Analysis of ubiquitin recognition by the HECT ligase E6AP provides insight into its linkage specificity.

Deregulation of the HECT-type ubiquitin ligase E6AP (UBE3A) is implicated in human papilloma virus-induced cervical tumorigenesis and several neurodevelopmental disorders. Yet the structural underpinnings of activity and specificity in this crucial ligase are incompletely understood. Here, we unravel the determinants of ubiquitin recognition by the catalytic domain of E6AP and assign them to particular steps in the catalytic cycle. We identify a functionally critical interface that is specifically required during the initial formation of a thioester-linked intermediate between the C terminus of ubiquitin and the ligase-active site. This interface resembles the one utilized by NEDD4-type enzymes, indicating that it is widely conserved across HECT ligases, independent of their linkage specificities. Moreover, we uncover surface regions in ubiquitin and E6AP, both in the N- and C-terminal portions of the catalytic domain, that are important for the subsequent reaction step of isopeptide bond formation between two ubiquitin molecules. We decipher key elements of linkage specificity, including the C-terminal tail of E6AP and a hydrophilic surface region of ubiquitin in proximity to the acceptor site Lys-48. Intriguingly, mutation of Glu-51, a single residue within this region, permits formation of alternative chain types, thus pointing to a key role of ubiquitin in conferring linkage specificity to E6AP. We speculate that substrate-assisted catalysis, as described previously for certain RING-associated ubiquitin-conjugating enzymes, constitutes a common principle during linkage-specific ubiquitin chain assembly by diverse classes of ubiquitination enzymes, including HECT ligases.

Comparative biochemical and structural analysis of the flavin-binding dodecins from Streptomyces davaonensis and Streptomyces coelicolor reveals striking differences with regard to multimerization.

Dodecins are small flavin-binding proteins that are widespread amongst haloarchaeal and bacterial species. Haloarchaeal dodecins predominantly bind riboflavin, while bacterial dodecins have been reported to bind riboflavin-5'-phosphate, also called flavin mononucleotide (FMN), and the FMN derivative, flavin adenine dinucleotide (FAD). Dodecins form dodecameric complexes and represent buffer systems for cytoplasmic flavins. In this study, dodecins of the bacteria Streptomyces davaonensis (SdDod) and Streptomyces coelicolor (ScDod) were investigated. Both dodecins showed an unprecedented low affinity for riboflavin, FMN and FAD when compared to other bacterial dodecins. Significant binding of FMN and FAD occurred at relatively low temperatures and under acidic conditions. X-ray diffraction analyses of SdDod and ScDod revealed that the structures of both Streptomyces dodecins are highly similar, which explains their similar binding properties for FMN and FAD. In contrast, SdDod and ScDod showed very different properties with regard to the stability of their dodecameric complexes. Site-directed mutagenesis experiments revealed that a specific salt bridge (D10-K62) is responsible for this difference in stability.

The release of host-derived antibodies bound to the variant surface glycoprotein (VSG) of Trypanosoma brucei cannot be explained by pH-dependent conformational changes of the VSG dimer.

Background: Trypanosoma brucei is a protozoan parasite that evades the mammalian host's adaptive immune response by antigenic variation of the highly immunogenic variant surface glycoprotein (VSG). VSGs form a dense surface coat that is constantly recycled through the endosomal system. Bound antibodies are separated in the endosome from the VSG and destroyed in the lysosome. For VSGs it has been hypothesized that pH-dependent structural changes of the VSG could occur in the more acidic environment of the endosome and hence, facilitate the separation of the antibody from the VSG. Methods: We used size exclusion chromatography, where molecules are separated according to their hydrodynamic radius to see if the VSG is present as a homodimer at both pH values. To gain information about the structural integrity of the protein we used circular dichroism spectroscopy by exposing the VSG in solution to a mixture of right- and left-circularly polarized light and analysing the absorbed UV spectra. Evaluation of protein stability and molecular dynamics simulations at different pH values was performed using different computational methods. Results: We show, for an A2-type VSG, that the dimer size is only slightly larger at pH 5.2 than at pH 7.4. Moreover, the dimer was marginally more stable at lower pH due to the higher affinity (ΔG = 353.37 kcal/mol) between the monomers. Due to the larger size, the predicted epitopes were more exposed to the solvent at low pH. Moderate conformational changes (ΔRMSD = 0.35 nm) in VSG were detected between the dimers at pH 5.2 and pH 7.4 in molecular dynamics simulations, and no significant differences in the protein secondary structure were observed by circular dichroism spectroscopy. Conclusions: Thus, the dissociation of anti-VSG-antibodies in endosomes cannot be explained by changes in pH.

Structural characterization of gephyrin by AFM and SAXS reveals a mixture of compact and extended states.

Gephyrin is a trimeric protein involved in the final steps of molybdenum-cofactor (Moco) biosynthesis and in the clustering of inhibitory glycine and GABAA receptors at postsynaptic specializations. Each protomer consists of stably folded domains (referred to as the G and E domains) located at either terminus and connected by a proteolytically sensitive linker of ∼150 residues. Both terminal domains can oligomerize in their isolated forms; however, in the context of the full-length protein only the G-domain trimer is permanently present, whereas E-domain dimerization is prevented. Atomic force microscopy (AFM) and small-angle X-ray scattering (SAXS) reveal a high degree of flexibility in the structure of gephyrin. The results imply an equilibrium between compact and extended conformational states in solution, with a preference for compact states. CD spectroscopy suggests that a partial compaction is achieved by interactions of the linker with the G and E domains. Taken together, the data provide a rationale for the role of the linker in the overall structure and the conformational dynamics of gephyrin.

Identification of an atypical interaction site in the BTB domain of the MYC-interacting zinc-finger protein 1.

The repurposing of structurally conserved protein domains in different functional contexts is thought to be a driving force in the evolution of complex protein interaction networks. The BTB/POZ domain is such a versatile binding module that occurs over 200 times in the human proteome with diverse protein-specific adaptations. In BTB-zinc-finger transcription factors, the BTB domain drives homo- and heterodimerization as well as interactions with non-BTB-domain-containing proteins. Which mechanisms encode specificity in these interactions at a structural level is incompletely understood. Here, we uncover an atypical peptide-binding site in the BTB domain of the MYC-interacting zinc-finger protein 1 (MIZ1) that arises from local flexibility of the core BTB fold and may provide a target site for MIZ1-directed therapeutic approaches. Intriguingly, the identified binding mode requires the BTB domain to be in a homodimeric state, thus holding opportunities for functional discrimination between homo- and heterodimers of MIZ1 in the cell.

A conformational switch regulates the ubiquitin ligase HUWE1.

The human ubiquitin ligase HUWE1 has key roles in tumorigenesis, yet it is unkown how its activity is regulated. We present the crystal structure of a C-terminal part of HUWE1, including the catalytic domain, and reveal an asymmetric auto-inhibited dimer. We show that HUWE1 dimerizes in solution and self-associates in cells, and that both occurs through the crystallographic dimer interface. We demonstrate that HUWE1 is inhibited in cells and that it can be activated by disruption of the dimer interface. We identify a conserved segment in HUWE1 that counteracts dimer formation by associating with the dimerization region intramolecularly. Our studies reveal, intriguingly, that the tumor suppressor p14ARF binds to this segment and may thus shift the conformational equilibrium of HUWE1 toward the inactive state. We propose a model, in which the activity of HUWE1 underlies conformational control in response to physiological cues-a mechanism that may be exploited for cancer therapy.

The LisH motif of muskelin is crucial for oligomerization and governs intracellular localization.

Neurons regulate the number of surface receptors by balancing the transport to and from the plasma membrane to adjust their signaling properties. The protein muskelin was recently identified as a key factor guiding the transport of α1 subunit-containing GABAA receptors. Here we present the crystal structure of muskelin, comprising its N-terminal discoidin domain and Lis1-homology (LisH) motif. The molecule crystallized as a dimer with the LisH motif exclusively mediating oligomerization. Our subsequent biochemical analyses confirmed that the LisH motif acts as a dimerization element in muskelin. Together with an intermolecular head-to-tail interaction, the LisH-dependent dimerization is required to assemble a muskelin tetramer. Intriguingly, our cellular studies revealed that the loss of this dimerization results in a complete redistribution of muskelin from the cytoplasm to the nucleus and impairs muskelin's function in GABAA receptor transport. These studies demonstrate that the LisH-dependent dimerization is a crucial factor for muskelin function.