Darcie J. Miller

Insights into Ubiquitin Transfer Cascades from a Structure of a UbcH5B∼Ubiquitin-HECTNEDD4L Complex

In E1-E2-E3 ubiquitin (Ub) conjugation cascades, the E2 first forms a transient E2 approximately Ub covalent complex and then interacts with an E3 for Ub transfer. For cascades involving E3s in the HECT class, Ub is transferred from an associated E2 to the acceptor cysteine in the HECT domain C lobe. To gain insights into this process, we determined the crystal structure of a complex between the HECT domain of NEDD4L and the E2 UbcH5B bearing a covalently linked Ub at its active site (UbcH5B approximately Ub). Noncovalent interactions between UbcH5B and the HECT N lobe and between Ub and the HECT domain C lobe lead to an overall compact structure, with the Ub C terminus sandwiched between UbcH5B and HECT domain active sites. The structure suggests a model for E2-to-HECT Ub transfer, in which interactions between a donor Ub and an acceptor domain constrain upstream and downstream enzymes for conjugation.

Mechanism of ubiquitin ligation and lysine prioritization by a HECT E3

Ubiquitination by HECT E3 enzymes regulates myriad processes, including tumor suppression, transcription, protein trafficking, and degradation. HECT E3s use a two-step mechanism to ligate ubiquitin to target proteins. The first step is guided by interactions between the catalytic HECT domain and the E2∼ubiquitin intermediate, which promote formation of a transient, thioester-bonded HECT∼ubiquitin intermediate. Here we report that the second step of ligation is mediated by a distinct catalytic architecture established by both the HECT E3 and its covalently linked ubiquitin. The structure of a chemically trapped proxy for an E3∼ubiquitin-substrate intermediate reveals three-way interactions between ubiquitin and the bilobal HECT domain orienting the E3∼ubiquitin thioester bond for ligation, and restricting the location of the substrate-binding domain to prioritize target lysines for ubiquitination. The data allow visualization of an E2-to-E3-to-substrate ubiquitin transfer cascade, and show how HECT-specific ubiquitin interactions driving multiple reactions are repurposed by a major E3 conformational change to promote ligation. DOI: http://dx.doi.org/10.7554/eLife.00828.001

Structural basis for the transcriptional regulation of membrane lipid homeostasis.

DesT is a transcriptional repressor that regulates the genes that control the unsaturated:saturated fatty acid ratio available for membrane lipid synthesis. DesT bound to unsaturated acyl-CoA has a high affinity for its cognate palindromic DNA-binding site, whereas DesT bound to saturated acyl-CoA does not bind this site. Structural analyses of the DesT–oleoyl-CoA–DNA and DesT–palmitoyl-CoA complexes reveal that acyl chain shape directly influences the packing of hydrophobic core residues within the DesT ligand-binding domain. These changes are propagated to the paired DNA-binding domains via conformational changes to modulate DNA binding. These structural interpretations are supported by the in vitro and in vivo characterization of site-directed mutants. The regulation of DesT by the unsaturated:saturated ratio of acyl chains rather than the concentration of a single ligand is a paradigm for understanding transcriptional regulation of membrane lipid homeostasis.

Structural Conservation of Distinctive N-terminal Acetylation-Dependent Interactions across a Family of Mammalian NEDD8 Ligation Enzymes

Little is known about molecular recognition of acetylated N termini, despite prevalence of this modification among eukaryotic cytosolic proteins. We report that the family of human DCN-like (DCNL) co-E3s, which promote ligation of the ubiquitin-like protein NEDD8 to cullin targets, recognizes acetylated N termini of the E2 enzymes UBC12 and UBE2F. Systematic biochemical and biophysical analyses reveal 40- and 10-fold variations in affinities among different DCNL-cullin and DCNL-E2 complexes, contributing to varying efficiencies of different NEDD8 ligation cascades. Structures of DCNL2 and DCNL3 complexes with N-terminally acetylated peptides from UBC12 and UBE2F illuminate a common mechanism by which DCNL proteins recognize N-terminally acetylated E2s and how selectivity for interactions dependent on N-acetyl-methionine are established through side chains recognizing distal residues. Distinct preferences of UBC12 and UBE2F peptides for inhibiting different DCNLs, including the oncogenic DCNL1 protein, suggest it may be possible to develop small molecules blocking specific N-acetyl-methionine-dependent protein interactions.

Analysis of the Staphylococcus aureus DgkB structure reveals a common catalytic mechanism for the soluble diacylglycerol kinases.

Soluble diacylglycerol (DAG) kinases function as regulators of diacylglycerol metabolism in cell signaling and intermediary metabolism. We report the structure of a DAG kinase, DgkB from Staphylococcus aureus, both as the free enzyme and in complex with ADP. The molecule is a tight homodimer, and each monomer comprises two domains with the catalytic center located within the interdomain cleft. Two distinctive features of DkgB are a structural Mg2+ site and an associated Asp*water*Mg2+ network that extends toward the active site locale. Site-directed mutagenesis revealed that these features play important roles in the catalytic mechanism. The key active site residues and the components of the Asp*water*Mg2+ network are conserved in the catalytic cores of the mammalian signaling DAG kinases, indicating that these enzymes use the same mechanism and have similar structures as DgkB.

Dual RING E3 Architectures Regulate Multiubiquitination and Ubiquitin Chain Elongation by APC/C.

Protein ubiquitination involves E1, E2, and E3 trienzyme cascades. E2 and RING E3 enzymes often collaborate to first prime a substrate with a single ubiquitin (UB) and then achieve different forms of polyubiquitination: multiubiquitination of several sites and elongation of linkage-specific UB chains. Here, cryo-EM and biochemistry show that the human E3 anaphase-promoting complex/cyclosome (APC/C) and its two partner E2s, UBE2C (aka UBCH10) and UBE2S, adopt specialized catalytic architectures for these two distinct forms of polyubiquitination. The APC/C RING constrains UBE2C proximal to a substrate and simultaneously binds a substrate-linked UB to drive processive multiubiquitination. Alternatively, during UB chain elongation, the RING does not bind UBE2S but rather lures an evolving substrate-linked UB to UBE2S positioned through a cullin interaction to generate a Lys11-linked chain. Our findings define mechanisms of APC/C regulation, and establish principles by which specialized E3-E2-substrate-UB architectures control different forms of polyubiquitination.

How the MccB bacterial ancestor of ubiquitin E1 initiates biosynthesis of the microcin C7 antibiotic

The 39‐kDa Escherichia coli enzyme MccB catalyses a remarkable posttranslational modification of the MccA heptapeptide during the biosynthesis of microcin C7 (MccC7), a ‘Trojan horse’ antibiotic. The approximately 260‐residue C‐terminal region of MccB is homologous to ubiquitin‐like protein (UBL) activating enzyme (E1) adenylation domains. Accordingly, MccB‐catalysed C‐terminal MccA‐acyl‐adenylation is reminiscent of the E1‐catalysed activation reaction. However, unlike E1 substrates, which are UBLs with a C‐terminal di‐glycine sequence, MccBs substrate, MccA, is a short peptide with an essential C‐terminal Asn. Furthermore, after an intramolecular rearrangement of MccA‐acyl‐adenylate, MccB catalyses a second, unique reaction, producing a stable phosphoramidate‐linked analogue of acyl‐adenylated aspartic acid. We report six‐crystal structures of MccB in apo, substrate‐, intermediate‐, and inhibitor‐bound forms. Structural and kinetic analyses reveal a novel‐peptide clamping mechanism for MccB binding to heptapeptide substrates and a dynamic‐active site for catalysing dual adenosine triphosphate‐consuming reactions. The results provide insight into how a distinctive member of the E1 superfamily carries out two‐step activation for generating the peptidyl‐antibiotic MccC7.

Crystal Structure of UBA2ufd-Ubc9: Insights into E1-E2 Interactions in Sumo Pathways

Canonical ubiquitin-like proteins (UBLs) such as ubiquitin, Sumo, NEDD8, and ISG15 are ligated to targets by E1-E2-E3 multienzyme cascades. The Sumo cascade, conserved among all eukaryotes, regulates numerous biological processes including protein localization, transcription, DNA replication, and mitosis. Sumo conjugation is initiated by the heterodimeric Aos1-Uba2 E1 enzyme (in humans called Sae1-Uba2), which activates Sumos C-terminus, binds the dedicated E2 enzyme Ubc9, and promotes Sumo C-terminal transfer between the Uba2 and Ubc9 catalytic cysteines. To gain insights into details of E1-E2 interactions in the Sumo pathway, we determined crystal structures of the C-terminal ubiquitin fold domain (ufd) from yeast Uba2 (Uba2ufd), alone and in complex with Ubc9. The overall structures of both yeast Uba2ufd and Ubc9 superimpose well on their individual human counterparts, suggesting conservation of fundamental features of Sumo conjugation. Docking the Uba2ufd-Ubc9 and prior full-length human Uba2 structures allows generation of models for steps in Sumo transfer from Uba2 to Ubc9, and supports the notion that Uba2 undergoes remarkable conformational changes during the reaction. Comparisons to previous structures from the NEDD8 cascade demonstrate that UBL cascades generally utilize some parallel E1-E2 interaction surfaces. In addition, the structure of the Uba2ufd-Ubc9 complex reveals interactions unique to Sumo E1 and E2. Comparison with a previous Ubc9-E3 complex structure demonstrates overlap between Uba2 and E3 binding sites on Ubc9, indicating that loading with Sumo and E3-catalyzed transfer to substrates are strictly separate steps. The results suggest mechanisms establishing specificity and order in Sumo conjugation cascades.

Structure of RhlG, an Essential β-Ketoacyl Reductase in the Rhamnolipid Biosynthetic Pathway of Pseudomonas aeruginosa

Rhamnolipids are extracellular biosurfactants and virulence factors secreted by the opportunistic human pathogen Pseudomonas aeruginosa that are required for swarming motility. The rhlG gene is essential for rhamnolipid formation, and the RhlG enzyme is thought to divert fatty acid synthesis intermediates into the rhamnolipid biosynthetic pathway based on its similarity to FabG, the β-ketoacyl-acyl carrier protein (ACP) reductase of type II fatty acid synthesis. Crystallographic analysis reveals that the overall structures of the RhlG·NADP+ and FabG·NADP+ complexes are indeed similar, but there are key differences related to function. RhlG does not undergo the conformational changes upon NADP(H) binding at the active site that in FabG are the structural basis of negative allostery. Also, the acyl chain-binding pocket of RhlG is narrow and rigid compared with the larger, flexible substrate-binding subdomain in FabG. Finally, RhlG lacks a positively charged/hydrophobic surface feature adjacent to the active site that is found on enzymes like FabG that recognize the ACP of fatty acid synthesis. RhlG catalyzed the NADPH-dependent reduction of β-ketodecanoyl-ACP to β-d-hydroxydecanoyl-ACP. However, the enzyme was 2000-fold less active than FabG in carrying out the same reaction. These structural and biochemical studies establish RhlG as a NADPH-dependent β-ketoacyl reductase of the SDR protein superfamily and further suggest that the ACP of fatty acid synthesis does not carry the substrates for RhlG.

Structural Studies of HHARI/UbcH7∼Ub Reveal Unique E2∼Ub Conformational Restriction by RBR RING1

Summary RING-between-RING (RBR) E3s contain RING1 domains that are structurally similar yet mechanistically distinct from canonical RING domains. Both types of E3 bind E2∼ubiquitin (E2∼Ub) via their RINGs but canonical RING E3s promote closed E2∼Ub conformations required for direct Ub transfer from the E2 to substrate, while RBR RING1s promote open E2∼Ub to favor Ub transfer to the E3 active site. This different RING/E2∼Ub conformation determines its direct target, which for canonical RING E3s is typically a substrate or substrate-linked Ub, but is the E3 active-site cysteine in the case of RBR-type E3s. Here we show that a short extension of HHARI RING1, namely Zn2+-loop II, not present in any RING E3s, acts as a steric wedge to disrupt closed E2∼Ub, providing a structural explanation for the distinctive RING1-dependent conformational restriction mechanism utilized by RBR E3s.