PDBsum entry 1o6w

Nuclear protein

PDB id

1o6w

Contents

			Protein chain

					75 a.a. *

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Solution structure and ligand recognition of the ww domain pair of the yeast splicing factor prp40.

Authors

S.Wiesner, G.Stier, M.Sattler, M.J.Macias.

Ref.

J Mol Biol, 2002, 324, 807-822. [DOI no: 10.1016/S0022-2836(02)01145-2]

PubMed id

12460579

Abstract

The yeast splicing factor pre-mRNA processing protein 40 (Prp40) comprises two N-terminal WW domains, separated by a ten-residue linker, and six consecutive FF domains. In the spliceosome, the Prp40 WW domains participate in cross-intron bridging by interacting with proline-rich regions present in the branch-point binding protein (BBP) and the U5 small nuclear ribonucleoprotein component Prp8. Furthermore, binding of Prp40 to the phosphorylated C-terminal domain (CTD) of the largest subunit of RNA polymerase II is thought to link splicing to transcription. To gain insight into this complex interaction network we have determined the solution structure of the tandem Prp40 WW domains by NMR spectroscopy and performed chemical shift mapping experiments with different proline-rich peptides. The WW domains each adopt the characteristic triple-stranded beta-sheet structure and are connected by a stable alpha-helical linker. On the basis of a detailed analysis of residual dipolar couplings (RDC) and 15N relaxation data we show that the tandem Prp40 WW domains behave in solution as a single folded unit with unique alignment and diffusion tensor, respectively. Using [1H-15N]-RDCs, we were able to accurately define the relative orientation of the WW domains revealing that the binding pockets of each domain face opposite sides of the structure. Furthermore, we found that both Prp40 WW domains interact with PPxY motifs (where x is any residue) present in peptides derived from the splicing factors BBP and Prp8. Moreover, the Prp40 WW domains are shown to bind proline-rich peptides devoid of aromatic residues, which are also recognised by the Abl-SH3 domain and the WW domain of the mammalian Prp40 orthologue formin binding protein 11. In contrast, no interaction was observed between the Prp40 WW domains and the CTD repeats used in this work.



	Figure 5. Figure 5. Superposition of representative regions of the 1H, 15N correlation spectra for the interaction of the Prp40 tandem WW domains and the second Rsp5 WW domain with different proline-rich peptides. The free WW domains are shown in black (reference spectra without ligand). In (a)-(c), G* corresponds to a glycine residue resulting from the TEV protease cleavage site (see Materials and Methods). (a) Addition of PPxY/F motif containing peptides from BBP Image in green) and Prp8 Image in blue and Image in red) to the Prp40 tandem WW domains. (b) Addition of PPQQP motif containing peptides from mouse formin Image in blue) and the Abl-SH3 3BP-10 peptide Image in red) to the Prp40 tandem WW domains. (c) Addition of Prp8 peptide (PPPPSNFE in green), the unphosphorylated tandem CTD repeat (YSPTSPSYSPTSPS in blue) and the doubly phosphorylated CTD repeat (SYpSPTpSPS in red) to the Prp40 tandem WW domains. (d) Addition of the unphosphorylated tandem CTD repeat (YSPTSPSYSPTSPS in red) and the doubly phosphorylated CTD repeat (SYpSPTpSPS in cyan) to the second WW domain of Rsp5. All peptide/protein ratios refer to the WW domain pair for Prp40 and to the single domain for Rsp5.		Figure 7. Figure 7. Intermolecular NOEs observed in the Prp40 WW2-PSPPPVYDA complex. The Figure is based on a schematic representation of the interaction produced using the program LIGPLOT[57.] and a model of the Prp40 WW2-PSPPPVYDA complex. Residues exhibiting inter-molecular NOEs (broken lines) are shown in grey for the Prp40 WW2 and in green for the BBP peptide. For reasons of clarity, protons have been removed from the illustration, but proton-proton NOEs are implied. Where NOEs involved diastereotopic protons degenerate in their chemical shifts, only one of the possible interactions is shown.

PROCHECK

	Headers