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1.  SUMO Chain-Induced Dimerization Activates RNF4 
Molecular Cell  2014;53(6):880-892.
Summary
Dimeric RING E3 ligases interact with protein substrates and conformationally restrain the ubiquitin-E2-conjugating enzyme thioester complex such that it is primed for catalysis. RNF4 is an E3 ligase containing an N-terminal domain that binds its polySUMO substrates and a C-terminal RING domain responsible for dimerization. To investigate how RNF4 activity is controlled, we increased polySUMO substrate concentration by ablating expression of SUMO protease SENP6. Accumulation of SUMO chains in vivo leads to ubiquitin-mediated proteolysis of RNF4. In vitro we demonstrate that at concentrations equivalent to those found in vivo RNF4 is predominantly monomeric and inactive as an ubiquitin E3 ligase. However, in the presence of SUMO chains, RNF4 is activated by dimerization, leading to both substrate ubiquitylation and autoubiquitylation, responsible for degradation of RNF4. Thus the ubiquitin E3 ligase activity of RNF4 is directly linked to the availability of its polySUMO substrates.
Graphical Abstract
Highlights
•Characterization of the mechanism of activation of the ubiquitin E3 ligase RNF4•First indication of E3 ligase activation by SUMO chains
RNF4 is an E3 ligase containing an N-terminal domain that binds its polySUMO substrates and a C-terminal RING domain responsible for dimerization. Rojas-Fernandez et al. show that in the presence of SUMO chains RNF4 is activated by dimerization, leading to both substrate ubiquitylation and autoubiquitylation.
doi:10.1016/j.molcel.2014.02.031
PMCID: PMC3991395  PMID: 24656128
2.  Ube2W conjugates ubiquitin to α-amino groups of protein N-termini 
Biochemical Journal  2013;453(Pt 1):137-145.
The covalent attachment of the protein ubiquitin to intracellular proteins by a process known as ubiquitylation regulates almost all major cellular systems, predominantly by regulating protein turnover. Ubiquitylation requires the co-ordinated action of three enzymes termed E1, E2 and E3, and typically results in the formation of an isopeptide bond between the C-terminal carboxy group of ubiquitin and the ϵ-amino group of a target lysine residue. However, ubiquitin is also known to conjugate to the thiol of cysteine residue side chains and the α-amino group of protein N-termini, although the enzymes responsible for discrimination between different chemical groups have not been defined. In the present study, we show that Ube2W (Ubc16) is an E2 ubiquitin-conjugating enzyme with specific protein N-terminal mono-ubiquitylation activity. Ube2W conjugates ubiquitin not only to its own N-terminus, but also to that of the small ubiquitin-like modifier SUMO (small ubiquitin-related modifier) in a manner dependent on the SUMO-targeted ubiquitin ligase RNF4 (RING finger protein 4). Furthermore, N-terminal mono-ubiquitylation of SUMO-2 primes it for poly-ubiquitylation by the Ubc13–UEV1 (ubiquitin-conjugating enzyme E2 variant 1) heterodimer, showing that N-terminal ubiquitylation regulates protein fate. The description in the present study is the first of an E2-conjugating enzyme with N-terminal ubiquitylation activity, and highlights the importance of E2 enzymes in the ultimate outcome of E3-mediated ubiquitylation.
doi:10.1042/BJ20130244
PMCID: PMC3778709  PMID: 23560854
E2 ubiquitin-conjugating enzyme; N-terminal modification; RNF4 (RING finger protein 4); SUMO (small ubiquitin-related modifier); ubiquitin; ubiquitin-conjugating enzyme E2W (Ube2W); CHIP, C-terminus of the Hsc (heat-shock cognate) 70-interacting protein; E1 enzyme, ubiquitin-activating enzyme; E2 enzyme, ubiquitin-conjugating enzyme; HCD, higher energy collisional dissociation; Isopep.6His-SUMO-2×4, isopeptide bond-linked His6-polySUMO-2 construct; Isopep.SUMO-2×4, isopeptide bond-linked polySUMO-2 construct; Pep.6His-SUMO-2×4, peptide bond-linked His6-polySUMO-2 construct; RNF4, RING finger protein 4; SENP1, sentrin-specific protease 1; SUMO, small ubiquitin-related modifier; TEV, tobacco etch virus; Ube2W, ubiquitin-conjugating enzyme E2W; UEV1, ubiquitin-conjugating enzyme E2 variant 1
3.  Structure of a RING E3 ligase and ubiquitin-loaded E2 primed for catalysis 
Nature  2012;489(7414):115-120.
SUMMARY
Ubiquitin modification is mediated by a large family of specificity determining ubiquitin E3 ligases. To facilitate ubiquitin transfer, RING E3 ligases bind both substrate and a ubiquitin E2 conjugating enzyme linked to ubiquitin via a thioester bond, but the mechanism of transfer has remained elusive. Here we report the crystal structure of the dimeric RING of RNF4 in complex with E2 (UbcH5a) linked by an isopeptide bond to ubiquitin. While the E2 contacts a single protomer of the RING, ubiquitin is folded back onto the E2 by contacts from both RING protomers. The C-terminal tail of ubiquitin is locked into an active site groove on the E2 by an intricate network of interactions, resulting in changes at the E2 active site. This arrangement is primed for catalysis as it can deprotonate the incoming substrate lysine residue and stabilise the consequent tetrahedral transition state intermediate.
doi:10.1038/nature11376
PMCID: PMC3442243  PMID: 22842904
4.  Novel substrate-based inhibitors of human glutamate carboxypeptidase II with enhanced lipophilicity 
Journal of medicinal chemistry  2011;54(21):7535-7546.
Virtually all low molecular weight inhibitors of human glutamate carboxypeptidase II (GCPII) are highly polar compounds that have limited use in settings where more lipophilic molecules are desired. Here we report the identification and characterization of GCPII inhibitors with enhanced liphophilicity that are derived from a series of newly identified dipeptidic GCPII substrates featuring non-polar aliphatic side chains at the C-terminus. To analyze the interactions governing the substrate recognition by GCPII, we determined crystal structures of the inactive GCPII(E424A) mutant in complex with selected dipeptides and complemented the structural data with quantum mechanics/molecular mechanics calculations. Results reveal the importance of non-polar interactions governing GCPII affinity towards novel substrates as well as formerly unnoticed plasticity of the S1′ specificity pocket. Based on those data, we designed, synthesized and evaluated a series of novel GCPII inhibitors with enhanced lipophilicity, with the best candidates having low nanomolar inhibition constants and clogD > -0.3. Our findings offer new insights into the design of more lipophilic inhibitors targeting GCPII.
doi:10.1021/jm200807m
PMCID: PMC3222833  PMID: 21923190
PSMA; NAALADase; GCPII; zinc peptidase; folate hydrolase; inhibition; quantum mechanics/molecular mechanics (QM/MM)
5.  Mechanism of ubiquitylation by dimeric RING ligase RNF4 
Mammalian RNF4 is a dimeric RING ubiquitin E3 ligase that ubiquitylates poly-SUMOylated proteins. We found that RNF4 bound ubiquitin-charged UbcH5a tightly but free UbcH5a weakly. To provide insight into the mechanism of RING-mediated ubiquitylation we docked the UbcH5~ubiquitin thioester onto the RNF4 RING structure. This revealed that with E2 bound to one monomer of RNF4, the thioester-linked ubiquitin could reach across the dimer to engage the other monomer. In this model the “Ile44 hydrophobic patch” of ubiquitin is predicted to engage a conserved tyrosine located at the dimer interface of the RING and mutation of these residues blocked ubiquitylation activity. Thus, dimeric RING ligases are not simply inert scaffolds that bring substrate and E2-loaded ubiquitin into close proximity. Instead, they facilitate ubiquitin transfer by preferentially binding the E2~ubiquitin thioester across the dimer and activating the thioester bond for catalysis.
doi:10.1038/nsmb.2108
PMCID: PMC3326525  PMID: 21857666
6.  Glycosylation Directs Targeting and Activation of Cystatin F from Intracellular and Extracellular Sources 
Traffic (Copenhagen, Denmark)  2009;10(4):425-437.
Cystatin F is a cysteine protease inhibitor that is selectively expressed in immune cells and unlike other cystatin family members is targeted to a significant extent to intracellular compartments. Initially made as an inactive glycosylated disulfide-linked dimer, cystatin F is converted to an active monomer by proteolytic cleavage following transport to the endosomal/lysosomal system. This active form of cystatin F targets cathepsin C/DPPI and probably other cathepsins in immune cells. We show that efficient targeting of cystatin F to the endocytic pathway is dependent not on its unique dimeric conformation but rather on its oligosaccharide chains. We demonstrate the unusual addition of N-linked sugars to an Asn-X-Cys motif in cystatin F and provide evidence that the mannose 6-phosphate sorting machinery is used to divert cystatin F from the secretory pathway and to mediate its uptake from extracellular pools. These studies identify a function for the oligosaccharides on cystatin F and raise the possibility that cystatin F might regulate proteases in transby secretion in an inactive form by one cell and subsequent internalization and activation by another cell.
doi:10.1111/j.1600-0854.2009.00881.x
PMCID: PMC2691902  PMID: 19192250
cathepsin C; cystatin; endocytosis/internalization; glycosylation; mannose 6-phosphate
7.  Structural insight into SUMO chain recognition and manipulation by the ubiquitin ligase RNF4 
Nature Communications  2014;5:4217.
The small ubiquitin-like modifier (SUMO) can form polymeric chains that are important signals in cellular processes such as meiosis, genome maintenance and stress response. The SUMO-targeted ubiquitin ligase RNF4 engages with SUMO chains on linked substrates and catalyses their ubiquitination, which targets substrates for proteasomal degradation. Here we use a segmental labelling approach combined with solution nuclear magnetic resonance (NMR) spectroscopy and biochemical characterization to reveal how RNF4 manipulates the conformation of the SUMO chain, thereby facilitating optimal delivery of the distal SUMO domain for ubiquitin transfer.
SUMO forms flexible polymeric chains that can interact with ubiquitin ligases, such as RNF4. Here Xu et al. have used NMR spectroscopy and biochemical experiments to investigate the interaction between SUMO and RNF4, and propose a mechanism for delivery of substrates to the ubiquitination machinery.
doi:10.1038/ncomms5217
PMCID: PMC4083429  PMID: 24969970

Results 1-7 (7)