Solution structure of the yeast URN1 splicing factor FF domain: comparative analysis of charge distributions in FF domain structures-FFs and SURPs, two domains with a similar fold.

FF domains are present in three protein families: the splicing factors formin binding protein 11 (FBP11), Prp40, and URN1, the transcription factor CA150, and the p190RhoGTPase-related proteins. This simplicity in distribution, however, is contrasted by the difficulty in defining their biological role. At best, the group of ligand FF domains can bind to form a motley crew with binding reports pointing also to negative/aromatic sequences, the tetratricopeptide repeat, the transcription factor TFII-I and even to RNA. To expand our knowledge on the FF domain, we selected the FF domain present in the URN1 yeast splicing factor as the subject for structural studies. The URN1 protein is one of the two known proteins containing only one FF domain, making it the most simplified representative of FF domain-containing splicing factors. The solution structure reveals that the domain adopts the classical FF fold, with a distinctive negatively charged patch on its surface. All available FF structures have a well-conserved fold but variable electrostatic patches on their surfaces. These patches are unconserved, even for domains with similar pK(a)s. To investigate potential binding sites in FF domains, we performed structural comparisons to other proteins with similar folds. In addition to the structures detected by SCOP, we included SURP domains, which also adopt the alpha1-alpha2-3(10)-alpha3 architecture. We observed that the main difference between all these structures resides in the orientation of the second helix. Remarkably, in DEK, SURP, and Prp40FF1 structures (the exception is the FBP11FF1 domain), the second helix participates in ligand recognition. Furthermore, SURP and Prp40FF1 binding sites also include the 3(10) helix, which forms a partially exposed hydrophobic cavity. This cavity is also present in at least CA150FF1 and FF2 structures. Thus, as with WW domains, the FF fold seems to have developed binding-site variations to accommodate an abundant and variable set of ligands.

Structural characterization of a new binding motif and a novel binding mode in group 2 WW domains.

Formin homology 1 (FH1), is a long proline-rich region of formins, shown to bind to five WW containing proteins named formin binding proteins (FBPs). FH1 has several potential binding regions but only the PPLPx motif and its interaction with FBP11WW1 has been characterized structurally. To detect whether additional motifs exist in FH1, we synthesized five peptides and investigated their interaction with FBP28WW2, FBP11WW1 and FBP11WW2 domains. Peptides of sequence PTPPPLPP (positive control), PPPLIPPPP and PPLIPPPP (new motifs) interact with the domains with micromolar affinity. We observed that FBP28WW2 and FBP11WW2 behave differently from FBP11WW1 in terms of motif selection and affinity, since they prefer a doubly interrupted proline stretch of sequence PPLIPP. We determined the NMR structure of three complexes involving the FBP28WW2 domain and the three ligands. Depending on the peptide under study, the domain interacts with two proline residues accommodated in either the XP or the XP2 groove. This difference represents a one-turn displacement of the domain along the ligand sequence. To understand what drives this behavior, we performed further structural studies with the FBP11WW1 and a mutant of FBP28WW2 mimicking the XP2 groove of FBP11WW1. Our observations suggest that the nature of the XP2 groove and the balance of flexibility/rigidity around loop 1 of the domain contribute to the selection of the final ligand positioning in fully independent domains. Additionally, we analyzed the binding of a double WW domain region, FBP11WW1-2, to a long stretch of FH1 using fluorescence spectroscopy and NMR titrations. With this we show that the presence of two consecutive WW domains may also influence the selection of the binding mode, particularly if both domains can interact with consecutive motifs in the ligand. Our results represent the first observation of protein-ligand recognition where a pair of WW and two consecutive motifs in a ligand participate simultaneously.

NMR structural studies of the ItchWW3 domain reveal that phosphorylation at T30 inhibits the interaction with PPxY-containing ligands.

In this work, we study the role of phosphorylation as a regulatory mechanism for the interaction between the E3 ubiquitin ligase ItchWW3 domain and two PPxY motifs of one of its targets, the Epstein-Barr virus latent membrane protein 2A. Whereas ligand phosphorylation only diminishes binding, domain phosphorylation at residue T30 abrogates it. We show that two ItchWW domains can be phosphorylated at this position, using CK2 and PKA kinases and/or with stimulated T lymphocyte lysates. To better understand the regulation process, we determined the NMR structures of the ItchWW3-PPxY complex and of the phosphoT30-ItchWW3 variant. The peptide binds the domain using both XP and tyrosine grooves. A hydrogen bond from T30 to the ligand is also detected. This hydrogen-bond formation is precluded in the variant, explaining the inhibition upon phosphorylation. Our results suggest that phosphorylation at position 30 in ItchWW domains can be a mechanism to inhibit target recognition in vivo.

Structure of the dimeric exonuclease TREX1 in complex with DNA displays a proline-rich binding site for WW Domains.

TREX1 is the most abundant mammalian 3' --> 5' DNA exonuclease. It has been described to form part of the SET complex and is responsible for the Aicardi-Goutières syndrome in humans. Here we show that the exonuclease activity is correlated to the binding preferences toward certain DNA sequences. In particular, we have found three motifs that are selected, GAG, ACA, and CTGC. To elucidate how the discrimination occurs, we determined the crystal structures of two murine TREX1 complexes, with a nucleotide product of the exonuclease reaction, and with a single-stranded DNA substrate. Using confocal microscopy, we observed TREX1 both in nuclear and cytoplasmic subcellular compartments. Remarkably, the presence of TREX1 in the nucleus requires the loss of a C-terminal segment, which we named leucine-rich repeat 3. Furthermore, we detected the presence of a conserved proline-rich region on the surface of TREX1. This observation points to interactions with proline-binding domains. The potential interacting motif "PPPVPRPP" does not contain aromatic residues and thus resembles other sequences that select SH3 and/or Group 2 WW domains. By means of nuclear magnetic resonance titration experiments, we show that, indeed, a polyproline peptide derived from the murine TREX1 sequence interacted with the WW2 domain of the elongation transcription factor CA150. Co-immunoprecipitation studies confirmed this interaction with the full-length TREX1 protein, thereby suggesting that TREX1 participates in more functional complexes than previously thought.

Myotonia-related mutations in the distal C-terminus of ClC-1 and ClC-0 chloride channels affect the structure of a poly-proline helix.

Myotonia is a state of hyperexcitability of skeletal-muscle fibres. Mutations in the ClC-1 Cl- channel cause recessive and dominant forms of this disease. Mutations have been described throughout the protein-coding region, including three sequence variations (A885P, R894X and P932L) in a distal C-terminal stretch of residues [CTD (C-terminal domain) region] that are not conserved between CLC proteins. We show that surface expression of these mutants is reduced in Xenopus oocytes compared with wild-type ClC-1. Functional, biochemical and NMR spectroscopy studies revealed that the CTD region encompasses a segment conserved in most voltage-dependent CLC channels that folds with a secondary structure containing a short type II poly-proline helix. We found that the myotonia-causing mutation A885P disturbs this structure by extending the poly-proline helix. We hypothesize that this structural modification results in the observed alteration of the common gate that acts on both pores of the channel. We provide the first experimental investigation of structural changes resulting from myotonia-causing mutations.

The structure of Prp40 FF1 domain and its interaction with the crn-TPR1 motif of Clf1 gives a new insight into the binding mode of FF domains.

The yeast splicing factor Prp40 (pre-mRNA processing protein 40) consists of a pair of WW domains followed by several FF domains. The region comprising the FF domains has been shown to associate with the 5' end of U1 small nuclear RNA and to interact directly with two proteins, the Clf1 (Crooked neck-like factor 1) and the phosphorylated repeats of the C-terminal domain of RNA polymerase II (CTD-RNAPII). In this work we reported the solution structure of the first FF domain of Prp40 and the identification of a novel ligand-binding site in FF domains. By using chemical shift assays, we found a binding site for the N-terminal crooked neck tetratricopeptide repeat of Clf1 that is distinct and structurally separate from the previously identified CTD-RNAPII binding pocket of the FBP11 (formin-binding protein 11) FF1 domain. No interaction, however, was observed between the Prp40 FF1 domain and three different peptides derived from the CTD-RNAPII protein. Indeed, the equivalent CTD-RNAPII-binding site in the Prp40 FF1 domain is predominantly negatively charged and thus unfavorable for an interaction with phosphorylated peptide sequences. Sequence alignments and phylogenetic tree reconstructions using the FF domains of three functionally related proteins, Prp40, FBP11, and CA150, revealed that Prp40 and FBP11 are not orthologous proteins and supported the different ligand specificities shown by their respective FF1 domains. Our results also revealed that not all FF domains in Prp40 are functionally equivalent. We proposed that at least two different interaction surfaces exist in FF domains that have evolved to recognize distinct binding motifs.