@article{SanderXuEilersetal.2017, author = {Sander, Bodo and Xu, Wenshan and Eilers, Martin and Popov, Nikita and Lorenz, Sonja}, title = {A conformational switch regulates the ubiquitin ligase HUWE1}, series = {eLife}, volume = {6}, journal = {eLife}, doi = {10.7554/eLife.21036}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-171862}, year = {2017}, abstract = {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.}, language = {en} } @article{RiesSanderDeoletal.2019, author = {Ries, Lena K. and Sander, Bodo and Deol, Kirandeep K. and Letzelter, Marie-Annick and Strieter, Eric Robert and Lorenz, Sonja}, title = {Analysis of ubiquitin recognition by the HECT ligase E6AP provides insight into its linkage specificity}, series = {Journal of Biological Chemistry}, volume = {294}, journal = {Journal of Biological Chemistry}, number = {15}, doi = {10.1074/jbc.RA118.007014}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-226207}, pages = {6113-6129}, year = {2019}, abstract = {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.}, language = {en} } @article{LorenzBhattacharyyaFeileretal.2016, author = {Lorenz, Sonja and Bhattacharyya, Moitrayee and Feiler, Christian and Rape, Michael and Kuriyan, John}, title = {Crystal Structure of a Ube2S-Ubiquitin Conjugate}, series = {PLoS ONE}, volume = {11}, journal = {PLoS ONE}, number = {2}, doi = {10.1371/journal.pone.0147550}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-167265}, pages = {e0147550}, year = {2016}, abstract = {Protein ubiquitination occurs through the sequential formation and reorganization of specific protein-protein interfaces. Ubiquitin-conjugating (E2) enzymes, such as Ube2S, catalyze the formation of an isopeptide linkage between the C-terminus of a "donor" ubiquitin and a primary amino group of an "acceptor" ubiquitin molecule. This reaction involves an intermediate, in which the C-terminus of the donor ubiquitin is thioester-bound to the active site cysteine of the E2 and a functionally important interface is formed between the two proteins. A docked model of a Ube2S-donor ubiquitin complex was generated previously, based on chemical shift mapping by NMR, and predicted contacts were validated in functional studies. We now present the crystal structure of a covalent Ube2S-ubiquitin complex. The structure contains an interface between Ube2S and ubiquitin in trans that resembles the earlier model in general terms, but differs in detail. The crystallographic interface is more hydrophobic than the earlier model and is stable in molecular dynamics (MD) simulations. Remarkably, the docked Ube2S-donor complex converges readily to the configuration seen in the crystal structure in 3 out of 8 MD trajectories. Since the crystallographic interface is fully consistent with mutational effects, this indicates that the structure provides an energetically favorable representation of the functionally critical Ube2S-donor interface.}, language = {en} } @article{RiesLiessFeileretal.2020, author = {Ries, Lena K. and Liess, Anna K. L. and Feiler, Christian G. and Spratt, Donald E. and Lowe, Edward D. and Lorenz, Sonja}, title = {Crystal structure of the catalytic C-lobe of the HECT-type ubiquitin ligase E6AP}, series = {Protein Science}, volume = {29}, journal = {Protein Science}, number = {6}, doi = {10.1002/pro.3832}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-214812}, pages = {1550 -- 1554}, year = {2020}, abstract = {The HECT-type ubiquitin ligase E6AP (UBE3A) is critically involved in several neurodevelopmental disorders and human papilloma virus-induced cervical tumorigenesis; the structural mechanisms underlying the activity of this crucial ligase, however, are incompletely understood. Here, we report a crystal structure of the C-terminal lobe ("C-lobe") of the catalytic domain of E6AP that reveals two molecules in a domain-swapped, dimeric arrangement. Interestingly, the molecular hinge that enables this structural reorganization with respect to the monomeric fold coincides with the active-site region. While such dimerization is unlikely to occur in the context of full-length E6AP, we noticed a similar domain swap in a crystal structure of the isolated C-lobe of another HECT-type ubiquitin ligase, HERC6. This may point to conformational strain in the active-site region of HECT-type ligases with possible implications for catalysis. Significance Statement The HECT-type ubiquitin ligase E6AP has key roles in human papilloma virus-induced cervical tumorigenesis and certain neurodevelopmental disorders. Here, we present a crystal structure of the C-terminal, catalytic lobe of E6AP, providing basic insight into the conformational properties of this functionally critical region of HECT-type ligases.}, language = {en} } @article{DeolLorenzStrieter2019, author = {Deol, Kirandeep K. and Lorenz, Sonja and Strieter, Eric R.}, title = {Enzymatic logic of ubiquitin chain assembly}, series = {Frontiers in Physiology}, volume = {10}, journal = {Frontiers in Physiology}, number = {835}, doi = {10.3389/fphys.2019.00835}, url = {http://nbn-resolving.de/urn:nbn:de:bvb:20-opus-201731}, year = {2019}, abstract = {Protein ubiquitination impacts virtually every biochemical pathway in eukaryotic cells. The fate of a ubiquitinated protein is largely dictated by the type of ubiquitin modification with which it is decorated, including a large variety of polymeric chains. As a result, there have been intense efforts over the last two decades to dissect the molecular details underlying the synthesis of ubiquitin chains by ubiquitin-conjugating (E2) enzymes and ubiquitin ligases (E3s). In this review, we highlight these advances. We discuss the evidence in support of the alternative models of transferring one ubiquitin at a time to a growing substrate-linked chain (sequential addition model) versus transferring a pre-assembled ubiquitin chain (en bloc model) to a substrate. Against this backdrop, we outline emerging principles of chain assembly: multisite interactions, distinct mechanisms of chain initiation and elongation, optimal positioning of ubiquitin molecules that are ultimately conjugated to each other, and substrate-assisted catalysis. Understanding the enzymatic logic of ubiquitin chain assembly has important biomedical implications, as the misregulation of many E2s and E3s and associated perturbations in ubiquitin chain formation contribute to human disease. The resurgent interest in bifunctional small molecules targeting pathogenic proteins to specific E3s for polyubiquitination and subsequent degradation provides an additional incentive to define the mechanisms responsible for efficient and specific chain synthesis and harness them for therapeutic benefit.}, language = {en} }