Publications by authors named "Peter S Brzovic"

29 Publications

  • Page 1 of 1

BRCA1/BARD1 site-specific ubiquitylation of nucleosomal H2A is directed by BARD1.

Nat Struct Mol Biol 2021 Mar 15;28(3):268-277. Epub 2021 Feb 15.

Department of Biochemistry, University of Washington, Seattle, WA, USA.

Mutations in the E3 ubiquitin ligase RING domains of BRCA1/BARD1 predispose carriers to breast and ovarian cancers. We present the structure of the BRCA1/BARD1 RING heterodimer with the E2 enzyme UbcH5c bound to its cellular target, the nucleosome, along with biochemical data that explain how the complex selectively ubiquitylates lysines 125, 127 and 129 in the flexible C-terminal tail of H2A in a fully human system. The structure reveals that a novel BARD1-histone interface couples to a repositioning of UbcH5c compared to the structurally similar PRC1 E3 ligase Ring1b/Bmi1 that ubiquitylates H2A Lys119 in nucleosomes. This interface is sensitive to both H3 Lys79 methylation status and mutations found in individuals with cancer. Furthermore, NMR reveals an unexpected mode of E3-mediated substrate regulation through modulation of dynamics in the C-terminal tail of H2A. Our findings provide insight into how E3 ligases preferentially target nearby lysine residues in nucleosomes by a steric occlusion and distancing mechanism.
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http://dx.doi.org/10.1038/s41594-020-00556-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007219PMC
March 2021

Legionella effector MavC targets the Ube2N~Ub conjugate for noncanonical ubiquitination.

Nat Commun 2020 05 12;11(1):2365. Epub 2020 May 12.

Department of Chemistry, Purdue University, West Lafayette, IN, 47907, USA.

The bacterial effector MavC modulates the host immune response by blocking Ube2N activity employing an E1-independent ubiquitin ligation, catalyzing formation of a γ-glutamyl-ε-Lys (Gln40-Lys92) isopeptide crosslink using a transglutaminase mechanism. Here we provide biochemical evidence in support of MavC targeting the activated, thioester-linked Ube2N~ubiquitin conjugate, catalyzing an intramolecular transglutamination reaction, covalently crosslinking the Ube2N and Ub subunits effectively inactivating the E2~Ub conjugate. Ubiquitin exhibits weak binding to MavC alone, but shows an increase in affinity when tethered to Ube2N in a disulfide-linked substrate that mimics the charged E2~Ub conjugate. Crystal structures of MavC in complex with the substrate mimic and crosslinked product provide insights into the reaction mechanism and underlying protein dynamics that favor transamidation over deamidation, while revealing a crucial role for the structurally unique insertion domain in substrate recognition. This work provides a structural basis of ubiquitination by transglutamination and identifies this enzyme's true physiological substrate.
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http://dx.doi.org/10.1038/s41467-020-16211-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7217864PMC
May 2020

Salmonella Translocated Effectors Recruit OSBP1 to the Phagosome to Promote Vacuolar Membrane Integrity.

Cell Rep 2019 05;27(7):2147-2156.e5

Department of Microbiology, University of Washington, Seattle, WA 98195, USA; Department of Immunology, University of Washington, Seattle, WA 98195, USA; Department of Medicine, University of Washington, Seattle, WA 98195, USA; Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA. Electronic address:

Intracellular Salmonella use a type III secretion system (TTSS) to translocate effector proteins across the phagosome membrane and thus promote vacuole membrane tubulation, resulting in intracellular survival. This work demonstrates that the effector SseJ binds the eukaryotic lipid transporter oxysterol binding protein 1 (OSBP1). SseJ directs OSBP1 to the endosomal compartment in a manner dependent on the TTSS located on Salmonella pathogenicity island 2 (SPI2). OSBP1 localization is mediated by both SseJ and another OSBP1-binding SPI2 translocated effector, the deubiquitinase SseL. Deletion of both SseJ and SseL reduced vacuolar integrity with increased bacteria released into the eukaryotic cytoplasm of epithelial cells, indicating that their combined activities are necessary for vacuole membrane stability. Cells knocked down for OSBP1 or deleted for the OSBP1-binding proteins VAPA/B also demonstrate loss of vacuole integrity, consistent with the hypothesis that OSBP1 recruitment is required for SPI2-mediated alterations that promote vacuolar integrity of salmonellae.
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http://dx.doi.org/10.1016/j.celrep.2019.04.021DOI Listing
May 2019

The ubiquitin ligase SspH1 from uses a modular and dynamic E3 domain to catalyze substrate ubiquitylation.

J Biol Chem 2019 01 20;294(3):783-793. Epub 2018 Nov 20.

From the Departments of Biochemistry and

SspH/IpaH bacterial effector E3 ubiquitin (Ub) ligases, unrelated in sequence or structure to eukaryotic E3s, are utilized by a wide variety of Gram-negative bacteria during pathogenesis. These E3s function in a eukaryotic environment, utilize host cell E2 ubiquitin-conjugating enzymes of the Ube2D family, and target host proteins for ubiquitylation. Despite several crystal structures, details of Ube2D∼Ub binding and the mechanism of ubiquitin transfer are poorly understood. Here, we show that the catalytic E3 ligase domain of SspH1 can be divided into two subdomains: an N-terminal subdomain that harbors the active-site cysteine and a C-terminal subdomain containing the Ube2D∼Ub-binding site. SspH1 mutations designed to restrict subdomain motions show rapid formation of an E3∼Ub intermediate, but impaired Ub transfer to substrate. NMR experiments using paramagnetic spin labels reveal how SspH1 binds Ube2D∼Ub and targets the E2∼Ub active site. Unexpectedly, hydrogen/deuterium exchange MS shows that the E2∼Ub-binding region is dynamic but stabilized in the E3∼Ub intermediate. Our results support a model in which both subunits of an Ube2D∼Ub clamp onto a dynamic region of SspH1, promoting an E3 conformation poised for transthiolation. A conformational change is then required for Ub transfer from E3∼Ub to substrate.
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http://dx.doi.org/10.1074/jbc.RA118.004247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6341402PMC
January 2019

Mechanism of phosphoribosyl-ubiquitination mediated by a single Legionella effector.

Nature 2018 05 23;557(7707):729-733. Epub 2018 May 23.

Weill Institute for Cell and Molecular Biology and Department of Molecular Biology and Genetics, Cornell University, Ithaca, NY, USA.

Ubiquitination is a post-translational modification that regulates many cellular processes in eukaryotes. The conventional ubiquitination cascade culminates in a covalent linkage between the C terminus of ubiquitin (Ub) and a target protein, usually on a lysine side chain. Recent studies of the Legionella pneumophila SidE family of effector proteins revealed a ubiquitination method in which a phosphoribosyl ubiquitin (PR-Ub) is conjugated to a serine residue on substrates via a phosphodiester bond. Here we present the crystal structure of a fragment of the SidE family member SdeA that retains ubiquitination activity, and determine the mechanism of this unique post-translational modification. The structure reveals that the catalytic module contains two distinct functional units: a phosphodiesterase domain and a mono-ADP-ribosyltransferase domain. Biochemical analysis shows that the mono-ADP-ribosyltransferase domain-mediated conversion of Ub to ADP-ribosylated Ub (ADPR-Ub) and the phosphodiesterase domain-mediated ligation of PR-Ub to substrates are two independent activities of SdeA. Furthermore, we present two crystal structures of a homologous phosphodiesterase domain from the SidE family member SdeD in complexes with Ub and ADPR-Ub. The structures suggest a mechanism for how SdeA processes ADPR-Ub to PR-Ub and AMP, and conjugates PR-Ub to a serine residue in substrates. Our study establishes the molecular mechanism of phosphoribosyl-linked ubiquitination and will enable future studies of this unusual type of ubiquitination in eukaryotes.
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http://dx.doi.org/10.1038/s41586-018-0147-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5980775PMC
May 2018

Tuning BRCA1 and BARD1 activity to investigate RING ubiquitin ligase mechanisms.

Protein Sci 2017 03 23;26(3):475-483. Epub 2017 Jan 23.

Department of Biochemistry, University of Washington, Seattle, Washington, 98195.

The tumor-suppressor protein BRCA1 works with BARD1 to catalyze the transfer of ubiquitin onto protein substrates. The N-terminal regions of BRCA1 and BARD1 that contain their RING domains are responsible for dimerization and ubiquitin ligase activity. This activity is a common feature among hundreds of human RING domain-containing proteins. RING domains bind and activate E2 ubiquitin-conjugating enzymes to promote ubiquitin transfer to substrates. We show that the identity of residues at specific positions in the RING domain can tune activity levels up or down. We report substitutions that create a structurally intact BRCA1/BARD1 heterodimer that is inactive in vitro with all E2 enzymes. Other substitutions in BRCA1 or BARD1 RING domains result in hyperactivity, revealing that both proteins have evolved attenuated activity. Loss of attenuation results in decreased product specificity, providing a rationale for why nature has tuned BRCA1 activity. The ability to tune BRCA1 provides powerful tools for understanding its biological functions and provides a basis to assess mechanisms for rescuing the activity of cancer-associated variations. Beyond the applicability to BRCA1, we show the identity of residues at tuning positions that can be used to predict and modulate the activity of an unrelated RING E3 ligase. These findings provide valuable insights into understanding the mechanism and function of RING E3 ligases like BRCA1.
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http://dx.doi.org/10.1002/pro.3091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5326557PMC
March 2017

E2 enzymes: more than just middle men.

Cell Res 2016 Apr 22;26(4):423-40. Epub 2016 Mar 22.

Department of Biochemistry, University of Washington, Seattle, WA 98102 USA.

Ubiquitin-conjugating enzymes (E2s) are the central players in the trio of enzymes responsible for the attachment of ubiquitin (Ub) to cellular proteins. Humans have ∼40 E2s that are involved in the transfer of Ub or Ub-like (Ubl) proteins (e.g., SUMO and NEDD8). Although the majority of E2s are only twice the size of Ub, this remarkable family of enzymes performs a variety of functional roles. In this review, we summarize common functional and structural features that define unifying themes among E2s and highlight emerging concepts in the mechanism and regulation of E2s.
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http://dx.doi.org/10.1038/cr.2016.35DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4822130PMC
April 2016

Structure and Energetics of Allosteric Regulation of HCN2 Ion Channels by Cyclic Nucleotides.

J Biol Chem 2016 Jan 11;291(1):371-81. Epub 2015 Nov 11.

From the Departments of Chemistry, Physiology and Biophysics, and

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels play an important role in regulating electrical activity in the heart and brain. They are gated by the binding of cyclic nucleotides to a conserved, intracellular cyclic nucleotide-binding domain (CNBD), which is connected to the channel pore by a C-linker region. Binding of cyclic nucleotides increases the rate and extent of channel activation and shifts it to less hyperpolarized voltages. We probed the allosteric mechanism of different cyclic nucleotides on the CNBD and on channel gating. Electrophysiology experiments showed that cAMP, cGMP, and cCMP were effective agonists of the channel and produced similar increases in the extent of channel activation. In contrast, electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) on the isolated CNBD indicated that the induced conformational changes and the degrees of stabilization of the active conformation differed for the three cyclic nucleotides. We explain these results with a model where different allosteric mechanisms in the CNBD all converge to have the same effect on the C-linker and render all three cyclic nucleotides similarly potent activators of the channel.
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http://dx.doi.org/10.1074/jbc.M115.696450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4697172PMC
January 2016

Regulating the Regulators: Recent Revelations in the Control of E3 Ubiquitin Ligases.

J Biol Chem 2015 Aug 17;290(35):21244-51. Epub 2015 Jul 17.

From the Department of Biochemistry, University of Washington, Seattle, Washington 98195-7742

Since its discovery as a post-translational signal for protein degradation, our understanding of ubiquitin (Ub) has vastly evolved. Today, we recognize that the role of Ub signaling is expansive and encompasses diverse processes including cell division, the DNA damage response, cellular immune signaling, and even organismal development. With such a wide range of functions comes a wide range of regulatory mechanisms that control the activity of the ubiquitylation machinery. Ub attachment to substrates occurs through the sequential action of three classes of enzymes, E1s, E2s, and E3s. In humans, there are 2 E1s, ∼ 35 E2s, and hundreds of E3s that work to attach Ub to thousands of cellular substrates. Regulation of ubiquitylation can occur at each stage of the stepwise Ub transfer process, and substrates can also impact their own modification. Recent studies have revealed elegant mechanisms that have evolved to control the activity of the enzymes involved. In this minireview, we highlight recent discoveries that define some of the various mechanisms by which the activities of E3-Ub ligases are regulated.
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http://dx.doi.org/10.1074/jbc.R115.675165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4571856PMC
August 2015

Structural mechanism for the regulation of HCN ion channels by the accessory protein TRIP8b.

Structure 2015 Apr 19;23(4):734-44. Epub 2015 Mar 19.

Department of Chemistry, University of Washington, Seattle, WA 98195, USA. Electronic address:

Hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels underlie the cationic Ih current present in many neurons. The direct binding of cyclic AMP to HCN channels increases the rate and extent of channel opening and results in a depolarizing shift in the voltage dependence of activation. TRIP8b is an accessory protein that regulates the cell surface expression and dendritic localization of HCN channels and reduces the cyclic nucleotide dependence of these channels. Here, we use electron paramagnetic resonance (EPR) to show that TRIP8b binds to the apo state of the cyclic nucleotide binding domain (CNBD) of HCN2 channels without changing the overall domain structure. With EPR and nuclear magnetic resonance, we locate TRIP8b relative to the HCN channel and identify the binding interface on the CNBD. These data provide a structural framework for understanding how TRIP8b regulates the cyclic nucleotide dependence of HCN channels.
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http://dx.doi.org/10.1016/j.str.2015.02.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4394039PMC
April 2015

Intrinsic disorder drives N-terminal ubiquitination by Ube2w.

Nat Chem Biol 2015 Jan 1;11(1):83-9. Epub 2014 Dec 1.

Department of Biochemistry, University of Washington, Seattle, Washington, USA.

Ubiquitination of the αN-terminus of protein substrates has been reported sporadically since the early 1980s. However, the identity of an enzyme responsible for this unique ubiquitin (Ub) modification has only recently been elucidated. We show the Ub-conjugating enzyme (E2) Ube2w uses a unique mechanism to facilitate the specific ubiquitination of the α-amino group of its substrates that involves recognition of backbone atoms of intrinsically disordered N termini. We present the NMR-based solution ensemble of full-length Ube2w that reveals a structural architecture unlike that of any other E2 in which its C terminus is partly disordered and flexible to accommodate variable substrate N termini. Flexibility of the substrate is critical for recognition by Ube2w, and either point mutations in or the removal of the flexible C terminus of Ube2w inhibits substrate binding and modification. Mechanistic insights reported here provide guiding principles for future efforts to define the N-terminal ubiquitome in cells.
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http://dx.doi.org/10.1038/nchembio.1700DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4270946PMC
January 2015

E2~Ub conjugates regulate the kinase activity of Shigella effector OspG during pathogenesis.

EMBO J 2014 Mar 20;33(5):437-49. Epub 2014 Jan 20.

Department of Biochemistry, University of Washington, Seattle, WA, USA.

Pathogenic bacteria introduce effector proteins directly into the cytosol of eukaryotic cells to promote invasion and colonization. OspG, a Shigella spp. effector kinase, plays a role in this process by helping to suppress the host inflammatory response. OspG has been reported to bind host E2 ubiquitin-conjugating enzymes activated with ubiquitin (E2~Ub), a key enzyme complex in ubiquitin transfer pathways. A co-crystal structure of the OspG/UbcH5c~Ub complex reveals that complex formation has important ramifications for the activity of both OspG and the UbcH5c~Ub conjugate. OspG is a minimal kinase domain containing only essential elements required for catalysis. UbcH5c~Ub binding stabilizes an active conformation of the kinase, greatly enhancing OspG kinase activity. In contrast, interaction with OspG stabilizes an extended, less reactive form of UbcH5c~Ub. Recognizing conserved E2 features, OspG can interact with at least ten distinct human E2s~Ub. Mouse oral infection studies indicate that E2~Ub conjugates act as novel regulators of OspG effector kinase function in eukaryotic host cells.
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http://dx.doi.org/10.1002/embj.201386386DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3989626PMC
March 2014

Biochemical and structural characterization of the ubiquitin-conjugating enzyme UBE2W reveals the formation of a noncovalent homodimer.

Cell Biochem Biophys 2013 Sep;67(1):103-10

Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.

The biochemical and structural characterization of ubiquitin-conjugating enzymes (E2s) over the past 30 years has fostered important insights into ubiquitin transfer mechanisms. Although many of these enzymes share high sequence and structural conservation, their functional roles in the cell are decidedly diverse. Here, we report that the mono-ubiquitinating E2 UBE2W forms a homodimer using two distinct protein surfaces. Dimerization is primarily driven by residues in the ß-sheet region and Loops 4 and 7 of the catalytic domain. Mutation of two residues in the catalytic domain of UBE2W is capable of disrupting UBE2W homodimer formation, however, we find that dimerization of this E2 is not required for its ubiquitin transfer activity. In addition, residues in the C-terminal region, although not compulsory for the dimerization of UBE2W, play an ancillary role in the dimer interface. In all current E2 structures, the C-terminal helix of the UBC domain is at least 15Å away from the primary dimerization surface shown here for UBE2W. This leads to the proposal that the C-terminal region of UBE2W adopts a noncanonical position that places it closer to the UBC ß-sheet, providing the first indication that at least some E2s adopt C-terminal conformations different from the canonical structures observed to date.
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http://dx.doi.org/10.1007/s12013-013-9633-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3758794PMC
September 2013

Activity-enhancing mutations in an E3 ubiquitin ligase identified by high-throughput mutagenesis.

Proc Natl Acad Sci U S A 2013 Apr 18;110(14):E1263-72. Epub 2013 Mar 18.

Howard Hughes Medical Institute, Department of Genome Sciences, University of Washington, Seattle, WA 98195, USA.

Although ubiquitination plays a critical role in virtually all cellular processes, mechanistic details of ubiquitin (Ub) transfer are still being defined. To identify the molecular determinants within E3 ligases that modulate activity, we scored each member of a library of nearly 100,000 protein variants of the murine ubiquitination factor E4B (Ube4b) U-box domain for auto-ubiquitination activity in the presence of the E2 UbcH5c. This assay identified mutations that enhance activity both in vitro and in cellular p53 degradation assays. The activity-enhancing mutations fall into two distinct mechanistic classes: One increases the U-box:E2-binding affinity, and the other allosterically stimulates the formation of catalytically active conformations of the E2∼Ub conjugate. The same mutations enhance E3 activity in the presence of another E2, Ube2w, implying a common allosteric mechanism, and therefore the general applicability of our observations to other E3s. A comparison of the E3 activity with the two different E2s identified an additional variant that exhibits E3:E2 specificity. Our results highlight the general utility of high-throughput mutagenesis in delineating the molecular basis of enzyme activity.
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http://dx.doi.org/10.1073/pnas.1303309110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3619334PMC
April 2013

Structure of an E3:E2~Ub complex reveals an allosteric mechanism shared among RING/U-box ligases.

Mol Cell 2012 Sep 9;47(6):933-42. Epub 2012 Aug 9.

Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.

Despite the widespread importance of RING/U-box E3 ubiquitin ligases in ubiquitin (Ub) signaling, the mechanism by which this class of enzymes facilitates Ub transfer remains enigmatic. Here, we present a structural model for a RING/U-box E3:E2~Ub complex poised for Ub transfer. The model and additional analyses reveal that E3 binding biases dynamic E2~Ub ensembles toward closed conformations with enhanced reactivity for substrate lysines. We identify a key hydrogen bond between a highly conserved E3 side chain and an E2 backbone carbonyl, observed in all structures of active RING/U-Box E3/E2 pairs, as the linchpin for allosteric activation of E2~Ub. The conformational biasing mechanism is generalizable across diverse E2s and RING/U-box E3s, but is not shared by HECT-type E3s. The results provide a structural model for a RING/U-box E3:E2~Ub ligase complex and identify the long sought-after source of allostery for RING/U-Box activation of E2~Ub conjugates.
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http://dx.doi.org/10.1016/j.molcel.2012.07.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3462262PMC
September 2012

A Salmonella typhimurium-translocated glycerophospholipid:cholesterol acyltransferase promotes virulence by binding to the RhoA protein switch regions.

J Biol Chem 2012 Aug 27;287(35):29654-63. Epub 2012 Jun 27.

Department of Microbiology, University of Washington, Seattle, WA 98195, USA.

Salmonella enterica serovar typhimurium translocates a glycerophospholipid:cholesterol acyltransferase (SseJ) into the host cytosol after its entry into mammalian cells. SseJ is recruited to the cytoplasmic face of the host cell phagosome membrane where it is activated upon binding the small GTPase, RhoA. SseJ is regulated similarly to cognate eukaryotic effectors, as only the GTP-bound form of RhoA family members stimulates enzymatic activity. Using NMR and biochemistry, this work demonstrates that SseJ competes effectively with Rhotekin, ROCK, and PKN1 in binding to a similar RhoA surface. The RhoA surface that binds SseJ includes the regulatory switch regions that control activation of mammalian effectors. These data were used to create RhoA mutants with altered SseJ binding and activation. This structure-function analysis supports a model in which SseJ activation occurs predominantly through binding to residues within switch region II. We further defined the nature of the interaction between SseJ and RhoA by constructing SseJ mutants in the RhoA binding surface. These data indicate that SseJ binding to RhoA is required for recruitment of SseJ to the endosomal network and for full Salmonella virulence for inbred susceptible mice, indicating that regulation of SseJ by small GTPases is an important virulence strategy of this bacterial pathogen. The dependence of a bacterial effector on regulation by a mammalian GTPase defines further how intimately host pathogen interactions have coevolved through similar and divergent evolutionary strategies.
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http://dx.doi.org/10.1074/jbc.M112.363598DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3436183PMC
August 2012

OTUB1 co-opts Lys48-linked ubiquitin recognition to suppress E2 enzyme function.

Mol Cell 2012 Feb;45(3):384-97

Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, ON M5G 1X5, Canada.

Ubiquitylation entails the concerted action of E1, E2, and E3 enzymes. We recently reported that OTUB1, a deubiquitylase, inhibits the DNA damage response independently of its isopeptidase activity. OTUB1 does so by blocking ubiquitin transfer by UBC13, the cognate E2 enzyme for RNF168. OTUB1 also inhibits E2s of the UBE2D and UBE2E families. Here we elucidate the structural mechanism by which OTUB1 binds E2s to inhibit ubiquitin transfer. OTUB1 recognizes ubiquitin-charged E2s through contacts with both donor ubiquitin and the E2 enzyme. Surprisingly, free ubiquitin associates with the canonical distal ubiquitin-binding site on OTUB1 to promote formation of the inhibited E2 complex. Lys48 of donor ubiquitin lies near the OTUB1 catalytic site and the C terminus of free ubiquitin, a configuration that mimics the products of Lys48-linked ubiquitin chain cleavage. OTUB1 therefore co-opts Lys48-linked ubiquitin chain recognition to suppress ubiquitin conjugation and the DNA damage response.
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http://dx.doi.org/10.1016/j.molcel.2012.01.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3306812PMC
February 2012

The acidic transcription activator Gcn4 binds the mediator subunit Gal11/Med15 using a simple protein interface forming a fuzzy complex.

Mol Cell 2011 Dec;44(6):942-53

Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.

The structural basis for binding of the acidic transcription activator Gcn4 and one activator-binding domain of the Mediator subunit Gal11/Med15 was examined by NMR. Gal11 activator-binding domain 1 has a four-helix fold with a small shallow hydrophobic cleft at its center. In the bound complex, eight residues of Gcn4 adopt a helical conformation, allowing three Gcn4 aromatic/aliphatic residues to insert into the Gal11 cleft. The protein-protein interface is dynamic and surprisingly simple, involving only hydrophobic interactions. This allows Gcn4 to bind Gal11 in multiple conformations and orientations, an example of a "fuzzy" complex, where the Gcn4-Gal11 interface cannot be described by a single conformation. Gcn4 uses a similar mechanism to bind two other unrelated activator-binding domains. Functional studies in yeast show the importance of residues at the protein interface, define the minimal requirements for a functional activator, and suggest a mechanism by which activators bind to multiple unrelated targets.
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http://dx.doi.org/10.1016/j.molcel.2011.11.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3246216PMC
December 2011

UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids.

Nature 2011 Jun 1;474(7349):105-8. Epub 2011 May 1.

Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.

Although the functional interaction between ubiquitin-conjugating enzymes (E2s) and ubiquitin ligases (E3s) is essential in ubiquitin (Ub) signalling, the criteria that define an active E2-E3 pair are not well established. The human E2 UBCH7 (also known as UBE2L3) shows broad specificity for HECT-type E3s, but often fails to function with RING E3s in vitro despite forming specific complexes. Structural comparisons of inactive UBCH7-RING complexes with active UBCH5-RING complexes reveal no defining differences, highlighting a gap in our understanding of Ub transfer. Here we show that, unlike many E2s that transfer Ub with RINGs, UBCH7 lacks intrinsic, E3-independent reactivity with lysine, explaining its preference for HECTs. Despite lacking lysine reactivity, UBCH7 exhibits activity with the RING-in-between-RING (RBR) family of E3s that includes parkin (also known as PARK2) and human homologue of ariadne (HHARI; also known as ARIH1). Found in all eukaryotes, RBRs regulate processes such as translation and immune signalling. RBRs contain a canonical C3HC4-type RING, followed by two conserved Cys/His-rich Zn(2+)-binding domains, in-between-RING (IBR) and RING2 domains, which together define this E3 family. We show that RBRs function like RING/HECT hybrids: they bind E2s via a RING domain, but transfer Ub through an obligate thioester-linked Ub (denoted ∼Ub), requiring a conserved cysteine residue in RING2. Our results define the functional cadre of E3s for UBCH7, an E2 involved in cell proliferation and immune function, and indicate a novel mechanism for an entire class of E3s.
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http://dx.doi.org/10.1038/nature09966DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3444301PMC
June 2011

The essential Ubc4/Ubc5 function in yeast is HECT E3-dependent, and RING E3-dependent pathways require only monoubiquitin transfer by Ubc4.

J Biol Chem 2011 Apr 25;286(17):15165-70. Epub 2011 Feb 25.

Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.

The ubiquitin (Ub)-conjugating enzymes Ubc4 and Ubc5 are involved in a variety of ubiquitination pathways in yeast, including Rsp5- and anaphase-promoting complex (APC)-mediated pathways. We have found the double deletion of UBC4 and UBC5 genes in yeast to be lethal. To investigate the essential pathway disrupted by the ubc4/ubc5 deletion, several point mutations were inserted in Ubc4. The Ubc4 active site mutation C86A and the E3-binding mutations A97D and F63A were both unable to rescue the lethal phenotype, indicating that an active E3/E2∼Ub complex is required for the essential function of Ubc4/Ubc5. A mutation that specifically eliminates RING E3-catalyzed isopeptide formation but not HECT E3 transthiolation (N78S-Ubc4) rescued the lethal phenotype. Thus, the essential redundant function performed by Ubc4 and Ubc5 in yeast is with a HECT-type E3, likely the only essential HECT in yeast, Rsp5. Our results also suggest that Ubc1 can weakly replace Ubc4 to transfer mono-Ub with APC, but Ubc4 cannot replace Ubc1 for poly-Ub chain extension on APC substrates. Finally, the backside Ub-binding mutant S23R-Ubc4 has no observable effect in yeast. Together, our results are consistent with a model in which Ubc4 and Ubc5 are 1) the primary E2s for Rsp5 in yeast and 2) act as monoubiquitinating E2s in RING E3-catalyzed pathways, in contrast to the processive human ortholog UbcH5.
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http://dx.doi.org/10.1074/jbc.M110.203968DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3083232PMC
April 2011

Ubiquitin in motion: structural studies of the ubiquitin-conjugating enzyme∼ubiquitin conjugate.

Biochemistry 2011 Mar 21;50(10):1624-33. Epub 2011 Feb 21.

Department of Biochemistry, University of Washington, Seattle, Washington 98195, United States.

Ubiquitination of proteins provides a powerful and versatile post-translational signal in the eukaryotic cell. The formation of a thioester bond between ubiquitin (Ub) and the active site of a ubiquitin-conjugating enzyme (E2) is critical for the transfer of Ub to substrates. Assembly of a functional ubiquitin ligase (E3) complex poised for Ub transfer involves recognition and binding of an E2∼Ub conjugate. Therefore, full characterization of the structure and dynamics of E2∼Ub conjugates is required for further mechanistic understanding of Ub transfer reactions. Here we present characterization of the dynamic behavior of E2∼Ub conjugates of two human enzymes, UbcH5c∼Ub and Ubc13∼Ub, in solution as determined by nuclear magnetic resonance and small-angle X-ray scattering. Within each conjugate, Ub retains great flexibility with respect to the E2, indicative of highly dynamic species that adopt manifold orientations. The population distribution of Ub conformations is dictated by the identity of the E2: the UbcH5c∼Ub conjugate populates an array of extended conformations, and the population of Ubc13∼Ub conjugates favors a closed conformation in which the hydrophobic surface of Ub faces helix 2 of Ubc13. We propose that the varied conformations adopted by Ub represent available binding modes of the E2∼Ub species and thus provide insight into the diverse E2∼Ub protein interactome, particularly with regard to interaction with Ub ligases.
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http://dx.doi.org/10.1021/bi101913mDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3056393PMC
March 2011

Identification of an unconventional E3 binding surface on the UbcH5 ~ Ub conjugate recognized by a pathogenic bacterial E3 ligase.

Proc Natl Acad Sci U S A 2010 Feb 1;107(7):2848-53. Epub 2010 Feb 1.

Department of Immunology, University of Washington, Seattle, WA 98195, USA.

Gram-negative bacteria deliver a cadre of virulence factors directly into the cytoplasm of eukaryotic host cells to promote pathogenesis and/or commensalism. Recently, families of virulence proteins have been recognized that function as E3 Ubiquitin-ligases. How these bacterial ligases integrate into the ubiquitin (Ub) signaling pathways of the host and how they differ functionally from endogenous eukaryotic E3s is not known. Here we show that the bacterial E3 SspH2 from S. typhimurium selectively binds the human UbcH5 ~ Ub conjugate recognizing regions of both UbcH5 and Ub subunits. The surface of the E2 UbcH5 involved in this interaction differs substantially from that defined for other E2/E3 complexes involving eukaryotic E3-ligases. In vitro, SspH2 directs the synthesis of K48-linked poly-Ub chains, suggesting that cellular protein targets of SspH2-catalyzed Ub transfer are destined for proteasomal destruction. Unexpectedly, we found that intermediates in SspH2-directed reactions are activated poly-Ub chains directly tethered to the UbcH5 active site (UbcH5 ~ Ub(n)). Rapid generation of UbcH5 ~ Ub(n) may allow for bacterially directed modification of eukaryotic target proteins with a completed poly-Ub chain, efficiently tagging host targets for destruction.
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http://dx.doi.org/10.1073/pnas.0914821107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2840284PMC
February 2010

Structural and functional characterization of the monomeric U-box domain from E4B.

Biochemistry 2010 Jan;49(2):347-55

Department of Biochemistry, Vanderbilt University, Nashville,Tennessee 37232, USA.

Substantial evidence has accumulated indicating a significant role for oligomerization in the function of E3 ubiquitin ligases. Among the many characterized E3 ligases, the yeast U-box protein Ufd2 and its mammalian homologue E4B appear to be unique in functioning as monomers. An E4B U-box domain construct (E4BU) has been subcloned, overexpressed in Escherichia coli, and purified, which enabled determination of a high-resolution NMR solution structure and detailed biophysical analysis. E4BU is a stable monomeric protein that folds into the same structure observed for other structurally characterized U-box domain homodimers. Multiple sequence alignment combined with comparative structural analysis reveals substitutions in the sequence that inhibit dimerization. The interaction between E4BU and the E2 conjugating enzyme UbcH5c has been mapped using NMR, and these data have been used to generate a structural model for the complex. The E2 binding site is found to be similar to that observed for dimeric U-box and RING domain E3 ligases. Despite the inability to dimerize, E4BU was found to be active in a standard autoubiquitination assay. The structure of E4BU and its ability to function as a monomer are discussed in light of the ubiquitous observation of U-box and RING domain oligomerization.
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http://dx.doi.org/10.1021/bi901620vDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2806929PMC
January 2010

Solution structure of the cGMP binding GAF domain from phosphodiesterase 5: insights into nucleotide specificity, dimerization, and cGMP-dependent conformational change.

J Biol Chem 2008 Aug 4;283(33):22749-59. Epub 2008 Jun 4.

Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA.

Phosphodiesterase 5 (PDE5) controls intracellular levels of cGMP through its regulation of cGMP hydrolysis. Hydrolytic activity of the C-terminal catalytic domain is increased by cGMP binding to the N-terminal GAF A domain. We present the NMR solution structure of the cGMP-bound PDE5A GAF A domain. The cGMP orientation in the buried binding pocket was defined through 37 intermolecular nuclear Overhauser effects. Comparison with GAF domains from PDE2A and adenylyl cyclase cyaB2 reveals a conserved overall domain fold of a six-stranded beta-sheet and four alpha-helices that form a well defined cGMP binding pocket. However, the nucleotide coordination is distinct with a series of altered binding contacts. The structure suggests that nucleotide binding specificity is provided by Asp-196, which is positioned to form two hydrogen bonds to the guanine ring of cGMP. An alanine mutation of Asp-196 disrupts cGMP binding and increases cAMP affinity in constructs containing only GAF A causing an altered cAMP-bound structural conformation. NMR studies on the tandem GAF domains reveal a flexible GAF A domain in the absence of cGMP, and indicate a large conformational change upon ligand binding. Furthermore, we identify a region of approximately 20 residues directly N-terminal of GAF A as critical for tight dimerization of the tandem GAF domains. The features of the PDE5 regulatory domain revealed here provide an initial structural basis for future investigations of the regulatory mechanism of PDE5 and the design of GAF-specific regulators of PDE5 function.
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http://dx.doi.org/10.1074/jbc.M801577200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2504876PMC
August 2008

Crystal structure of the BARD1 ankyrin repeat domain and its functional consequences.

J Biol Chem 2008 Jul 14;283(30):21179-86. Epub 2008 May 14.

Department of Biochemistry, University of Washington, Seattle, WA 98195-7350, USA.

BARD1 is the constitutive nuclear partner to the breast and ovarian cancer-specific tumor suppressor BRCA1. Together, they form a heterodimeric complex responsible for maintaining genomic stability through nuclear functions involving DNA damage signaling and repair, transcriptional regulation, and cell cycle control. We report the 2.0A structure of the BARD1 ankyrin repeat domain. The structure includes four ankyrin repeats with a non-canonical C-terminal capping ankyrin repeat and a well ordered extended loop preceding the first repeat. Conserved surface features show an acidic patch and an acidic pocket along the surface typically used by ankyrin repeat domains for binding cognate proteins. We also demonstrate that two reported mutations, N470S and V507M, in the ankyrin repeat domain do not result in observable structural defects. These results provide a structural basis for exploring the biological function of the ankyrin repeat domain and for modeling BARD1 isoforms.
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http://dx.doi.org/10.1074/jbc.M802333200DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2475683PMC
July 2008

E2-BRCA1 RING interactions dictate synthesis of mono- or specific polyubiquitin chain linkages.

Nat Struct Mol Biol 2007 Oct 16;14(10):941-8. Epub 2007 Sep 16.

Department of Biochemistry, University of Washington, Box 357350, Seattle, Washington 98195-7350, USA.

An E3 ubiquitin ligase mediates the transfer of activated ubiquitin from an E2 ubiquitin-conjugating enzyme to its substrate lysine residues. Using a structure-based, yeast two-hybrid strategy, we discovered six previously unidentified interactions between the human heterodimeric RING E3 BRCA1-BARD1 and the human E2s UbcH6, Ube2e2, UbcM2, Ubc13, Ube2k and Ube2w. All six E2s bind directly to the BRCA1 RING motif and are active with BRCA1-BARD1 for autoubiquitination in vitro. Four of the E2s direct monoubiquitination of BRCA1. Ubc13-Mms2 and Ube2k direct the synthesis of Lys63- or Lys48-linked ubiquitin chains on BRCA1 and require an acceptor ubiquitin attached to BRCA1. Differences between the mono- and polyubiquitination activities of the BRCA1-interacting E2s correlate with their ability to bind ubiquitin noncovalently at a site distal to the active site. Thus, BRCA1 has the ability to direct the synthesis of specific polyubiquitin chain linkages, depending on the E2 bound to its RING.
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http://dx.doi.org/10.1038/nsmb1295DOI Listing
October 2007

Ubiquitin transfer from the E2 perspective: why is UbcH5 so promiscuous?

Cell Cycle 2006 Dec 8;5(24):2867-73. Epub 2006 Dec 8.

Department of Biochemistry, University of Washington, Seattle, Washington 98195-7742, USA.

Protein ubiquitination is a regulatory process that influences nearly every aspect of eukaryotic cell biology. Pathways that range from cell-cycle progression and differentiation to DNA repair to vesicle budding all rely on regulated modification of target proteins by ubiquitin. Target proteins can be tagged by a single molecule of ubiquitin or modified by ubiquitin polymers that can vary in length and linkage specificity, and these variations influence how ubiquitination signals are interpreted. Surprisingly, little is understood regarding mechanisms of protein ubiquitination and how poly-ubiquitin chains are synthesized. Simple models to explain ubiquitin transfer have dominated the literature, but recent work suggests basic assumptions as to how proteins assemble to facilitate protein ubiquitination and poly-ubiquitin chain synthesis should be reexamined. This is particularly necessary for understanding the roles played by E2 ubiquitin-conjugating enzymes, a central protein component in all ubiquitin transfer reactions. In particular, UbcH5, a canonical E2 protein that is active in a broad number of in vitro ubiquitin transfer reactions, is capable of binding ubiquitin noncovalently on a surface distinct from its active site. This unique property allows activated UbcH5 approximately Ub complexes to self-assemble and has a profound influence on poly-ubiquitin chain synthesis.
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http://dx.doi.org/10.4161/cc.5.24.3592DOI Listing
December 2006

A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination.

Mol Cell 2006 Mar;21(6):873-80

Department of Biochemistry, University of Washington, Seattle, 98195, USA.

Protein ubiquitination is a powerful regulatory modification that influences nearly every aspect of eukaryotic cell biology. The general pathway for ubiquitin (Ub) modification requires the sequential activities of a Ub-activating enzyme (E1), a Ub transfer enzyme (E2), and a Ub ligase (E3). The E2 must recognize both the E1 and a cognate E3 in addition to carrying activated Ub. These central functions are performed by a topologically conserved alpha/beta-fold core domain of approximately 150 residues shared by all E2s. However, as presented herein, the UbcH5 family of E2s can also bind Ub noncovalently on a surface well removed from the E2 active site. We present the solution structure of the UbcH5c/Ub noncovalent complex and demonstrate that this noncovalent interaction permits self-assembly of activated UbcH5c approximately Ub molecules. Self-assembly has profound consequences for the processive formation of polyubiquitin (poly-Ub) chains in ubiquitination reactions directed by the breast and ovarian cancer tumor susceptibility protein BRCA1.
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http://dx.doi.org/10.1016/j.molcel.2006.02.008DOI Listing
March 2006

Binding and recognition in the assembly of an active BRCA1/BARD1 ubiquitin-ligase complex.

Proc Natl Acad Sci U S A 2003 May 5;100(10):5646-51. Epub 2003 May 5.

Department of Biochemistry, University of Washington, Seattle, WA 98195, USA.

BRCA1 is a breast and ovarian cancer tumor suppressor protein that associates with BARD1 to form a RINGRING heterodimer. The BRCA1BARD1 RING complex functions as an ubiquitin (Ub) ligase with activity substantially greater than individual BRCA1 or BARD1 subunits. By using NMR spectroscopy and site-directed mutagenesis, we have mapped the binding site on the BRCA1BARD1 heterodimer for the Ub-conjugating enzyme UbcH5c. The results demonstrate that UbcH5c binds only to the BRCA1 RING domain and not the BARD1 RING. The binding interface is formed by the first and second Zn(2+)-loops and central alpha-helix of the BRCA1 RING domain, a region disrupted by cancer-predisposing mutations. Unexpectedly, a second Ub-conjugating enzyme, UbcH7, also interacts with the BRCA1BARD1 complex with similar affinity, although it is not active in Ub-ligase activity assays. Thus, binding alone is not sufficient for BRCA1-dependent Ub-ligase activity.
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http://dx.doi.org/10.1073/pnas.0836054100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC156255PMC
May 2003