Crystal structure of the C-terminal domain of splicing factor Prp8 carrying retinitis pigmentosa mutants

Abstract

Prp8 is a critical pre-mRNA splicing factor. Prp8 is proposed to help form and stabilize the spliceosome catalytic core and to be an important regulator of spliceosome activation. Mutations in human Prp8 (hPrp8) cause a severe form of the genetic disorder retinitis pigmentosa, RP13. Understanding the molecular mechanism of Prp8's function in pre-mRNA splicing and RP13 has been hindered by its large size (over 2000 amino acids) and remarkably low-sequence similarity with other proteins. Here we present the crystal structure of the C-terminal domain (the last 273 residues) of Caenorhabditis elegans Prp8 (cPrp8). The core of the C-terminal domain is an alpha/beta structure that forms the MPN (Mpr1, Pad1 N-terminal) fold but without Zn(2+) coordination. We propose that the C-terminal domain is a protein interaction domain instead of a Zn(2+)-dependent metalloenzyme as proposed for some MPN proteins. Mapping of RP13 mutants on the Prp8 structure suggests that these residues constitute a binding surface between Prp8 and other partner(s), and the disruption of this interaction provides a plausible molecular mechanism for RP13.

Figure 1.

Structure of cC273. (A) Schematic view of the cC273 construct and its MPN core as well as the N- and C-terminal extensions in the context of the full-length C. elegans Prp8 (not drawn to proportion). (B) Overall structure of cC273 with secondary structures labeled. The structure is colored from blue to red in a rainbow spectrum from the N terminus to the C terminus. (C) Superimposition of the MPN domains of cC273 (blue) and Af2198 (yellow). The MPN domain is rotated 90° compared with B to best show the comparison. Labels 1–3 are examples of insertions that are much longer in cC273 than Af2198.

Figure 2.

Multiple sequence alignment among cC273, hC273, yC273, and Af2198. The three Prp8 sequences are aligned using MultAlin (Corpet 1988). Af2198 is aligned with cC273 based on structural superimposition. Secondary structures as they are observed in the crystal structures of cC273 and Af2198 are labeled on top of the cC273 sequence and the bottom of the Af2198 sequence, respectively. 3₁₀ helices are not differentiated from α helices and are both labeled as helices with prefix α for simplicity. Gray represents residues that are disordered in the crystal structure. Gray bars highlight residues in the JAMM motif. Boxed and underlined residues are missense and frameshift RP13 mutants, respectively.

Figure 3.

GST-yC273 can pull down in vitro-translated ySnu114 (residues 618–1008). GST alone and GST-yC273 were expressed in E. coli, purified with glutathione resin, incubated with in vitro-translated ySnu114 (³⁵S-labeled), washed, and separated by SDS-PAGE. The polyacrylamide gel was stained with Coomassie Blue to show equal loading of GST and GST-yC273 (bottom). The gel was dried, and bound ySnu114 was visualized by autoradiography (top).

Figure 4.

RP13 mutations. (A, left) Residues corresponding to the human RP13 mutations (ball and stick models) are located on the C-terminal extension of the cC273 structure (ribbon diagram). Dark blue, cyan, and purple backbones represent the N-terminal extension, MPN core, and C-terminal extension, respectively. (Middle) RP13 mutants on a surface representation (Nicholls et al. 1991) of the cC273 structure. Note that c-P2294 is partially exposed to the protein surface and c-Y2297 and c-H2302 are completely buried and invisible from the protein surface. (Right) Structural comparison of the cC273 R2303K mutant (silver backbone and silver residues) and wild-type cC273 (blue backbone and blue residues). (B) S200 gel filtration profiles and (C) CD spectra of four RP13 mutants indicate that these mutations do not affect the overall folding of the protein. All proteins were expressed and purified under identical conditions.