiting conformation 1 are depicted in different shades of red, whereas molecules belonging to conformation 2 are displayed in different shades of yellow. (Right) A zoomed-in view of the alternative conformations at the C-terminal end of the G-patch with one representative for each conformation. (B) Overview of hydrophobic interactions between ctSpp2211–254 and ctPrp2. Hydrophobic residues of ctSpp2 are shown in orange, while hydrophobic residues of ctPrp2 within 8 Å of the conserved ctSpp2211–254 hydrophobic residues are displayed in blue. Glycine residues of ctSpp2211–254 are highlighted as green spheres. (C) Sequence alignment of the G-patch domains of Spp2 from C. thermophilum, S. cerevisiae, and H. sapiens together with Ntr1, Gno1, and Pfa1 from S. cerevisiae. Conserved hydrophobic residues are highlighted in yellow, and glycine residues are shown in red. Secondary structure elements present in any of the five crystal forms are displayed on top of the corresponding segment of the sequence. The N-terminal amphipathic helix, as well as a hydrophobic stretch at the C-terminal end, are highly conserved. (D) Residues identified to cross-link to the lysines K236 and K250 of ctSpp2211–254 are shown as sticks and the cross-linked residues on ctPrp2 are shown in green.
- Fig. 3.
Transient helical conformations in the G-patch of ctSpp2 before complex formation. (A) Far-UV CD spectrum (mean residue ellipticity vs. wavelength, in nm) of ctSpp2208–254. (Inset) DLS measurements demonstrating that ctSpp2208–254 is predominantly monomeric in solution. (B) Two-dimensional 1H-15N-HSQC spectrum of ctSpp2208–254. The sequence-specific assignment of the backbone resonances is indicated. (C) Residue-specific ΔCα-ΔCβ secondary chemical shifts of ctSpp2208–254 together with S2 parameters derived by TALOS+. Positive values of ΔCα-ΔCβ indicate a propensity for α-helical conformations, while negative values indicate a propensity to form extended structures. The positions of α-helices observed in ctSpp2208–254 when in complex with Prp2 are shown on top. (D) Heteronuclear steady-state {1H,15N} NOE as a function of residue number. Error bars represent the SDs and were calculated as described in Materials and Methods. (E) Ensemble of α-helical conformations populated by residues D213 to F222 of ctSpp2208–254.
- Fig. 4.
Spp2 in the spliceosome and its conformational adaptability to different DEAH-box ATPase conformations. Using a combination of the ctPrp2-ctSpp2211–254 complex structure together with the cryo-EM and cross-linking data from Rauhut et al. (27), how Spp2 interacts with the spliceosome can be estimated. All spliceosomal factors are depicted as cartoon models, and cross-links of Spp2 are highlighted as pink spheres. The estimated path of Spp2 is displayed as a pink dashed line, and main contact sites are numbered starting from the most N-terminal cross-linked residue. (A) N-terminally from the G-patch, Spp2 cross-links with Brr2 and Rse1, as well as with Prp2. (B) The C-terminal end of the Spp2 G-patch cross-links with the OB-fold domain of Prp2 and parts of Bud13 and Pml1. Cross-linked regions of Bud13 and Pml1 are not part of the cryo-EM model and are symbolically depicted as dashed lines. C-terminally from the G-patch, Spp2 contacts another part of the RecA2 domain. (C) Overview of Spp2 cross-links numbered as in A and B. (D) Schematic representation of the catalytic states of Prp2 during one translocation cycle. The RecA2 domain is the most mobile domain during this process, and due to the versatile conformations of the conserved C-terminal stretch together with the flexible linker region, the Spp2 G-patch is able to adapt to the individual conformations of the RecA2 domain.
Data supplements
Supporting Information
Structural analysis of the intrinsically disordered splicing factor Spp2 and its binding to the DEAH-box ATPase Prp2
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