The WW1 Domain Enhances Autoinhibition in Smurf Ubiquitin Ligases.

Downregulation of ubiquitin (Ub) ligase activity prevents premature ubiquitination and is critical for cellular homeostasis. Nedd4 Ub ligases share a common domain architecture and yet are regulated in distinct ways through interactions of the catalytic HECT domain with the N-terminal C2 domain or the central WW domain region. Smurf1 and Smurf2 are two highly related Nedd4 ligases with ~70% overall sequence identity. Here, we show that the Smurf1 C2 domain interacts with the HECT domain and inhibits ligase activity in trans. However, in contrast to Smurf2, we find that full-length Smurf1 is a highly active Ub ligase, and we can attribute this striking difference in regulation to the lack of one WW domain (WW1) in Smurf1. Using NMR spectroscopy and biochemical assays, we identified the WW1 region as an additional inhibitory element in Smurf2 that cooperates with the C2 domain to enhance HECT domain binding and Smurf2 inhibition. Our work provides important insights into Smurf regulation and highlights that the activities of highly related proteins can be controlled in distinct ways.

Author Correction: The structure of the ubiquitin-like modifier FAT10 reveals an alternative targeting mechanism for proteasomal degradation.

The original version of the Supplementary Information associated with this Article inadvertently omitted Supplementary Table 3. The HTML version of the Article has been updated to include a corrected version of the Supplementary Information.

The structure of the ubiquitin-like modifier FAT10 reveals an alternative targeting mechanism for proteasomal degradation.

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.

ß-Sheet Augmentation Is a Conserved Mechanism of Priming HECT E3 Ligases for Ubiquitin Ligation.

Ubiquitin (Ub) ligases (E3s) catalyze the attachment of Ub chains to target proteins and thereby regulate a wide array of signal transduction pathways in eukaryotes. In HECT-type E3s, Ub first forms a thioester intermediate with a strictly conserved Cys in the C-lobe of the HECT domain and is then ligated via an isopeptide bond to a Lys residue in the substrate or a preceding Ub in a poly-Ub chain. To date, many key aspects of HECT-mediated Ub transfer have remained elusive. Here, we provide structural and functional insights into the catalytic mechanism of the HECT-type ligase Huwe1 and compare it to the unrelated, K63-specific Smurf2 E3, a member of the Nedd4 family. We found that the Huwe1 HECT domain, in contrast to Nedd4-family E3s, prioritizes K6- and K48-poly-Ub chains and does not interact with Ub in a non-covalent manner. Despite these mechanistic differences, we demonstrate that the architecture of the C-lobe~Ub intermediate is conserved between Huwe1 and Smurf2 and involves a reorientation of the very C-terminal residues. Moreover, in Nedd4 E3s and Huwe1, the individual sequence composition of the Huwe1 C-terminal tail modulates ubiquitination activity, without affecting thioester formation. In sum, our data suggest that catalysis of HECT ligases hold common features, such as the ß-sheet augmentation that primes the enzymes for ligation, and variable elements, such as the sequence of the HECT C-terminal tail, that fine-tune ubiquitination activity and may aid in determining Ub chain specificity by positioning the substrate or acceptor Ub.

Structural basis for the interaction between the cell polarity proteins Par3 and Par6.

Polarity is a fundamental property of most cell types. The Par protein complex is a major driving force in generating asymmetrically localized protein networks and consists of atypical protein kinase C (aPKC), Par3, and Par6. Dysfunction of this complex causes developmental abnormalities and diseases such as cancer. We identified a PDZ domain-binding motif in Par6 that was essential for its interaction with Par3 in vitro and for Par3-mediated membrane localization of Par6 in cultured cells. In fly embryos, we observed that the PDZ domain-binding motif was functionally redundant with the PDZ domain in targeting Par6 to the cortex of epithelial cells. Our structural analyses by x-ray crystallography and NMR spectroscopy showed that both the PDZ1 and PDZ3 domains but not the PDZ2 domain in Par3 engaged in a canonical interaction with the PDZ domain-binding motif in Par6. Par3 thus has the potential to recruit two Par6 proteins simultaneously, which may facilitate the assembly of polarity protein networks through multivalent PDZ domain interactions.

The adherens junction-associated LIM domain protein Smallish regulates epithelial morphogenesis.

In epithelia, cells adhere to each other in a dynamic fashion, allowing the cells to change their shape and move along each other during morphogenesis. The regulation of adhesion occurs at the belt-shaped adherens junction, the zonula adherens (ZA). Formation of the ZA depends on components of the Par-atypical PKC (Par-aPKC) complex of polarity regulators. We have identified the Lin11, Isl-1, Mec-3 (LIM) protein Smallish (Smash), the orthologue of vertebrate LMO7, as a binding partner of Bazooka/Par-3 (Baz), a core component of the Par-aPKC complex. Smash also binds to Canoe/Afadin and the tyrosine kinase Src42A and localizes to the ZA in a planar polarized fashion. Animals lacking Smash show loss of planar cell polarity (PCP) in the embryonic epidermis and reduced cell bond tension, leading to severe defects during embryonic morphogenesis of epithelial tissues and organs. Overexpression of Smash causes apical constriction of epithelial cells. We propose that Smash is a key regulator of morphogenesis coordinating PCP and actomyosin contractility at the ZA.

Methyl groups as NMR probes for biomolecular interactions.

Intermolecular interactions are indispensible for biological function. Here we discuss how novel NMR techniques can provide unique insights into the assembly, dynamics and regulation of biomolecular complexes. We focus on applications that exploit the methyl TROSY effect and show that methodological advances and biological insights go hand in hand. We envision that future methyl TROSY applications will continue to provide unique information regarding intermolecular interactions, even for very large eukaryotic protein complexes that are often highly asymmetric.

Structural and functional framework for the autoinhibition of Nedd4-family ubiquitin ligases.

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.

Methionine scanning as an NMR tool for detecting and analyzing biomolecular interaction surfaces.

Methyl NMR spectroscopy is a powerful tool for studying protein structure, dynamics, and interactions. Yet difficulties with resonance assignment and the low abundance of methyl groups can preclude detailed NMR studies, particularly the determination of continuous interaction surfaces. Here we present a straightforward strategy that overcomes these problems. We systematically substituted solvent-exposed residues with reporter methionines in the expected binding site and performed chemical shift perturbation (CSP) experiments using methyl-TROSY spectra. We demonstrate the utility of this approach for the interaction between the HECT domain of the Rsp5p ubiquitin ligase and its cognate E2, Ubc4. Using these mutants, we could instantaneously assign all newly arising reporter methyl signals, determine the Ubc4 interaction surface on a per-residue basis, and investigate the importance of each individual mutation for ligand binding. Our data show that methionine scanning significantly extends the applicability, information content, and spatial resolution of methyl CSP experiments.

The ubiquitin binding region of the Smurf HECT domain facilitates polyubiquitylation and binding of ubiquitylated substrates.

Mono- and polyubiquitylation of proteins are key steps in a wide range of biological processes. However, the molecular mechanisms that mediate these different events are poorly understood. Here, we employed NMR spectroscopy to map a non-covalent ubiquitin binding surface (UBS) on the Smurf ubiquitin ligase HECT domain. Analysis of mutants of the HECT UBS reveal that interfering with the UBS surface blocked Smurf-dependent degradation of its substrate RhoA in cells. In vitro analysis revealed that the UBS was not required for UbcH7-dependent charging of the HECT catalytic cysteine. Surprisingly, although the UBS was required for polyubiquitylation of both Smurf itself and the Smurf substrate RhoA, it was not required for monoubiquitylation. Furthermore, we show that mutating the UBS interfered with efficient binding of a monoubiquitylated form of RhoA to the Smurf HECT domain. Our findings suggest the UBS promotes polyubiquitylation by stabilizing ubiquitylated substrate binding to the HECT domain.

Computational design of ligand binding is not a solved problem.

Computational design has been very successful in recent years: multiple novel ligand binding proteins as well as enzymes have been reported. We wanted to know in molecular detail how precise the predictions of the interactions of protein and ligands are. Therefore, we performed a structural analysis of a number of published receptors designed onto the periplasmic binding protein scaffold that were reported to bind to the new ligands with nano- to micromolar affinities. It turned out that most of these designed proteins are not suitable for structural studies due to instability and aggregation. However, we were able to solve the crystal structure of an arabinose binding protein designed to bind serotonin to 2.2 A resolution. While crystallized in the presence of an excess of serotonin, the protein is in an open conformation with no serotonin bound, although the side-chain conformations in the empty binding pocket are very similar to the conformations predicted. During subsequent characterization using isothermal titration calorimetry, CD, and NMR spectroscopy, no indication of binding could be detected for any of the tested designed receptors, whereas wild-type proteins bound their ligands as expected. We conclude that although the computational prediction of side-chain conformations appears to be working, it does not necessarily confer binding as expected. Hence, the computational design of ligand binding is not a solved problem and needs to be revisited.

Structural studies of FF domains of the transcription factor CA150 provide insights into the organization of FF domain tandem arrays.

FF domains are poorly understood protein interaction modules that are present within eukaryotic transcription factors, such as CA150 (TCERG-1). The CA150 FF domains have been shown to mediate interactions with the phosphorylated C-terminal domain of RNA polymerase II (phosphoCTD) and a multitude of transcription factors and RNA processing proteins, and may therefore have a central role in organizing transcription. FF domains occur in tandem arrays of up to six domains, although it is not known whether they adopt higher-order structures. We have used the CA150 FF1+FF2 domains as a model system to examine whether tandem FF domains form higher-order structures in solution using NMR spectroscopy. In the solution structure of FF1 fused to the linker that joins FF1 to FF2, we observed that the highly conserved linker peptide is ordered and forms a helical extension of helix alpha3, suggesting that the interdomain linker might have a role in orientating FF1 relative to FF2. However, examination of the FF1+FF2 domains using relaxation NMR experiments revealed that although these domains are not rigidly orientated relative to one another, they do not tumble independently. Thus, the FF1+FF2 structure conforms to a dumbbell-shape in solution, where the helical interdomain linker maintains distance between the two dynamic FF domains without cementing their relative orientations. This model for FF domain organization within tandem arrays suggests a general mechanism by which individual FF domains can manoeuvre to achieve optimal recognition of flexible binding partners, such as the intrinsically-disordered phosphoCTD.

Interactions between the three CIN85 SH3 domains and ubiquitin: implications for CIN85 ubiquitination.

CIN85 is an adaptor protein linking the ubiquitin ligase Cbl and clathrin-binding proteins in clathrin-mediated receptor endocytosis. The SH3 domains of CIN85 bind to a proline-rich region of Cbl. Here we show that all three SH3 domains of CIN85 bind to ubiquitin. We also present a data-based structural model of the CIN85 SH3-C domain in complex with ubiquitin. In this complex, ubiquitin binds to the canonical interaction surface of the SH3 domain for proline-rich ligands and mimics the PPII helix, and we provide evidence that ubiquitin competes with these ligands for binding. We demonstrate that disruption of ubiquitin binding results in constitutive ubiquitination of CIN85 and an increased level of ubiquitination of EGFR in the absence of EGF stimulation. These results suggest that competition between Cbl and ubiquitin binding to CIN85 regulates Cbl function and EGFR endocytosis.

Autoinhibition of the HECT-type ubiquitin ligase Smurf2 through its C2 domain.

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.

An exchange-free measure of 15N transverse relaxation: an NMR spectroscopy application to the study of a folding intermediate with pervasive chemical exchange.

A series of experiments are presented that provide an exchange-free measure of dipole-dipole (15)N transverse relaxation, R(dd), that can then be substituted for (15)N R(1rho) or R(2) rates in the study of internal protein dynamics. The method is predicated on the measurement of a series of relaxation rates involving (1)H-(15)N longitudinal order, anti-phase (1)H and (15)N single-quantum coherences, and (1)H-(15)N multiple quantum coherences; the relaxation rates of all coherences are measured under conditions of spin-locking. Results from detailed simulations and experiments on a number of protein systems establish that R(dd) values are independent of exchange and systematic errors from dipolar interactions with proximal protons are calculated to be less than 1-2%, on average, for applications to perdeuterated proteins. Simulations further indicate that the methodology is rather insensitive to the exact level of deuteration so long as proteins are reasonably highly deuterated (>50%). The utility of the methodology is demonstrated with applications involving protein L, ubiquitin, and a stabilized folding intermediate of apocytochrome b(562) that shows large contributions to (15)N R(1rho) relaxation from chemical exchange.

Fractional 13C enrichment of isolated carbons using [1-13C]- or [2- 13C]-glucose facilitates the accurate measurement of dynamics at backbone Calpha and side-chain methyl positions in proteins.

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.

A change in conformational dynamics underlies the activation of Eph receptor tyrosine kinases.

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 auto-inhibited 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 alphaC 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 alphaC and the activation segment rather than a transition to a static active conformation underlies Eph RTK activation.

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.

Structural basis for the cytoskeletal association of Bcr-Abl/c-Abl.

The Bcr-Abl tyrosine kinase causes different forms of leukemia in humans. Depending on its position within the cell, Bcr-Abl differentially affects cellular growth. However, no structural and molecular details for the anticipated localization determinants are available. We present the NMR structure of the F-actin binding domain (FABD) of Bcr-Abl and its cellular counterpart c-Abl. The FABD forms a compact left-handed four-helix bundle in solution. We show that the nuclear export signal (NES) previously reported in this region is part of the hydrophobic core and nonfunctional in the intact protein. In contrast, we could identify the critical residues of helix alphaIII that are responsible for F-actin binding and cytoskeletal association. We propose that these interactions represent a major determinant for both Bcr-Abl and c-Abl localization.