Due to its small size and compact fold, the WW domain became an attractive model for studies of protein stability and design [7^11]. Speci¢c residues have been identi¢ed that play a critical role in the structure and function of the domain and also in modulating its stability. In fact, the WW domain is the ¢rst protein module that has been successfully designed de novo, demonstrating the signi¢cant insight we already have regarding its fold [12]. Besides, the WW domain sequence is well conserved in length, even in its loops, which is a remarkable feature of this domain, compared with others, making protein modeling a useful tool for generating three-dimensional representations of their sequences. Nevertheless, attempts to predict binding targets for a speci¢c WW domain sequence or even for one of its subgroups or classes, with a good probability, have not been made so far.Based on the pattern of semi-conserved residues, WW domain sequences have been classi¢ed into three groups as described previously [12]. Group I contains the C-terminal tryptophan and the N-terminal proline, Group II sequences lack the N-terminal proline and ¢nally Group III with sequences without the second tryptophan. In another classi¢cation, based on the ligand predilection, WW domains were divided into two major and two minor groups [5]. One major group (Group I) binds polypeptides with the minimal core consensus PPxY, whereas the other binds ligands with the PPLP motif usually embedded in a long stretch of prolines (Group II). Group III WW domains select poly-P motifs £anked by R or K, whereas Group IV WW domains bind to short sequences with phospho-S or phospho-T followed by P, in a phosphorylation-dependent manner [5]. A sequence alignment of some selected WW sequences combining binding preferences and sequence conservation is shown in Fig. 1.In this contribution we will review the structural characteristics of WW domain^ligand complexes determined so far. On the basis of four WW domain structures in complex with di¡erent peptides and two structures of free WW domains [3,12^16], a three-dimensional structure has been modeled for the Npw38 WW domain that allows us to compare binding properties of WW and SH3 domains. WW domain as a phosphate-dependent SH3 domain?WW domains have the ability to bind proline-rich cores and/or phospho-SP/phospho-TP-containing motifs [5]. It is interesting that such a small and well-conserved module has a surprisingly large repertoire of potential ligands. The dissociation constants (K d ) for WW^ligand complexes lie in the high nM to low mM range for proline-rich ligands, and in the low mM range for phospho-SP-or phospho-TP-containing ligands [5].

Ubiquitination of proteins is an abundant modification that controls numerous cellular processes. Many Ubiquitin (Ub) protein ligases (E3s) target both their substrates and themselves for degradation. However, the mechanisms regulating their catalytic activity are largely unknown. The C2-WW-HECT-domain E3 Smurf2 downregulates transforming growth factor-beta (TGF-beta) signaling by targeting itself, the adaptor protein Smad7, and TGF-beta receptor kinases for degradation. Here, we demonstrate that an intramolecular interaction between the C2 and HECT domains inhibits Smurf2 activity, stabilizes Smurf2 levels in cells, and similarly inhibits certain other C2-WW-HECT-domain E3s. Using NMR analysis the C2 domain was shown to bind in the vicinity of the catalytic cysteine, where it interferes with Ub thioester formation. The HECT-binding domain of Smad7, which activates Smurf2, antagonizes this inhibitory interaction. Thus, interactions between C2 and HECT domains autoinhibit a subset of HECT-type E3s to protect them and their substrates from futile degradation in cells.

A simple labeling approach is presented based on protein expression in [1-(13)C]- or [2-(13)C]-glucose containing media that produces molecules enriched at methyl carbon positions or backbone C(alpha) sites, respectively. All of the methyl groups, with the exception of Thr and Ile(delta1) are produced with isolated (13)C spins (i.e., no (13)C-(13)C one bond couplings), facilitating studies of dynamics through the use of spin-spin relaxation experiments without artifacts introduced by evolution due to large homonuclear scalar couplings. Carbon-alpha sites are labeled without concomitant labeling at C(beta) positions for 17 of the common 20 amino acids and there are no cases for which (13)C(alpha)-(13)CO spin pairs are observed. A large number of probes are thus available for the study of protein dynamics with the results obtained complimenting those from more traditional backbone (15)N studies. The utility of the labeling is established by recording (13)C R (1rho) and CPMG-based experiments on a number of different protein systems.

Eph receptor tyrosine kinases (RTKs) mediate numerous developmental processes. Their activity is regulated by auto-phosphorylation on two tyrosines within the juxtamembrane segment (JMS) immediately N-terminal to the kinase domain (KD). Here, we probe the molecular details of Eph kinase activation through mutational analysis, X-ray crystallography and NMR spectroscopy on autoinhibited and active EphB2 and EphA4 fragments. We show that a Tyr750Ala gain-of-function mutation in the KD and JMS phosphorylation independently induce disorder of the JMS and its dissociation from the KD. Our X-ray analyses demonstrate that this occurs without major conformational changes to the KD and with only partial ordering of the KD activation segment. However, conformational exchange for helix aC in the N-terminal KD lobe and for the activation segment, coupled with increased inter-lobe dynamics, is observed upon kinase activation in our NMR analyses. Overall, our results suggest that a change in inter-lobe dynamics and the sampling of catalytically competent conformations for helix aC and the activation segment rather than a transition to a static active conformation underlies Eph RTK activation.

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FAT10 is a ubiquitin-like modifier that directly targets proteins for proteasomal degradation. Here, we report the high-resolution structures of the two individual ubiquitin-like domains (UBD) of FAT10 that are joined by a flexible linker. While the UBDs of FAT10 show the typical ubiquitin-fold, their surfaces are entirely different from each other and from ubiquitin explaining their unique binding specificities. Deletion of the linker abrogates FAT10-conjugation while its mutation blocks auto-FAT10ylation of the FAT10-conjugating enzyme USE1 but not bulk conjugate formation. FAT10- but not ubiquitin-mediated degradation is independent of the segregase VCP/p97 in the presence but not the absence of FAT10’s unstructured N-terminal heptapeptide. Stabilization of the FAT10 UBDs strongly decelerates degradation suggesting that the intrinsic instability of FAT10 together with its disordered N-terminus enables the rapid, joint degradation of FAT10 and its substrates without the need for FAT10 de-conjugation and partial substrate unfolding.

Nedd4-family ubiquitin ligases are key regulators of cell surface receptor signaling. Their dysregulation is associated with several human diseases, including cancer. Under normal conditions, the activity of various Nedd4 E3s is controlled through an autoinhibitory interaction of the N-terminal C2 domain with the C-terminal catalytic HECT domain. Here, we report the structural and functional framework for this intramolecular interaction. Our nuclear magnetic resonance (NMR) data and biochemical analyses on Smurf2 and Nedd4 show that the C2 domain has the potential to regulate E3 activity by maintaining the HECT domain in a low-activity state where its ability for transthiolation and noncovalent Ub binding are impaired.