Development of an OTUD1 ubiquitin variant inhibitor.

OTUD1 (Ovarian tumor domain-containing deubiquitinase 1) is a member of the OTU domain-containing deubiquitinase family of enzymes involved in immunoregulation and tumorigenesis pathways. OTUD1 consists of three distinct regions: an unstructured N-terminal region, an OTU-fold catalytic domain, and a ubiquitin-interacting motif (UIM) containing region. Enhanced enzymatic activity and a strong preference for K63-linked substrates are imparted by the UIM containing region. We used phage display with a ubiquitin variant (UbV) library to identify binders for OTUD1 lacking the unstructured N-terminal region (OTUD1OTU + UIM) in an attempt to identify inhibitors bridging the catalytic domain and the UIM containing region. Two UbVs were identified (UbVOD.1 and UbVOD.2) with high affinity and specificity for OTUD1. Of the UbVs identified, UbVOD.1 inhibited OTUD1 activity towards mono-Ub and K63-linked di-Ub substrates in vitro with single-digit nanomolar IC50 and potently inhibited deubiquitinase activity with poly-Ub chains of other linkages. In vivo expression of UbVOD.1 alone was unstable, however as a di-UbV, global deubiquitination and deubiquitinase activity with the OTUD1 substrate RIPK1 were inhibited. Herein we describe the development of molecular tools for exploring the activity of OTUD1 in a cellular context, towards protein-based therapeutics.

Targeted Degradation of 53BP1 Using Ubiquitin Variant Induced Proximity.

In recent years, researchers have leveraged the ubiquitin-proteasome system (UPS) to induce selective degradation of proteins by E3 ubiquitin ligases, which has great potential as novel therapeutics for human diseases, including cancer and neurodegenerative disorders. However, despite extensive efforts, only a handful of ~600 human E3 ligases were utilized, and numerous protein-protein interaction surfaces on E3 ligases were not explored. To tackle these problems, we leveraged a structure-based protein engineering technology to develop a multi-domain fusion protein bringing functional E3 ligases to the proximity of a target protein to trigger its proteasomal degradation, which we termed Ubiquitin Variant Induced Proximity (UbVIP). We first generated non-inhibitory synthetic UbV binders for a selected group of human E3 ligases. With these UbVs employed as E3 ligase engagers, we designed a library of UbVIPs targeting a DNA damage response protein 53BP1. We observed that two UbVIPs recruiting RFWD3 and NEDD4L could effectively induce proteasome degradation of 53BP1 in human cell lines. This provides a proof-of-principle that UbVs can act as a means of targeted degradation for nucleus-localized proteins. Our work demonstrated that UbV technology is suitable to develop protein-based molecules for targeted degradation and can help identify novel E3 ligases for future therapeutic development.

Structural and functional characterization of ubiquitin variant inhibitors for the JAMM-family deubiquitinases STAMBP and STAMBPL1.

Ubiquitination is a crucial posttranslational protein modification involved in a myriad of biological pathways. This modification is reversed by deubiquitinases (DUBs) that deconjugate the single ubiquitin (Ub) moiety or poly-Ub chains from substrates. In the past decade, tremendous efforts have been focused on targeting DUBs for drug discovery. However, most chemical compounds with inhibitory activity for DUBs suffer from mild potency and low selectivity. To overcome these obstacles, we developed a phage display-based protein engineering strategy for generating Ub variant (UbV) inhibitors, which was previously successfully applied to the Ub-specific protease (USP) family of cysteine proteases. In this work, we leveraged the UbV platform to selectively target STAMBP, a member of the JAB1/MPN/MOV34 (JAMM) metalloprotease family of DUB enzymes. We identified two UbVs (UbVSP.1 and UbVSP.3) that bind to STAMBP with high affinity but differ in their selectivity for the closely related paralog STAMBPL1. We determined the STAMBPL1-UbVSP.1 complex structure by X-ray crystallography, revealing hotspots of the JAMM-UbV interaction. Finally, we show that UbVSP.1 and UbVSP.3 are potent inhibitors of STAMBP isopeptidase activity, far exceeding the reported small-molecule inhibitor BC-1471. This work demonstrates that UbV technology is suitable to develop molecules as tools to target metalloproteases, which can be used to further understand the cellular function of JAMM family DUBs.

Targeted modulation of E3 ligases using engineered ubiquitin variants.

Ubiquitination plays an essential role in signal transduction to regulate most if not all cellular processes. Among the enzymes that are involved in the ubiquitin (Ub) signaling cascade, tremendous efforts have been focused on elucidating the roles of E3 Ub ligases as they determine the complexity and specificity of ubiquitination. Not surprisingly, the malfunction of E3 ligases is directly implicated in many human diseases, including cancer. Therefore, there is an urgent need to develop potent and specific molecules to modulate E3 ligase activity as intracellular probes for target validation and as pharmacological agents in preclinical research. Unfortunately, the progress has been hampered by the dynamic regulation mechanisms for different types of E3 ligases. Here, we summarize the progress of using protein engineering to develop Ub variant (UbV) inhibitors for all major families of E3 ligases and UbV activators for homologous with E6-associated protein C terminus E3s and homodimeric RING E3s. We believe that this provides a general strategy and a valuable toolkit for the research community to inhibit or activate E3 ligases and these synthetic molecules have important implications in exploring protein degradation for drug discovery.

A Key Glycine in Bacterial Steroid-Degrading Acyl-CoA Dehydrogenases Allows Flavin-Ring Repositioning and Modulates Substrate Side Chain Specificity.

A wide variety of steroid metabolites synthesized by eukaryotes are all ultimately catabolized by bacteria; while generally saprophytic, pathogenic Mycobacteria have repurposed these pathways to utilize host intracellular cholesterol pools. Steroid degradation is complex, but a recurring theme is that cycles of ß-oxidation are used to iteratively remove acetyl- or propanoyl-CoA groups. These ß-oxidation cycles are initiated by the FAD-dependent oxidation of acyl groups, catalyzed by acyl-CoA dehydrogenases (ACADs). We show here that the tcur3481 and tcur3483 genes of Thermomonospora curvata encode subunits of a single ACAD that degrades steroid side chains with a preference for three-carbon over five-carbon substituents. The structure confirms that this enzyme is heterotetrameric, with active sites only in the Tcur3483 subunits. In comparison with the steroid ACAD FadE26-FadE27 from Mycobacterium tuberculosis, the active site is narrower and closed at the steroid-binding end, suggesting that Tcur3481-Tcur3483 is in a catalytically productive state, while FadE26-FadE27 is opened up to allow substrate entry. The flavin rings in Tcur3481-Tcur3483 sit in an unusual pocket created by Gly363, a residue conserved as Ala in steroid ACADs narrowly specific for five-carbon side chains, including FadE34. A Gly363Ala variant of Tcur3481-Tcur3483 prefers five-carbon side chains, while an inverse Ala691Gly FadE34 variant enables three-carbon side chain steroid oxidation. We determined the structure of the Tcur3483 Gly363Ala variant, showing that the flavin rings shift into the more conventional position. Modeling suggests that the shifted flavin position made possible by Gly363 is required to allow the bulky, inflexible three-carbon steroid to bind productively in the active site.

A bifunctional O-antigen polymerase structure reveals a new glycosyltransferase family.

Lipopolysaccharide O-antigen is an attractive candidate for immunotherapeutic strategies targeting antibiotic-resistant Klebsiella pneumoniae. Several K. pneumoniae O-serotypes are based on a shared O2a-antigen backbone repeating unit: (→ 3)-α-Galp-(1 → 3)-ß-Galf-(1 →). O2a antigen is synthesized on undecaprenol diphosphate in a pathway involving the O2a polymerase, WbbM, before its export by an ATP-binding cassette transporter. This dual domain polymerase possesses a C-terminal galactopyranosyltransferase resembling known GT8 family enzymes, and an N-terminal DUF4422 domain identified here as a galactofuranosyltransferase defining a previously unrecognized family (GT111). Functional assignment of DUF4422 explains how galactofuranose is incorporated into various polysaccharides of importance in vaccine production and the food industry. In the 2.1-Å resolution structure, three WbbM protomers associate to form a flattened triangular prism connected to a central stalk that orients the active sites toward the membrane. The biochemical, structural and topological properties of WbbM offer broader insight into the mechanisms of assembly of bacterial cell-surface glycans.

The steroid side-chain-cleaving aldolase Ltp2-ChsH2DUF35 is a thiolase superfamily member with a radically repurposed active site.

An aldolase from the bile acid-degrading actinobacterium Thermomonospora curvata catalyzes the C-C bond cleavage of an isopropyl-CoA side chain from the D-ring of the steroid metabolite 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA (17-HOPC-CoA). Like its homolog from Mycobacterium tuberculosis, the T. curvata aldolase is a protein complex of Ltp2 with a DUF35 domain derived from the C-terminal domain of a hydratase (ChsH2DUF35) that catalyzes the preceding step in the pathway. We determined the structure of the Ltp2-ChsH2DUF35 complex at 1.7 Å resolution using zinc-single anomalous diffraction. The enzyme adopts an αßßα organization, with the two Ltp2 protomers forming a central dimer, and the two ChsH2DUF35 protomers being at the periphery. Docking experiments suggested that Ltp2 forms a tight complex with the hydratase but that each enzyme retains an independent CoA-binding site. Ltp2 adopted a fold similar to those in thiolases; however, instead of forming a deep tunnel, the Ltp2 active site formed an elongated cleft large enough to accommodate 17-HOPC-CoA. The active site lacked the two cysteines that served as the nucleophile and general base in thiolases and replaced a pair of oxyanion-hole histidine residues with Tyr-246 and Tyr-344. Phenylalanine replacement of either of these residues decreased aldolase catalytic activity at least 400-fold. On the basis of a 17-HOPC-CoA -docked model, we propose a catalytic mechanism where Tyr-294 acts as the general base abstracting a proton from the D-ring hydroxyl of 17-HOPC-CoA and Tyr-344 as the general acid that protonates the propionyl-CoA anion following C-C bond cleavage.

Biosynthesis of a conserved glycolipid anchor for Gram-negative bacterial capsules.

Several important Gram-negative bacterial pathogens possess surface capsular layers composed of hypervariable long-chain polysaccharides linked via a conserved 3-deoxy-ß-D-manno-oct-2-ulosonic acid (ß-Kdo) oligosaccharide to a phosphatidylglycerol residue. The pathway for synthesis of the terminal glycolipid was elucidated by determining the structures of reaction intermediates. In Escherichia coli, KpsS transfers a single Kdo residue to phosphatidylglycerol; this primer is extended using a single enzyme (KpsC), possessing two cytidine 5'-monophosphate (CMP)-Kdo-dependent glycosyltransferase catalytic centers with different linkage specificities. The structure of the N-terminal ß-(2→4) Kdo transferase from KpsC reveals two α/ß domains, supplemented by several helices. The N-terminal Rossmann-like domain, typically responsible for acceptor binding, is severely reduced in size compared with canonical GT-B folds in glycosyltransferases. The similar structure of the C-terminal ß-(2→7) Kdo transferase indicates a past gene duplication event. Both Kdo transferases have a narrow active site tunnel, lined with key residues shared with GT99 ß-Kdo transferases. This enzyme provides the prototype for the GT107 family.

Structural and kinetic characterization of (S)-1-amino-2-propanol kinase from the aminoacetone utilization microcompartment of Mycobacterium smegmatis.

Bacterial microcompartments encapsulate enzymatic pathways that generate small, volatile, aldehyde intermediates. The Rhodococcus and Mycobacterium microcompartment (RMM) operon from Mycobacterium smegmatis encodes four enzymes, including (S)-1-amino-2-propanol dehydrogenase and a likely propionaldehyde dehydrogenase. We show here that a third enzyme (and its nonmicrocompartment-associated paralog) is a moderately specific (S)-1-amino-2-propanol kinase. We determined the structure of apo-aminopropanol kinase at 1.35 Å, revealing that it has structural similarity to hexosamine kinases, choline kinases, and aminoglycoside phosphotransferases. We modeled substrate binding, and tested our model by characterizing key enzyme variants. Bioinformatics analysis established that this enzyme is widespread in Actinobacteria, Proteobacteria, and Firmicutes, and is very commonly associated with a candidate phospholyase. In Rhizobia, aminopropanol kinase is generally associated with aromatic degradation pathways. In the RMM (and the parallel pathway that includes the second paralog), aminopropanol kinase likely degrades aminoacetone through a propanolamine-phosphate phospho-lyase-dependent pathway. These enzymatic activities were originally described in Pseudomonas, but the proteins responsible have not been previously identified. Bacterial microcompartments typically co-encapsulate enzymes which can regenerate required co-factors, but the RMM enzymes require four biochemically distinct co-factors with no overlap. This suggests that either the RMM shell can uniquely transport multiple co-factors in stoichiometric quantities, or that all enzymes except the phospho-lyase reside outside of the shell. In summary, aminopropanol kinase is a novel enzyme found in diverse bacteria and multiple metabolic pathways; its presence in the RMM implies that this microcompartment degrades aminoacetone, using a pathway that appears to violate some established precepts as to how microcompartments function.

Structure and Kinetics of the S-(+)-1-Amino-2-propanol Dehydrogenase from the RMM Microcompartment of Mycobacterium smegmatis.

S-(+)-1-Amino-2-propanol dehydrogenase (APDH) is a short-chain dehydrogenase/reductase associated with the incompletely characterized Rhodococcus and Mycobacterium bacterial microcompartment (RMM). We enzymatically characterized the APDH from M. smegmatis and showed it is highly selective, with a low micromolar Km for S-(+)-1-amino-2-propanol and specificity for NADP(H). A paralogous enzyme from a nonmicrocompartment-associated operon in the same organism was also shown to have a similar activity. We determined the structure of APDH in both apo form (at 1.7 Å) and as a ternary enzyme complex with NADP+ and aminoacetone (at 1.9 Å). Recognition of aminoacetone was mediated by strong hydrogen bonds to the amino group by Thr145 and by Glu251 from the C-terminus of an adjacent protomer. The substrate binding site entirely encloses the substrate, with close contacts between the aminoacetone methyl group and Phe95, Trp154, and Leu195. Kinetic characterization of several of these residues confirm their importance in enzyme functioning. Bioinformatics analysis of APDH homologues implies that many nonmicrocompartment APDH orthologues partake in an aminoacetone degradation pathway that proceeds via an aminopropanol O-phosphate phospholyase. RMM microcompartments may mediate a similar pathway, though possibly with differences in the details of the pathway that necessitates encapsulation behind a shell.

A Complete Structural Inventory of the Mycobacterial Microcompartment Shell Proteins Constrains Models of Global Architecture and Transport.

Bacterial microcompartments are bacterial analogs of eukaryotic organelles in that they spatially segregate aspects of cellular metabolism, but they do so by building not a lipid membrane but a thin polyhedral protein shell. Although multiple shell protein structures are known for several microcompartment types, additional uncharacterized components complicate systematic investigations of shell architecture. We report here the structures of all four proteins proposed to form the shell of an uncharacterized microcompartment designated the Rhodococcus and Mycobacterium microcompartment (RMM), which, along with crystal interactions and docking studies, suggests possible models for the particle's vertex and edge organization. MSM0272 is a typical hexameric ß-sandwich shell protein thought to form the bulk of the facet. MSM0273 is a pentameric ß-barrel shell protein that likely plugs the vertex of the particle. MSM0271 is an unusual double-ringed bacterial microcompartment shell protein whose rings are organized in an offset position relative to all known related proteins. MSM0275 is related to MSM0271 but self-organizes as linear strips that may line the facet edge; here, the presence of a novel extendable loop may help ameliorate poor packing geometry of the rigid main particle at the angled edges. In contrast to previously characterized homologs, both of these proteins show closed pores at both ends. This suggests a model where key interactions at the vertex and edges are mediated at the inner layer of the shell by MSM0271 (encircling MSM0273) and MSM0275, and the facet is built from MSM0272 hexamers tiling in the outer layer of the shell.

Bacterial ß-Kdo glycosyltransferases represent a new glycosyltransferase family (GT99).

Kdo (3-deoxy-d-manno-oct-2-ulosonic acid) is an eight-carbon sugar mostly confined to Gram-negative bacteria. It is often involved in attaching surface polysaccharides to their lipid anchors. α-Kdo provides a bridge between lipid A and the core oligosaccharide in all bacterial LPSs, whereas an oligosaccharide of ß-Kdo residues links "group 2" capsular polysaccharides to (lyso)phosphatidylglycerol. ß-Kdo is also found in a small number of other bacterial polysaccharides. The structure and function of the prototypical cytidine monophosphate-Kdo-dependent α-Kdo glycosyltransferase from LPS assembly is well characterized. In contrast, the ß-Kdo counterparts were not identified as glycosyltransferase enzymes by bioinformatics tools and were not represented among the 98 currently recognized glycosyltransferase families in the Carbohydrate-Active Enzymes database. We report the crystallographic structure and function of a prototype ß-Kdo GT from WbbB, a modular protein participating in LPS O-antigen synthesis in Raoultella terrigena The ß-Kdo GT has dual Rossmann-fold motifs typical of GT-B enzymes, but extensive deletions, insertions, and rearrangements result in a unique architecture that makes it a prototype for a new GT family (GT99). The cytidine monophosphate-binding site in the C-terminal α/ß domain closely resembles the corresponding site in bacterial sialyltransferases, suggesting an evolutionary connection that is not immediately evident from the overall fold or sequence similarities.

The Klebsiella pneumoniae O12 ATP-binding Cassette (ABC) Transporter Recognizes the Terminal Residue of Its O-antigen Polysaccharide Substrate.

Export of the Escherichia coli serotype O9a O-antigenic polysaccharides (O-PS) involves an ATP-binding cassette (ABC) transporter. The process requires a non-reducing terminal residue, which is recognized by a carbohydrate-binding module (CBM) appended to the C terminus of the nucleotide-binding domain of the transporter. Here, we investigate the process in Klebsiella pneumoniae serotype O12 (and Raoultella terrigena ATCC 33257). The O12 polysaccharide is terminated at the non-reducing end by a ß-linked 3-deoxy-d-manno-oct-2-ulosonic acid (Kdo) residue. The O12 ABC transporter also binds its cognate O-PS via a CBM, and export is dependent on the presence of the terminal ß-Kdo residue. The overall structural architecture of the O12 CBM resembles the O9a prototype, but they share only weak sequence similarity, and the putative binding pocket for the O12 glycan is different. Removal of the CBM abrogated O-PS transport, but export was restored when the CBM was expressed in trans with the mutant CBM-deficient ABC transporter. These results demonstrate that the CBM-mediated substrate-recognition mechanism is evolutionarily conserved and can operate with glycans of widely differing structures.