Scaffolding in the Spliceosome via Single α Helices Alexander K.C. Ulrich, Martin Seeger, Tonio Schütze, Natascha Bartlick, Markus C. Wahl  Structure  Volume 24, Issue 11, Pages 1972-1983 (November 2016) DOI: 10.1016/j.str.2016.09.007 Copyright © 2016 Elsevier Ltd Terms and Conditions

Structure 2016 24, 1972-1983DOI: (10.1016/j.str.2016.09.007) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 1 Characterization of Structural Contents and Interactions (A) Secondary structure predictions of Prp38, MFAP1, and Snu23 proteins. NTD, N-terminal domain; AL, acidic linker; RS, arginine-serine repeat region; ZnF, zinc finger. Red/gray bars, predicted α helices/β strands; blue/yellow bars, predicted solvent-exposed/buried regions; green bars, regions of predicted structural disorder. (B) Reference CD spectra of poly-L-lysine at 22°C in pure α helix, α sheet, or random coil conformations (Greenfield and Fasman, 1969) and a reference CD melting curve of the globular selenocysteine synthase from mouse at 222 nm. (C) CD spectra at 4°C or 7°C (solid lines) and 90°C (dashed lines) and CD melting curves at 222 nm of hsMFAP130-344 and hsSnu23. (D) SDS-PAGE gels of gel filtration runs of identified interaction regions of Prp38, MFAP1, and Snu23 proteins. Structure 2016 24, 1972-1983DOI: (10.1016/j.str.2016.09.007) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 2 Prp38 Interacts with Long α Helices of MFAP1 and Snu23 (A) Crystal structure of a hsPrp38NTD (red)-hsMFAP1267-344 (blue) complex. Charged side chains of MFAP1 are indicated as sticks. Inset: zoom of boxed region. Charged side chains stabilize SAHs by engaging in fluctuating salt bridges (black arrows), hydrophobic contacts (yellow ovals), and by preventing water from approaching the backbone of the α helix (red symbol). (B) Crystal structure of a ctPrp38NTD+ (red)-ctMFAP1217-296 (blue) complex. Charged side chains of MFAP1 are indicated as sticks. The AL region forms the loop following α10 and the terminal α11 helix that engages in additional electrostatic and hydrophobic contacts with MFAP1. CtMFAP1217-296 forms two elongated α helices (α1 and α2) separated by a kink. (C) Crystal structure of a ctSnu23131-164 (yellow)-ctPrp38NTD+ (red)-ctMFAP1217-258 (blue) complex. Charged side chains of Snu23 and MFAP1 are indicated as sticks. For electron densities of all structures see Figure S1. For details on intramolecular salt bridges observed in the Prp38-binding helices of MFAP1 and Snu23 see Figure S2. (D–F) Monitoring the interaction of hsMFAP1267-344 (titrant) and hsPrp38NTD (analyte) (D), hsPrp38NTD+ (titrant) and hsMFAP267-344 (analyte) (E), and ctPrp38NTD+ (titrant) and ctSnu23131-164 (analyte) (F) by ITC at 10°C. Structure 2016 24, 1972-1983DOI: (10.1016/j.str.2016.09.007) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 3 Analysis of the hsPrp38-hsMFAP1 and ctSnu23-ctPrp38 Interactions (A and C) Molecular details of the hsPrp38 (blue)-hsMFAP1 (red) (A) and ctSnu23 (yellow)-ctPrp38 (blue) (C) interaction. Key residues are shown as sticks; residues mutated in (B) or (D) are highlighted in cyan. Salt bridges are indicated by dashed black lines. (B and D) Chromatograms and SDS-PAGE gels of analytical gel filtration experiments with the indicated hsPrp38NTD and hsMFAP1267-344 (B), or ctSnu23131-164 and ctPrp38NTD+ (D) variants. Vertical red lines indicate the elution volume of the respective WT complexes. Structure 2016 24, 1972-1983DOI: (10.1016/j.str.2016.09.007) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 4 SAHs in the Prp38-Binding Regions of MFAP1 and Snu23 (A and B) CD spectra at 5°C or 6°C (solid lines) and 90°C (dashed lines) and CD melting curves recorded at 222 nm of hsMFAP1267-344 (A) and ctSnu23131-164 (B) in the absence of binding partners. Significant fractions of isolated hsMFAP1267-344 (57.9% at 5°C and 20.3% at 90°C) and ctSnu23131-164 (20.3% at 6°C and 17.2% at 90°C) are α-helical at 5°C or 6°C. Structure 2016 24, 1972-1983DOI: (10.1016/j.str.2016.09.007) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 5 ER/K Motifs Stabilize SAHs and Modulate Binding Strength (A) CD spectra of the WT hsMFAP1267-344 and of the hsMFAP1267-344,ΔER/K variants collected between 5°C and 90°C in steps of 5°C. (B) α Helix content as a function of temperature of the WT hsMFAP1267-344 variant (closed circles) and the hsMFAP1267-344,ΔER/K variant (open circles). (C) Analysis of the interaction between hsPrp38NTD (analyte) and WT hsMFAP1267-344 (titrant; left panel) or hsMFAP1267-344,ΔER/K (titrant; right panel) by ITC at 10°C. Results shown are from one representative run. Thermodynamic parameters are means ± SD of four independent experiments. Structure 2016 24, 1972-1983DOI: (10.1016/j.str.2016.09.007) Copyright © 2016 Elsevier Ltd Terms and Conditions

Figure 6 ER/K Motif-Stabilized Single α Helices in the Spliceosome (A and B) Surface representation of the post-catalytic spliceosome (PDB: 3JB9) (A) and of the tri-snRNP (PDB: 5GAN) (B) with proteins in gray and RNAs in black. Proteins that exhibit elongated α helices not involved in an intramolecular tertiary structure are shown in cartoon representation for spPrp45 (orange), spCdc5 (cyan), scPrp3 (salmon), and scSnu66 (lime green). α Helices highlighted in boxes contain ER/K motifs and are putative SAHs. (C) Ribbon plots of putative SAHs with high ER/K content, for which side chains have been assigned in the structures. Glutamate, arginine, and lysine side chains are shown as sticks; dashed black lines represent intramolecular salt bridges. Sequences of the putative SAHs are given below the plots; blue residues, arginine/lysine side chains; red residues, glutamate side chains. Solid black/red lines above the sequences, ER/K motifs in the sequences not forming/forming salt bridges in the structures; dashed red lines, additional Asp-Arg salt bridges present in the structures. See also Figure S3 and Table S1. Structure 2016 24, 1972-1983DOI: (10.1016/j.str.2016.09.007) Copyright © 2016 Elsevier Ltd Terms and Conditions