Donald E. Spratt

RBR E3 ubiquitin ligases: new structures, new insights, new questions

The RBR (RING-BetweenRING-RING) or TRIAD [two RING fingers and a DRIL (double RING finger linked)] E3 ubiquitin ligases comprise a group of 12 complex multidomain enzymes. This unique family of E3 ligases includes parkin, whose dysfunction is linked to the pathogenesis of early-onset Parkinsons disease, and HOIP (HOIL-1-interacting protein) and HOIL-1 (haem-oxidized IRP2 ubiquitin ligase 1), members of the LUBAC (linear ubiquitin chain assembly complex). The RBR E3 ligases share common features with both the larger RING and HECT (homologous with E6-associated protein C-terminus) E3 ligase families, directly catalysing ubiquitin transfer from an intrinsic catalytic cysteine housed in the C-terminal domain, as well as recruiting thioester-bound E2 enzymes via a RING domain. Recent three-dimensional structures and biochemical findings of the RBRs have revealed novel protein domain folds not previously envisioned and some surprising modes of regulation that have raised many questions. This has required renaming two of the domains in the RBR E3 ligases to more accurately reflect their structures and functions: the C-terminal Rcat (required-for-catalysis) domain, essential for catalytic activity, and a central BRcat (benign-catalytic) domain that adopts the same fold as the Rcat, but lacks a catalytic cysteine residue and ubiquitination activity. The present review discusses how three-dimensional structures of RBR (RING1-BRcat-Rcat) E3 ligases have provided new insights into our understanding of the biochemical mechanisms of these important enzymes in ubiquitin biology.

A molecular explanation for the recessive nature of parkin -linked Parkinson’s disease

Mutations in the park2 gene, encoding the RING-inBetweenRING-RING E3 ubiquitin ligase parkin, cause 50% of autosomal recessive juvenile Parkinsonism cases. More than 70 known pathogenic mutations occur throughout parkin, many of which cluster in the inhibitory amino-terminal ubiquitin-like domain, and the carboxy-terminal RING2 domain that is indispensable for ubiquitin transfer. A structural rationale showing how autosomal recessive juvenile Parkinsonism mutations alter parkin function is still lacking. Here we show that the structure of parkin RING2 is distinct from canonical RING E3 ligases and lacks key elements required for E2-conjugating enzyme recruitment. Several pathogenic mutations in RING2 alter the environment of a single surface-exposed catalytic cysteine to inhibit ubiquitination. Native parkin adopts a globular inhibited conformation in solution facilitated by the association of the ubiquitin-like domain with the RING-inBetweenRING-RING C-terminus. Autosomal recessive juvenile Parkinsonism mutations disrupt this conformation. Finally, parkin autoubiquitinates only in cis, providing a molecular explanation for the recessive nature of autosomal recessive juvenile Parkinsonism.

Disruption of the autoinhibited state primes the E3 ligase parkin for activation and catalysis

The PARK2 gene is mutated in 50% of autosomal recessive juvenile parkinsonism (ARJP) cases. It encodes parkin, an E3 ubiquitin ligase of the RBR family. Parkin exists in an autoinhibited state that is activated by phosphorylation of its N‐terminal ubiquitin‐like (Ubl) domain and binding of phosphoubiquitin. We describe the 1.8 Å crystal structure of human parkin in its fully inhibited state and identify the key interfaces to maintain parkin inhibition. We identify the phosphoubiquitin‐binding interface, provide a model for the phosphoubiquitin–parkin complex and show how phosphorylation of the Ubl domain primes parkin for optimal phosphoubiquitin binding. Furthermore, we demonstrate that the addition of phosphoubiquitin leads to displacement of the Ubl domain through loss of structure, unveiling a ubiquitin‐binding site used by the E2~Ub conjugate, thus leading to active parkin. We find the role of the Ubl domain is to prevent parkin activity in the absence of the phosphorylation signals, and propose a model for parkin inhibition, optimization for phosphoubiquitin recruitment, release of inhibition by the Ubl domain and engagement with an E2~Ub conjugate. Taken together, this model provides a mechanistic framework for activating parkin.

Binding and activation of nitric oxide synthase isozymes by calmodulin EF hand pairs

Calmodulin (CaM) is a cytosolic Ca2+ signal‐transducing protein that binds and activates many different cellular enzymes with physiological relevance, including the nitric oxide synthase (NOS) isozymes. CaM consists of two globular domains joined by a central linker; each domain contains an EF hand pair. Four different mutant CaM proteins were used to investigate the role of the two CaM EF hand pairs in the binding and activation of the mammalian inducible NOS (iNOS) and the constitutive NOS (cNOS) enzymes, endothelial NOS (eNOS) and neuronal NOS (nNOS). The role of the CaM EF hand pairs in different aspects of NOS enzymatic function was monitored using three assays that monitor electron transfer within a NOS homodimer. Gel filtration studies were used to determine the effect of Ca2+ on the dimerization of iNOS when coexpressed with CaM and the mutant CaM proteins. Gel mobility shift assays were performed to determine binding stoichiometries of CaM proteins to synthetic NOS CaM‐binding domain peptides. Our results show that the N‐terminal EF hand pair of CaM contains important binding and activating elements for iNOS, whereas the N‐terminal EF hand pair in conjunction with the central linker region is required for cNOS enzyme binding and activation. The iNOS enzyme must be coexpressed with wild‐type CaM in vitro because of its propensity to aggregate when residues of the highly hydrophobic CaM‐binding domain are exposed to an aqueous environment. A possible role for iNOS aggregation in vivo is also discussed.

Selective Recruitment of an E2∼Ubiquitin Complex by an E3 Ubiquitin Ligase

Background: Rbx1/ROC1 is an E3 ligase adaptor protein that functions with the E2 enzyme CDC34. Results: NMR and biochemical data show that Rbx1/ROC1 binds CDC34∼ubiquitin 50-fold tighter than CDC34. Conclusion: Rbx1/ROC1 selectively recruits E2∼ubiquitin and releases the E2 after ubiquitin transfer. Significance: Direct evidence is shown for preferential recognition of an E2∼ubiquitin complex by an E3 ligase. RING E3 ligases are proteins that must selectively recruit an E2-conjugating enzyme and facilitate ubiquitin transfer to a substrate. It is not clear how a RING E3 ligase differentiates a naked E2 enzyme from the E2∼ubiquitin-conjugated form or how this is altered upon ubiquitin transfer. RING-box protein 1 (Rbx1/ROC1) is a key protein found in the Skp1/Cullin-1/F-box (SCF) E3 ubiquitin ligase complex that functions with the E2 ubiquitin conjugating enzyme CDC34. The solution structure of Rbx1/ROC1 revealed a globular RING domain (residues 40–108) stabilized by three structural zinc ions (root mean square deviation 0.30 ± 0.04 Å) along with a disordered N terminus (residues 12–39). Titration data showed that Rbx1/ROC1 preferentially recruits CDC34 in its ubiquitin-conjugated form and favors this interaction by 50-fold compared with unconjugated CDC34. Furthermore, NMR and biochemical assays identified residues in helix α2 of Rbx1/ROC1 that are essential for binding and activating CDC34∼ubiquitin for ubiquitylation. Taken together, this work provides the first direct structural and biochemical evidence showing that polyubiquitylation by the RING E3 ligase Rbx1/ROC1 requires the preferential recruitment of an E2∼ubiquitin complex and subsequent release of the unconjugated E2 protein upon ubiquitin transfer to a substrate or ubiquitin chain.

Structure and Dynamics of Calmodulin (CaM) Bound to Nitric Oxide Synthase Peptides: Effects of a Phosphomimetic CaM Mutation.

Nitric oxide synthase (NOS) plays a major role in a number of key physiological and pathological processes. Knowledge of how this is regulated is important. The small acidic calcium binding protein, calmodulin (CaM), is required to fully activate the enzyme. The exact mechanism of how CaM activates NOS is not fully understood. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the transfer of an electron between the reductase and oxygenase domains through a process that is thought to be highly dynamic. To investigate the dynamic properties of CaM-NOS interactions, we determined the solution structure of CaM bound to the inducible NOS (iNOS) and endothelial NOS (eNOS) CaM binding region peptides. In addition, we investigated the effect of CaM phosphorylation. Tyrosine 99 (Y99) of CaM is reported to be phosphorylated in vivo. We have produced a phosphomimetic Y99E CaM to investigate the structural and functional effects that the phosphorylation of this residue may have on nitric oxide production. All three mammalian NOS isoforms were included in the investigation. Our results show that a phosphomimetic Y99E CaM significantly reduces the maximal synthase activity of eNOS by 40% while having little effect on nNOS or iNOS activity. A comparative nuclear magnetic resonance study between phosphomimetic Y99E CaM and wild-type CaM bound to the eNOS CaM binding region peptide was performed. This investigation provides important insights into how the increased electronegativity of a phosphorylated CaM protein affects the binding, dynamics, and activation of the NOS enzymes.

Generation of phospho-ubiquitin variants by orthogonal translation reveals codon skipping

The activity of the Parkinsons disease‐linked E3 ligase parkin is stimulated by phosphorylation at ubiquitin Ser65 (pUbS65). The role of other ubiquitin phospho‐sites and their kinases are unknown. We produced pUb variants (pS7, pS12, pS20, pS57, pS65) by genetically encoding phosphoserine with the UAG codon. In release factor‐deficient Escherichia coli (ΔRF1), intended to enhance UAG read‐through, we discovered ubiquitin variants lacking the UAG‐encoded residue, demonstrating previously undocumented +3 frame shifting. We successfully purified each pUb variant from mistranslated products. While pUbS20 failed to stimulate parkin, parkin was partially active with pUbS12. We observed significant ubiquitination when pUbS65 was the sole substrate.

Association of the Disordered C-terminus of CDC34 with a Catalytically Bound Ubiquitin

Cell division cycle protein 34 (CDC34) is a key E2 ubiquitin (Ub)-conjugating enzyme responsible for the polyubiquitination of proteins controlling the G1/S stages of cell division. The acidic C-terminus of the enzyme is required for this function, although there is little structural information providing details for a mechanism. One logical time point involving the C-terminus is the CDC34-Ub thiolester complex that precedes Ub transfer to a substrate. To examine this, we used a CDC34-Ub disulfide complex that structurally mimics the thiolester intermediate. NMR spectroscopy was used to show that the CDC34 C-terminus is disordered but can intramolecularly interact with the catalytically bound Ub. Using chemical shift perturbation analysis, we mapped two interacting regions on the surface of Ub in the CDC34-Ub complex. The first site comprises a hydrophobic patch (typical of other Ub complexes) that associates with the CDC34 catalytic domain. A novel second site, dependent on the C-terminus of CDC34, comprises a lysine-rich surface (K6, K11, K29, and K33) on the opposite face of Ub. Further, NMR experiments show that this interaction is described by two slowly exchanging states-a compact conformation where the C-terminus of CDC34 interacts with bound Ub and an extended structure where the C-terminus is released. This work provides the first structural details that show how the C-terminus of CDC34 might direct a thiolester-bound Ub to control polyubiquitin chain formation.

Codon Optimization for Enhanced Escherichia Coli Expression of Human S100A11 and S100A1 Proteins

The cloning, expression and purification for the recombinant full-length human proteins S100A11 and human S100A1 is described. The genes were synthesized by overlapping complementary single-stranded oligonucleotides of various lengths. The coding sequence for both genes were codon optimized by selecting only the most preferential codons according to the Escherichia coli bias. In order to assemble the various oligonucleotides into the correct full-length genes, a unique one-step PCR procedure was implemented. The expression and purification procedures were also optimized for each protein. A single phenyl-Sepharose column was sufficient for the purification of human S100A11 whereas HiTrap Q anion exchange followed by phenyl-Sepharose columns were required for the purification of S100A1. By optimizing the S100A1 and S100A11 gene, expression and purification protocols, more than 45 and 150mg, respectively of the purified human proteins were obtained per litre of media. Protein identity was verified by both SDS-PAGE and mass spectrometry (MS) and further characterized by NMR spectroscopy. These results have established an efficient method for the expression and purification of large quantities of human S100A1 and S100A11 proteins for biophysical characterization.

Suramin inhibits cullin-RING E3 ubiquitin ligases.

Significance Interactions between E2 and E3 enzymes are key for ubiquitination, but whether such a dynamic association is susceptible to perturbation by small-molecule modulators remains elusive. By demonstrating that suramin can inhibit cullin-RING E3 ubiquitin ligase by disrupting its ability to recruit E2 Cdc34, this work suggests that the E2–E3 interface may be druggable. In addition, suramin is an antitrypansomal drug that also possesses antitumor activity. Our findings have linked the ubiquitin-proteasome pathway to suramin and suggest additional biochemical mode of action for this century-old drug. Cullin-RING E3 ubiquitin ligases (CRL) control a myriad of biological processes by directing numerous protein substrates for proteasomal degradation. Key to CRL activity is the recruitment of the E2 ubiquitin-conjugating enzyme Cdc34 through electrostatic interactions between E3′s cullin conserved basic canyon and the acidic C terminus of the E2 enzyme. This report demonstrates that a small-molecule compound, suramin, can inhibit CRL activity by disrupting its ability to recruit Cdc34. Suramin, an antitrypansomal drug that also possesses antitumor activity, was identified here through a fluorescence-based high-throughput screen as an inhibitor of ubiquitination. Suramin was shown to target cullin 1’s conserved basic canyon and to block its binding to Cdc34. Suramin inhibits the activity of a variety of CRL complexes containing cullin 2, 3, and 4A. When introduced into cells, suramin induced accumulation of CRL substrates. These observations help develop a strategy of regulating ubiquitination by targeting an E2–E3 interface through small-molecule modulators.