Cofactorless oxygenases guide anthraquinone-fused enediyne biosynthesis.

The anthraquinone-fused enediynes (AFEs) combine an anthraquinone moiety and a ten-membered enediyne core capable of generating a cytotoxic diradical species. AFE cyclization is triggered by opening the F-ring epoxide, which is also the site of the most structural diversity. Previous studies of tiancimycin A, a heavily modified AFE, have revealed a cryptic aldehyde blocking installation of the epoxide, and no unassigned oxidases could be predicted within the tnm biosynthetic gene cluster. Here we identify two consecutively acting cofactorless oxygenases derived from methyltransferase and α/ß-hydrolase protein folds, TnmJ and TnmK2, respectively, that are responsible for F-ring tailoring in tiancimycin biosynthesis by comparative genomics. Further biochemical and structural characterizations reveal that the electron-rich AFE anthraquinone moiety assists in catalyzing deformylation, epoxidation and oxidative ring cleavage without exogenous cofactors. These enzymes therefore fill important knowledge gaps for the biosynthesis of this class of molecules and the underappreciated family of cofactorless oxygenases.

Sel1-like proteins and peptides are the major Oxalobacter formigenes-derived factors stimulating oxalate transport by human intestinal epithelial cells.

Kidney stones (KSs) are very common, excruciating, and associated with tremendous healthcare cost, chronic kidney disease (CKD), and kidney failure (KF). Most KSs are composed of calcium oxalate and small increases in urinary oxalate concentration significantly enhance the stone risk. Oxalate also potentially contributes to CKD progression, kidney disease-associated cardiovascular diseases, and poor renal allograft survival. This emphasizes the urgent need for plasma and urinary oxalate lowering therapies, which can be achieved by enhancing enteric oxalate secretion. We previously identified Oxalobacter formigenes (O. formigenes)-derived factors secreted in its culture-conditioned medium (CM), which stimulate oxalate transport by human intestinal Caco2-BBE (C2) cells and reduce urinary oxalate excretion in hyperoxaluric mice by enhancing colonic oxalate secretion. Given their remarkable therapeutic potential, we now identified Sel1-like proteins as the major O. formigenes-derived secreted factors using mass spectrometry and functional assays. Crystal structures for six proteins were determined to confirm structures and better understand functions. OxBSel1-14-derived small peptides P8 and P9 were identified as the major factors, with P8 + 9 closely recapitulating the CM's effects, acting through the oxalate transporters SLC26A2 and SLC26A6 and PKA activation. Besides C2 cells, P8 + 9 also stimulate oxalate transport by human ileal and colonic organoids, confirming that they work in human tissues. In conclusion, P8 and P9 peptides are identified as the major O. formigenes-derived secreted factors and they have significant therapeutic potential for hyperoxalemia, hyperoxaluria, and related disorders, impacting the outcomes of patients suffering from KSs, enteric hyperoxaluria, primary hyperoxaluria, CKD, KF, and renal transplant recipients.NEW & NOTEWORTHY We previously identified Oxalobacter formigenes-derived secreted factors stimulating oxalate transport by human intestinal epithelial cells in vitro and reducing urinary oxalate excretion in hyperoxaluric mice by enhancing colonic oxalate secretion. We now identified Sel1-like proteins and small peptides as the major secreted factors and they have significant therapeutic potential for hyperoxalemia and hyperoxaluria, impacting the outcomes of patients suffering from kidney stones, primary and secondary hyperoxaluria, chronic kidney disease, kidney failure, and renal transplant recipients.

Mycobacterium tuberculosis Phe-tRNA synthetase: structural insights into tRNA recognition and aminoacylation.

Tuberculosis, caused by Mycobacterium tuberculosis, responsible for ∼1.5 million fatalities in 2018, is the deadliest infectious disease. Global spread of multidrug resistant strains is a public health threat, requiring new treatments. Aminoacyl-tRNA synthetases are plausible candidates as potential drug targets, because they play an essential role in translating the DNA code into protein sequence by attaching a specific amino acid to their cognate tRNAs. We report structures of M. tuberculosis Phe-tRNA synthetase complexed with an unmodified tRNAPhe transcript and either L-Phe or a nonhydrolyzable phenylalanine adenylate analog. High-resolution models reveal details of two modes of tRNA interaction with the enzyme: an initial recognition via indirect readout of anticodon stem-loop and aminoacylation ready state involving interactions of the 3' end of tRNAPhe with the adenylate site. For the first time, we observe the protein gate controlling access to the active site and detailed geometry of the acyl donor and tRNA acceptor consistent with accepted mechanism. We biochemically validated the inhibitory potency of the adenylate analog and provide the most complete view of the Phe-tRNA synthetase/tRNAPhe system to date. The presented topography of amino adenylate-binding and editing sites at different stages of tRNA binding to the enzyme provide insights for the rational design of anti-tuberculosis drugs.

Atomic structure of the Leishmania spp. Hsp100 N-domain.

Hsp100 is an ATP-dependent unfoldase that promotes protein disaggregation or facilitates the unfolding of aggregation-prone polypeptides marked for degradation. Recently, new Hsp100 functions are emerging. In Plasmodium, an Hsp100 drives malaria protein export, presenting a novel drug target. Whether Hsp100 has a similar function in other protists is unknown. We present the 1.06 Å resolution crystal structure of the Hsp100 N-domain from Leishmania spp., the causative agent of leishmaniasis in humans. Our structure reveals a network of methionines and aromatic amino acids that define the putative substrate-binding site and likely evolved to protect Hsp100 from oxidative damage in host immune cells.

Intramolecular C-C Bond Formation Links Anthraquinone and Enediyne Scaffolds in Tiancimycin Biosynthesis.

First discovered in 1989, the anthraquinone-fused enediynes are a class of DNA-cleaving bacterial natural products composed of a DNA-intercalating anthraquinone moiety and a 10-membered enediyne warhead. However, until recently, there has been a lack of genetically amenable hosts and sequenced biosynthetic gene clusters available for solving the biosynthetic questions surrounding these molecules. Herein, we have identified and biochemically and structurally characterized TnmK1, a member of the α/ß-hydrolase fold superfamily responsible for the C-C bond formation linking the anthraquinone moiety and enediyne core together in tiancimycin (TNM) biosynthesis. In doing so, two intermediates, TNM H and TNM I, in anthraquinone-fused enediyne biosynthesis, containing an unprecedented cryptic C16 aldehyde group, were identified. This aldehyde plays a key role in the TnmK1-catalyzed C-C bond formation via a Michael addition, representing the first example of this chemistry for the α/ß-hydrolase fold superfamily. Additionally, TNM I shows sub-nanomolar cytotoxicity against selected cancer cell lines, indicating a new mechanism of action compared to previously known anthraquinone-fused enediynes. Together, the findings from this study are expected to impact enzymology, natural product biosynthesis, and future efforts at enediyne discovery and drug development.

Fixed-target serial crystallography at the Structural Biology Center.

Serial synchrotron crystallography enables the study of protein structures under physiological temperature and reduced radiation damage by collection of data from thousands of crystals. The Structural Biology Center at Sector 19 of the Advanced Photon Source has implemented a fixed-target approach with a new 3D-printed mesh-holder optimized for sample handling. The holder immobilizes a crystal suspension or droplet emulsion on a nylon mesh, trapping and sealing a near-monolayer of crystals in its mother liquor between two thin Mylar films. Data can be rapidly collected in scan mode and analyzed in near real-time using piezoelectric linear stages assembled in an XYZ arrangement, controlled with a graphical user interface and analyzed using a high-performance computing pipeline. Here, the system was applied to two ß-lactamases: a class D serine ß-lactamase from Chitinophaga pinensis DSM 2588 and L1 metallo-ß-lactamase from Stenotrophomonas maltophilia K279a.

Characterization and Crystal Structure of a Nonheme Diiron Monooxygenase Involved in Platensimycin and Platencin Biosynthesis.

Nonheme diiron monooxygenases make up a rapidly growing family of oxygenases that are rarely identified in secondary metabolism. Herein, we report the in vivo, in vitro, and structural characterizations of a nonheme diiron monooxygenase, PtmU3, that installs a C-5 ß-hydroxyl group in the unified biosynthesis of platensimycin and platencin, two highly functionalized diterpenoids that act as potent and selective inhibitors of bacterial and mammalian fatty acid synthases. This hydroxylation sets the stage for the subsequent A-ring cleavage step key to the unique diterpene-derived scaffolds of platensimycin and platencin. PtmU3 adopts an unprecedented triosephosphate isomerase (TIM) barrel structural fold for this class of enzymes and possesses a noncanonical diiron active site architecture with a saturated six-coordinate iron center lacking a µ-oxo bridge. This study reveals the first member of a previously unidentified superfamily of TIM-barrel-fold enzymes for metal-dependent dioxygen activation, with the majority predicted to act on CoA-linked substrates, thus expanding our knowledge of nature's repertoire of nonheme diiron monooxygenases and TIM-barrel-fold enzymes.

Mitochondrial Hsp90 is a ligand-activated molecular chaperone coupling ATP binding to dimer closure through a coiled-coil intermediate.

Heat-shock protein of 90 kDa (Hsp90) is an essential molecular chaperone that adopts different 3D structures associated with distinct nucleotide states: a wide-open, V-shaped dimer in the apo state and a twisted, N-terminally closed dimer with ATP. Although the N domain is known to mediate ATP binding, how Hsp90 senses the bound nucleotide and facilitates dimer closure remains unclear. Here we present atomic structures of human mitochondrial Hsp90N (TRAP1N) and a composite model of intact TRAP1 revealing a previously unobserved coiled-coil dimer conformation that may precede dimer closure and is conserved in intact TRAP1 in solution. Our structure suggests that TRAP1 normally exists in an autoinhibited state with the ATP lid bound to the nucleotide-binding pocket. ATP binding displaces the ATP lid that signals the cis-bound ATP status to the neighboring subunit in a highly cooperative manner compatible with the coiled-coil intermediate state. We propose that TRAP1 is a ligand-activated molecular chaperone, which couples ATP binding to dramatic changes in local structure required for protein folding.

Structural and evolutionary relationships of "AT-less" type I polyketide synthase ketosynthases.

Acyltransferase (AT)-less type I polyketide synthases (PKSs) break the type I PKS paradigm. They lack the integrated AT domains within their modules and instead use a discrete AT that acts in trans, whereas a type I PKS module minimally contains AT, acyl carrier protein (ACP), and ketosynthase (KS) domains. Structures of canonical type I PKS KS-AT didomains reveal structured linkers that connect the two domains. AT-less type I PKS KSs have remnants of these linkers, which have been hypothesized to be AT docking domains. Natural products produced by AT-less type I PKSs are very complex because of an increased representation of unique modifying domains. AT-less type I PKS KSs possess substrate specificity and fall into phylogenetic clades that correlate with their substrates, whereas canonical type I PKS KSs are monophyletic. We have solved crystal structures of seven AT-less type I PKS KS domains that represent various sequence clusters, revealing insight into the large structural and subtle amino acid residue differences that lead to unique active site topologies and substrate specificities. One set of structures represents a larger group of KS domains from both canonical and AT-less type I PKSs that accept amino acid-containing substrates. One structure has a partial AT-domain, revealing the structural consequences of a type I PKS KS evolving into an AT-less type I PKS KS. These structures highlight the structural diversity within the AT-less type I PKS KS family, and most important, provide a unique opportunity to study the molecular evolution of substrate specificity within the type I PKSs.

Co-occurrence of analogous enzymes determines evolution of a novel (ßα)8-isomerase sub-family after non-conserved mutations in flexible loop.

We investigate the evolution of co-occurring analogous enzymes involved in L-tryptophan and L-histidine biosynthesis in Actinobacteria Phylogenetic analysis of trpF homologues, a missing gene in certain clades of this lineage whose absence is complemented by a dual-substrate HisA homologue, termed PriA, found that they fall into three categories: (i) trpF-1, an L-tryptophan biosynthetic gene horizontally acquired by certain Corynebacterium species; (ii) trpF-2, a paralogue known to be involved in synthesizing a pyrrolopyrrole moiety and (iii) trpF-3, a variable non-conserved orthologue of trpF-1 We previously investigated the effect of trpF-1 upon the evolution of PriA substrate specificity, but nothing is known about the relationship between trpF-3 and priA After in vitro steady-state enzyme kinetics we found that trpF-3 encodes a phosphoribosyl anthranilate isomerase. However, mutation of this gene in Streptomyces sviceus did not lead to auxothrophy, as expected from the biosynthetic role of trpF-1 Biochemical characterization of a dozen co-occurring TrpF-2 or TrpF-3, with PriA homologues, explained the prototrophic phenotype, and unveiled an enzyme activity trade-off between TrpF and PriA. X-ray structural analysis suggests that the function of these PriA homologues is mediated by non-conserved mutations in the flexible L5 loop, which may be responsible for different substrate affinities. Thus, the PriA homologues that co-occur with TrpF-3 represent a novel enzyme family, termed PriB, which evolved in response to PRA isomerase activity. The characterization of co-occurring enzymes provides insights into the influence of functional redundancy on the evolution of enzyme function, which could be useful for enzyme functional annotation.

A novel transcriptional regulator of L-arabinose utilization in human gut bacteria.

Carbohydrate metabolism plays a crucial role in the ecophysiology of human gut microbiota. Mechanisms of transcriptional regulation of sugar catabolism in commensal and prevalent human gut bacteria such as Bacteroides thetaiotaomicron remain mostly unknown. By a combination of bioinformatics and experimental approaches, we have identified an NrtR family transcription factor (BT0354 in B. thetaiotaomicron, BtAraR) as a novel regulator controlling the arabinose utilization genes. L-arabinose was confirmed to be a negative effector of BtAraR. We have solved the crystal structures of the apo and L-arabinose-bound BtAraR proteins, as well as the complex of apo-protein with a specific DNA operator. BtAraR forms a homodimer with each subunit comprised of the ligand-binding Nudix hydrolase-like domain and the DNA-binding winged-helix-turn-helix (wHTH) domain. We have identified the residues involved in binding of L-arabinose and recognition of DNA. The majority of these residues are well conserved in the AraR orthologs in Bacteroidetes. In the structure of the BtAraR-DNA complex, we found the unique interaction of arginine intercalating its guanidinum moiety into the base pair stacking of B-DNA. L-arabinose binding induces movement of wHTH domains, resulting in a conformation unsuitable for DNA binding. Our analysis facilitates reconstruction of the metabolic and regulatory networks involved in carbohydrate utilization in human gut Bacteroides.

Crystal Structure of the Zorbamycin-Binding Protein ZbmA, the Primary Self-Resistance Element in Streptomyces flavoviridis ATCC21892.

The bleomycins (BLMs), tallysomycins (TLMs), phleomycin, and zorbamycin (ZBM) are members of the BLM family of glycopeptide-derived antitumor antibiotics. The BLM-producing Streptomyces verticillus ATCC15003 and the TLM-producing Streptoalloteichus hindustanus E465-94 ATCC31158 both possess at least two self-resistance elements, an N-acetyltransferase and a binding protein. The N-acetyltransferase provides resistance by disrupting the metal-binding domain of the antibiotic that is required for activity, while the binding protein confers resistance by sequestering the metal-bound antibiotic and preventing drug activation via molecular oxygen. We recently established that the ZBM producer, Streptomyces flavoviridis ATCC21892, lacks the N-acetyltransferase resistance gene and that the ZBM-binding protein, ZbmA, is sufficient to confer resistance in the producing strain. To investigate the resistance mechanism attributed to ZbmA, we determined the crystal structures of apo and Cu(II)-ZBM-bound ZbmA at high resolutions of 1.90 and 1.65 Å, respectively. A comparison and contrast with other structurally characterized members of the BLM-binding protein family revealed key differences in the protein-ligand binding environment that fine-tunes the ability of ZbmA to sequester metal-bound ZBM and supports drug sequestration as the primary resistance mechanism in the producing organisms of the BLM family of antitumor antibiotics.

Biochemical and structural analysis of an Eis family aminoglycoside acetyltransferase from bacillus anthracis.

Proteins from the enhanced intracellular survival (Eis) family are versatile acetyltransferases that acetylate amines at multiple positions of several aminoglycosides (AGs). Their upregulation confers drug resistance. Homologues of Eis are present in diverse bacteria, including many pathogens. Eis from Mycobacterium tuberculosis (Eis_Mtb) has been well characterized. In this study, we explored the AG specificity and catalytic efficiency of the Eis family protein from Bacillus anthracis (Eis_Ban). Kinetic analysis of specificity and catalytic efficiency of acetylation of six AGs indicates that Eis_Ban displays significant differences from Eis_Mtb in both substrate binding and catalytic efficiency. The number of acetylated amines was also different for several AGs, indicating a distinct regiospecificity of Eis_Ban. Furthermore, most recently identified inhibitors of Eis_Mtb did not inhibit Eis_Ban, underscoring the differences between these two enzymes. To explain these differences, we determined an Eis_Ban crystal structure. The comparison of the crystal structures of Eis_Ban and Eis_Mtb demonstrates that critical residues lining their respective substrate binding pockets differ substantially, explaining their distinct specificities. Our results suggest that acetyltransferases of the Eis family evolved divergently to garner distinct specificities while conserving catalytic efficiency, possibly to counter distinct chemical challenges. The unique specificity features of these enzymes can be utilized as tools for developing AGs with novel modifications and help guide specific AG treatments to avoid Eis-mediated resistance.

Crystal structure of the Legionella pneumophila Lem10 effector reveals a new member of the HD protein superfamily.

Legionella pneumophila, the intracellular pathogen that can cause severe pneumonia known as Legionnaire's disease, translocates close to 300 effectors inside the host cell using Dot/Icm type IVB secretion system. The structure and function for the majority of these effector proteins remains unknown. Here, we present the crystal structure of the L. pneumophila effector Lem10. The structure reveals a multidomain organization with the largest C-terminal domain showing strong structural similarity to the HD protein superfamily representatives. However, Lem10 lacks the catalytic His-Asp residue pair and does not show any in vitro phosphohydrolase enzymatic activity, typical for HD proteins. While the biological function of Lem10 remains elusive, our analysis shows that similar distinct features are shared by a significant number of HD domains found in Legionella proteins, including the SidE family of effectors known to play an important role during infection. Taken together our data point to the presence of a specific group of non-catalytic Legionella HD domains, dubbed LHDs, which are involved in pathogenesis.

In situ X-ray data collection and structure phasing of protein crystals at Structural Biology Center 19-ID.

A prototype of a 96-well plate scanner for in situ data collection has been developed at the Structural Biology Center (SBC) beamline 19-ID, located at the Advanced Photon Source, USA. The applicability of this instrument for protein crystal diffraction screening and data collection at ambient temperature has been demonstrated. Several different protein crystals, including selenium-labeled, were used for data collection and successful SAD phasing. Without the common procedure of crystal handling and subsequent cryo-cooling for data collection at T = 100 K, crystals in a crystallization buffer show remarkably low mosaicity (<0.1°) until deterioration by radiation damage occurs. Data presented here show that cryo-cooling can cause some unexpected structural changes. Based on the results of this study, the integration of the plate scanner into the 19-ID end-station with automated controls is being prepared. With improvement of hardware and software, in situ data collection will become available for the SBC user program including remote access.

Epitopes recognition of SARS-CoV-2 nucleocapsid RNA binding domain by human monoclonal antibodies.

Coronavirus nucleocapsid protein (NP) of SARS-CoV-2 plays a central role in many functions important for virus proliferation including packaging and protecting genomic RNA. The protein shares sequence, structure, and architecture with nucleocapsid proteins from betacoronaviruses. The N-terminal domain (NPRBD) binds RNA and the C-terminal domain is responsible for dimerization. After infection, NP is highly expressed and triggers robust host immune response. The anti-NP antibodies are not protective and not neutralizing but can effectively detect viral proliferation soon after infection. Two structures of SARS-CoV-2 NPRBD were determined providing a continuous model from residue 48 to 173, including RNA binding region and key epitopes. Five structures of NPRBD complexes with human mAbs were isolated using an antigen-bait sorting. Complexes revealed a distinct complement-determining regions and unique sets of epitope recognition. This may assist in the early detection of pathogens and designing peptide-based vaccines. Mutations that significantly increase viral load were mapped on developed, full length NP model, likely impacting interactions with host proteins and viral RNA.

Structural and functional characterization of solute binding proteins for aromatic compounds derived from lignin: p-coumaric acid and related aromatic acids.

Lignin comprises 15-25% of plant biomass and represents a major environmental carbon source for utilization by soil microorganisms. Access to this energy resource requires the action of fungal and bacterial enzymes to break down the lignin polymer into a complex assortment of aromatic compounds that can be transported into the cells. To improve our understanding of the utilization of lignin by microorganisms, we characterized the molecular properties of solute binding proteins of ATP-binding cassette transporter proteins that interact with these compounds. A combination of functional screens and structural studies characterized the binding specificity of the solute binding proteins for aromatic compounds derived from lignin such as p-coumarate, 3-phenylpropionic acid and compounds with more complex ring substitutions. A ligand screen based on thermal stabilization identified several binding protein clusters that exhibit preferences based on the size or number of aromatic ring substituents. Multiple X-ray crystal structures of protein-ligand complexes for these clusters identified the molecular basis of the binding specificity for the lignin-derived aromatic compounds. The screens and structural data provide new functional assignments for these solute-binding proteins which can be used to infer their transport specificity. This knowledge of the functional roles and molecular binding specificity of these proteins will support the identification of the specific enzymes and regulatory proteins of peripheral pathways that funnel these compounds to central metabolic pathways and will improve the predictive power of sequence-based functional annotation methods for this family of proteins.

Structural basis of impaired disaggregase function in the oxidation-sensitive SKD3 mutant causing 3-methylglutaconic aciduria.

Mitochondria are critical to cellular and organismal health. To prevent damage, mitochondria have evolved protein quality control machines to survey and maintain the mitochondrial proteome. SKD3, also known as CLPB, is a ring-forming, ATP-fueled protein disaggregase essential for preserving mitochondrial integrity and structure. SKD3 deficiency causes 3-methylglutaconic aciduria type VII (MGCA7) and early death in infants, while mutations in the ATPase domain impair protein disaggregation with the observed loss-of-function correlating with disease severity. How mutations in the non-catalytic N-domain cause disease is unknown. Here, we show that the disease-associated N-domain mutation, Y272C, forms an intramolecular disulfide bond with Cys267 and severely impairs SKD3Y272C function under oxidizing conditions and in living cells. While Cys267 and Tyr272 are found in all SKD3 isoforms, isoform-1 features an additional α-helix that may compete with substrate-binding as suggested by crystal structure analyses and in silico modeling, underscoring the importance of the N-domain to SKD3 function.

A Structural Systems Biology Approach to High-Risk CG23 Klebsiella pneumoniae.

Klebsiella pneumoniae is a leading cause of antibiotic-resistant-associated deaths in the world. Here, we report the deposition of 14 structures of enzymes from both the core and accessory genomes of sequence type 23 (ST23) K1 hypervirulent K. pneumoniae.

Tipiracil binds to uridine site and inhibits Nsp15 endoribonuclease NendoU from SARS-CoV-2.

SARS-CoV-2 Nsp15 is a uridine-specific endoribonuclease with C-terminal catalytic domain belonging to the EndoU family that is highly conserved in coronaviruses. As endoribonuclease activity seems to be responsible for the interference with the innate immune response, Nsp15 emerges as an attractive target for therapeutic intervention. Here we report the first structures with bound nucleotides and show how the enzyme specifically recognizes uridine moiety. In addition to a uridine site we present evidence for a second base binding site that can accommodate any base. The structure with a transition state analog, uridine vanadate, confirms interactions key to catalytic mechanisms. In the presence of manganese ions, the enzyme cleaves unpaired RNAs. This acquired knowledge was instrumental in identifying Tipiracil, an FDA approved drug that is used in the treatment of colorectal cancer, as a potential anti-COVID-19 drug. Using crystallography, biochemical, and whole-cell assays, we demonstrate that Tipiracil inhibits SARS-CoV-2 Nsp15 by interacting with the uridine binding pocket in the enzyme's active site. Our findings provide new insights for the development of uracil scaffold-based drugs.