Biochemical and Structural Analysis of the Bacterial Enzyme Succinyl-Diaminopimelate Desuccinylase (DapE) from Acinetobacter baumannii.

There is an urgent need for new antibiotics given the rise of antibiotic resistance, and succinyl-diaminopimelate desuccinylase (DapE, E.C. 3.5.1.18) has emerged as a promising bacterial enzyme target. DapE from Haemophilus influenzae (HiDapE) has been studied and inhibitors identified, but it is essential to explore DapE from different species to assess selective versus broad-spectrum therapeutics. We have determined the structure of DapE from the ESKAPE pathogen Acinetobacter baumannii (AbDapE) and studied inhibition by known inhibitors of HiDapE. AbDapE is inhibited by captopril and sulfate comparable to HiDapE, but AbDapE was not significantly inhibited by a known indoline sulfonamide HiDapE inhibitor. Captopril and sulfate both stabilize HiDapE by increasing the thermal melting temperature (Tm) in thermal shift assays. By contrast, sulfate decreases the stability of the AbDapE enzyme, whereas captopril increases the stability. Further, we report two crystal structures of selenomethionine-substituted AbDapE in the closed conformation, one with AbDapE in complex with succinate derived from enzymatic hydrolysis of N6-methyl-l,l-SDAP substrate and acetate (PDB code 7T1Q, 2.25 Å resolution), and a crystal structure of AbDapE with bound succinate along with l-(S)-lactate, a product of degradation of citric acid from the crystallization buffer during X-ray irradiation (PDB code 8F8O, 2.10 Å resolution).

A high-throughput structural system biology approach to increase structure representation of proteins from Clostridioides difficile.

Clostridioides difficile causes life-threatening gastrointestinal infections. It is a high-risk pathogen due to a lack of effective treatments, antimicrobial resistance, and a poorly conserved genomic core. Herein, we report 30 X-ray structures from a structure genomics pipeline spanning 13 years, representing 10.2% of the X-ray structures for this important pathogen.

Discovery of a Druggable, Cryptic Pocket in SARS-CoV-2 nsp16 Using Allosteric Inhibitors.

A collaborative, open-science team undertook discovery of novel small molecule inhibitors of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase using a high throughput screening approach with the potential to reveal new inhibition strategies. This screen yielded compound 5a, a ligand possessing an electron-deficient double bond, as an inhibitor of SARS-CoV-2 nsp16 activity. Surprisingly, X-ray crystal structures revealed that 5a covalently binds within a previously unrecognized cryptic pocket near the S-adenosylmethionine binding cleft in a manner that prevents occupation by S-adenosylmethionine. Using a multidisciplinary approach, we examined the mechanism of binding of compound 5a to the nsp16 cryptic pocket and developed 5a derivatives that inhibited nsp16 activity and murine hepatitis virus replication in rat lung epithelial cells but proved cytotoxic to cell lines canonically used to examine SARS-CoV-2 infection. Our study reveals the druggability of this newly discovered SARS-CoV-2 nsp16 cryptic pocket, provides novel tool compounds to explore the site, and suggests a new approach for discovery of nsp16 inhibition-based pan-coronavirus therapeutics through structure-guided drug design.

Cytochrome c lysine acetylation regulates cellular respiration and cell death in ischemic skeletal muscle.

Skeletal muscle is more resilient to ischemia-reperfusion injury than other organs. Tissue specific post-translational modifications of cytochrome c (Cytc) are involved in ischemia-reperfusion injury by regulating mitochondrial respiration and apoptosis. Here, we describe an acetylation site of Cytc, lysine 39 (K39), which was mapped in ischemic porcine skeletal muscle and removed by sirtuin5 in vitro. Using purified protein and cellular double knockout models, we show that K39 acetylation and acetylmimetic K39Q replacement increases cytochrome c oxidase (COX) activity and ROS scavenging while inhibiting apoptosis via decreased binding to Apaf-1, caspase cleavage and activity, and cardiolipin peroxidase activity. These results are discussed with X-ray crystallography structures of K39 acetylated (1.50 Å) and acetylmimetic K39Q Cytc (1.36 Å) and NMR dynamics. We propose that K39 acetylation is an adaptive response that controls electron transport chain flux, allowing skeletal muscle to meet heightened energy demand while simultaneously providing the tissue with robust resilience to ischemia-reperfusion injury.

Drosophila melanogaster frataxin: protein crystal and predicted solution structure with identification of the iron-binding regions.

Friedreich's ataxia (FRDA) is a hereditary cardiodegenerative and neurodegenerative disease that affects 1 in 50 000 Americans. FRDA arises from either a cellular inability to produce sufficient quantities or the production of a nonfunctional form of the protein frataxin, a key molecule associated with mitochondrial iron-sulfur cluster biosynthesis. Within the mitochondrial iron-sulfur cluster (ISC) assembly pathway, frataxin serves as an allosteric regulator for cysteine desulfurase, the enzyme that provides sulfur for [2Fe-2S] cluster assembly. Frataxin is a known iron-binding protein and is also linked to the delivery of ferrous ions to the scaffold protein, the ISC molecule responsible for the direct assembly of [2Fe-2S] clusters. The goal of this report is to provide structural details of the Drosophila melanogaster frataxin ortholog (Dfh), using both X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy, in order to provide the foundational insight needed to understand the structure-function correlation of the protein. Additionally, NMR iron(II) titrations were used to provide metal contacts on the protein to better understand how it binds iron and aids its delivery to the ISC scaffold protein. Here, the structural and functional similarities of Dfh to its orthologs are also outlined. Structural data show that bacterial, yeast, human and Drosophila frataxins are structurally similar, apart from a structured C-terminus in Dfh that is likely to aid in protein stability. The iron-binding location on helix 1 and strand 1 of Dfh is also conserved across orthologs.

Structure and Enzymatic Activity of an Intellectual Disability-Associated Ornithine Decarboxylase Variant, G84R.

Ornithine decarboxylase (ODC) is a rate-limiting enzyme for the synthesis of polyamines (PAs). PAs are required for proliferation, and increased ODC activity is associated with cancer and neural over-proliferation. ODC levels and activity are therefore tightly regulated, including through the ODC-specific inhibitor, antizyme AZ1. Recently, ODC G84R has been reported as a partial loss-of-function variant that is associated with intellectual disability and seizures. However, G84 is distant from both the catalytic center and the ODC homodimerization interface. To understand how G84R modulates ODC activity, we have determined the crystal structure of ODC G84R in both the presence and the absence of the cofactor pyridoxal 5-phosphate. The structures show that the replacement of G84 by arginine leads to hydrogen bond formation of R84 with F420, the last residue of the ODC C-terminal helix, a structural element that is involved in the AZ1-mediated proteasomal degradation of ODC. In contrast, the catalytic center is essentially indistinguishable from that of wildtype ODC. We therefore reanalyzed the catalytic activity of ODC G84R and found that it is rescued when the protein is purified in the presence of a reducing agent to mimic the reducing environment of the cytoplasm. This suggests that R84 may exert its neurological effects not through reducing ODC catalytic activity but through misregulation of its AZ1-mediated proteasomal degradation.

Functional and Structural Characterization of OXA-935, a Novel OXA-10-Family ß-Lactamase from Pseudomonas aeruginosa.

Resistance to antipseudomonal penicillins and cephalosporins is often driven by the overproduction of the intrinsic ß-lactamase AmpC. However, OXA-10-family ß-lactamases are a rich source of resistance in Pseudomonas aeruginosa. OXA ß-lactamases have a propensity for mutation that leads to extended spectrum cephalosporinase and carbapenemase activity. In this study, we identified isolates from a subclade of the multidrug-resistant (MDR) high risk P. aeruginosa clonal complex CC446 with a resistance to ceftazidime. A genomic analysis revealed that these isolates harbored a plasmid containing a novel allele of blaOXA-10, named blaOXA-935, which was predicted to produce an OXA-10 variant with two amino acid substitutions: an aspartic acid instead of a glycine at position 157 and a serine instead of a phenylalanine at position 153. The G157D mutation, present in OXA-14, is associated with the resistance of P. aeruginosa to ceftazidime. Compared to OXA-14, OXA-935 showed increased catalytic efficiency for ceftazidime. The deletion of blaOXA-935 restored the sensitivity to ceftazidime, and susceptibility profiling of P. aeruginosa laboratory strains expressing blaOXA-935 revealed that OXA-935 conferred ceftazidime resistance. To better understand the impacts of the variant amino acids, we determined the crystal structures of OXA-14 and OXA-935. Compared to OXA-14, the F153S mutation in OXA-935 conferred increased flexibility in the omega (Ω) loop. Amino acid changes that confer extended spectrum cephalosporinase activity to OXA-10-family ß-lactamases are concerning, given the rising reliance on novel ß-lactam/ß-lactamase inhibitor combinations, such as ceftolozane-tazobactam and ceftazidime-avibactam, to treat MDR P. aeruginosa infections.

The histone H3.1 variant regulates TONSOKU-mediated DNA repair during replication.

The tail of replication-dependent histone H3.1 varies from that of replication-independent H3.3 at the amino acid located at position 31 in plants and animals, but no function has been assigned to this residue to demonstrate a unique and conserved role for H3.1 during replication. We found that TONSOKU (TSK/TONSL), which rescues broken replication forks, specifically interacts with H3.1 via recognition of alanine 31 by its tetratricopeptide repeat domain. Our results indicate that genomic instability in the absence of ATXR5/ATXR6-catalyzed histone H3 lysine 27 monomethylation in plants depends on H3.1, TSK, and DNA polymerase theta (Pol θ). This work reveals an H3.1-specific function during replication and a common strategy used in multicellular eukaryotes for regulating post-replicative chromatin maturation and TSK, which relies on histone monomethyltransferases and reading of the H3.1 variant.

Structural analysis of Atopobium parvulum SufS cysteine desulfurase linked to Crohn's disease.

Crohn's disease (CD) is characterized by the chronic inflammation of the gastrointestinal tract. A dysbiotic microbiome and a defective immune system are linked to CD, where hydrogen sulfide (H2 S) microbial producers positively correlate with the severity of the disease. Atopobium parvulum is a key H2 S producer from the microbiome of CD patients. In this study, the biochemical characterization of two Atopobium parvulum cysteine desulfurases, ApSufS and ApCsdB, shows that the enzymes are allosterically regulated. Structural analyses reveal that ApSufS forms a dimer with conserved characteristics observed in type II cysteine desulfurases. Four residues surrounding the active site are essential to catalyse cysteine desulfurylation, and a segment of short-chain residues grant access for substrate binding. A better understanding of ApSufS will help future avenues for CD treatment.

Structural basis of binding and inhibition of ornithine decarboxylase by 1-amino-oxy-3-aminopropane.

Ornithine decarboxylase (ODC) is the rate-limiting enzyme for the synthesis of polyamines (PAs). PAs are oncometabolites that are required for proliferation, and pharmaceutical ODC inhibition is pursued for the treatment of hyperproliferative diseases, including cancer and infectious diseases. The most potent ODC inhibitor is 1-amino-oxy-3-aminopropane (APA). A previous crystal structure of an ODC-APA complex indicated that APA non-covalently binds ODC and its cofactor pyridoxal 5-phosphate (PLP) and functions by competing with the ODC substrate ornithine for binding to the catalytic site. We have revisited the mechanism of APA binding and ODC inhibition through a new crystal structure of APA-bound ODC, which we solved at 2.49 Šresolution. The structure unambiguously shows the presence of a covalent oxime between APA and PLP in the catalytic site, which we confirmed in solution by mass spectrometry. The stable oxime makes extensive interactions with ODC but cannot be catabolized, explaining APA's high potency in ODC inhibition. In addition, we solved an ODC/PLP complex structure with citrate bound at the substrate-binding pocket. These two structures provide new structural scaffolds for developing more efficient pharmaceutical ODC inhibitors.

Structure of an AMPK complex in an inactive, ATP-bound state.

Adenosine monophosphate (AMP)-activated protein kinase (AMPK) regulates metabolism in response to the cellular energy states. Under energy stress, AMP stabilizes the active AMPK conformation, in which the kinase activation loop (AL) is protected from protein phosphatases, thus keeping the AL in its active, phosphorylated state. At low AMP:ATP (adenosine triphosphate) ratios, ATP inhibits AMPK by increasing AL dynamics and accessibility. We developed conformation-specific antibodies to trap ATP-bound AMPK in a fully inactive, dynamic state and determined its structure at 3.5-angstrom resolution using cryo-electron microscopy. A 180° rotation and 100-angstrom displacement of the kinase domain fully exposes the AL. On the basis of the structure and supporting biophysical data, we propose a multistep mechanism explaining how adenine nucleotides and pharmacological agonists modulate AMPK activity by altering AL phosphorylation and accessibility.

Mn2+ coordinates Cap-0-RNA to align substrates for efficient 2'-O-methyl transfer by SARS-CoV-2 nsp16.

Capping of viral messenger RNAs is essential for efficient translation, for virus replication, and for preventing detection by the host cell innate response system. The SARS-CoV-2 genome encodes the 2'-O-methyltransferase nsp16, which, when bound to the coactivator nsp10, uses S-adenosylmethionine (SAM) as a donor to transfer a methyl group to the first ribonucleotide of the mRNA in the final step of viral mRNA capping. Here, we provide biochemical and structural evidence that this reaction requires divalent cations, preferably Mn2+, and a coronavirus-specific four-residue insert. We determined the x-ray structures of the SARS-CoV-2 2'-O-methyltransferase (the nsp16-nsp10 heterodimer) in complex with its reaction substrates, products, and divalent metal cations. These structural snapshots revealed that metal ions and the insert stabilize interactions between the capped RNA and nsp16, resulting in the precise alignment of the ribonucleotides in the active site. Comparison of available structures of 2'-O-methyltransferases with capped RNAs from different organisms revealed that the four-residue insert unique to coronavirus nsp16 alters the backbone conformation of the capped RNA in the binding groove, thereby promoting catalysis. This insert is highly conserved across coronaviruses, and its absence in mammalian methyltransferases makes this region a promising site for structure-guided drug design of selective coronavirus inhibitors.

Lysine 53 Acetylation of Cytochrome c in Prostate Cancer: Warburg Metabolism and Evasion of Apoptosis.

Prostate cancer is the second leading cause of cancer-related death in men. Two classic cancer hallmarks are a metabolic switch from oxidative phosphorylation (OxPhos) to glycolysis, known as the Warburg effect, and resistance to cell death. Cytochrome c (Cytc) is at the intersection of both pathways, as it is essential for electron transport in mitochondrial respiration and a trigger of intrinsic apoptosis when released from the mitochondria. However, its functional role in cancer has never been studied. Our data show that Cytc is acetylated on lysine 53 in both androgen hormone-resistant and -sensitive human prostate cancer xenografts. To characterize the functional effects of K53 modification in vitro, K53 was mutated to acetylmimetic glutamine (K53Q), and to arginine (K53R) and isoleucine (K53I) as controls. Cytochrome c oxidase (COX) activity analyzed with purified Cytc variants showed reduced oxygen consumption with acetylmimetic Cytc compared to the non-acetylated Cytc (WT), supporting the Warburg effect. In contrast to WT, K53Q Cytc had significantly lower caspase-3 activity, suggesting that modification of Cytc K53 helps cancer cells evade apoptosis. Cardiolipin peroxidase activity, which is another proapoptotic function of the protein, was lower in acetylmimetic Cytc. Acetylmimetic Cytc also had a higher capacity to scavenge reactive oxygen species (ROS), another pro-survival feature. We discuss our experimental results in light of structural features of K53Q Cytc, which we crystallized at a resolution of 1.31 Å, together with molecular dynamics simulations. In conclusion, we propose that K53 acetylation of Cytc affects two hallmarks of cancer by regulating respiration and apoptosis in prostate cancer xenografts.

High-resolution structures of the SARS-CoV-2 2'-O-methyltransferase reveal strategies for structure-based inhibitor design.

There are currently no antiviral therapies specific for SARS-CoV-2, the virus responsible for the global pandemic disease COVID-19. To facilitate structure-based drug design, we conducted an x-ray crystallographic study of the SARS-CoV-2 nsp16-nsp10 2'-O-methyltransferase complex, which methylates Cap-0 viral mRNAs to improve viral protein translation and to avoid host immune detection. We determined the structures for nsp16-nsp10 heterodimers bound to the methyl donor S-adenosylmethionine (SAM), the reaction product S-adenosylhomocysteine (SAH), or the SAH analog sinefungin (SFG). We also solved structures for nsp16-nsp10 in complex with the methylated Cap-0 analog m7GpppA and either SAM or SAH. Comparative analyses between these structures and published structures for nsp16 from other betacoronaviruses revealed flexible loops in open and closed conformations at the m7GpppA-binding pocket. Bound sulfates in several of the structures suggested the location of the ribonucleic acid backbone phosphates in the ribonucleotide-binding groove. Additional nucleotide-binding sites were found on the face of the protein opposite the active site. These various sites and the conserved dimer interface could be exploited for the development of antiviral inhibitors.

Structural basis of Fusarium myosin I inhibition by phenamacril.

Fusarium is a genus of filamentous fungi that includes species that cause devastating diseases in major staple crops, such as wheat, maize, rice, and barley, resulting in severe yield losses and mycotoxin contamination of infected grains. Phenamacril is a novel fungicide that is considered environmentally benign due to its exceptional specificity; it inhibits the ATPase activity of the sole class I myosin of only a subset of Fusarium species including the major plant pathogens F. graminearum, F. asiaticum and F. fujikuroi. To understand the underlying mechanisms of inhibition, species specificity, and resistance mutations, we have determined the crystal structure of phenamacril-bound F. graminearum myosin I. Phenamacril binds in the actin-binding cleft in a new allosteric pocket that contains the central residue of the regulatory Switch 2 loop and that is collapsed in the structure of a myosin with closed actin-binding cleft, suggesting that pocket occupancy blocks cleft closure. We have further identified a single, transferable phenamacril-binding residue found exclusively in phenamacril-sensitive myosins to confer phenamacril selectivity.

A non-canonical monovalent zinc finger stabilizes the integration of Cfp1 into the H3K4 methyltransferase complex COMPASS.

COMPlex ASsociating with SET1 (COMPASS) is a histone H3 Lys-4 methyltransferase that typically marks the promoter region of actively transcribed genes. COMPASS is a multi-subunit complex in which the catalytic unit, SET1, is required for H3K4 methylation. An important subunit known to regulate SET1 methyltransferase activity is the CxxC zinc finger protein 1 (Cfp1). Cfp1 binds to COMPASS and is critical to maintain high level of H3K4me3 in cells but the mechanisms underlying its stimulatory activity is poorly understood. In this study, we show that Cfp1 only modestly activates COMPASS methyltransferase activity in vitro. Binding of Cfp1 to COMPASS is in part mediated by a new type of monovalent zinc finger (ZnF). This ZnF interacts with the COMPASS's subunits RbBP5 and disruption of this interaction blunts its methyltransferase activity in cells and in vivo. Collectively, our studies reveal that a novel form of ZnF on Cfp1 enables its integration into COMPASS and contributes to epigenetic signaling.

Serine-47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase-3 activity.

Cytochrome c (Cytc) is a multifunctional protein that operates as an electron carrier in the mitochondrial electron transport chain and plays a key role in apoptosis. We have previously shown that tissue-specific phosphorylations of Cytc in the heart, liver, and kidney play an important role in the regulation of cellular respiration and cell death. Here, we report that Cytc purified from mammalian brain is phosphorylated on S47 and that this phosphorylation is lost during ischemia. We have characterized the functional effects in vitro using phosphorylated Cytc purified from pig brain tissue and a recombinant phosphomimetic mutant (S47E). We crystallized S47E phosphomimetic Cytc at 1.55 Å and suggest that it spatially matches S47-phosphorylated Cytc, making it a good model system. Both S47-phosphorylated and phosphomimetic Cytc showed a lower oxygen consumption rate in reaction with isolated Cytc oxidase, which we propose maintains intermediate mitochondrial membrane potentials under physiologic conditions, thus minimizing production of reactive oxygen species. S47-phosphorylated and phosphomimetic Cytc showed lower caspase-3 activity. Furthermore, phosphomimetic Cytc had decreased cardiolipin peroxidase activity and is more stable in the presence of H2O2. Our data suggest that S47 phosphorylation of Cytc is tissue protective and promotes cell survival in the brain.-Kalpage, H. A., Vaishnav, A., Liu, J., Varughese, A., Wan, J., Turner, A. A., Ji, Q., Zurek, M. P., Kapralov, A. A., Kagan, V. E., Brunzelle, J. S., Recanati, M.-A., Grossman, L. I., Sanderson, T. H., Lee, I., Salomon, A. R., Edwards, B. F. P, Hüttemann, M. Serine-47 phosphorylation of cytochrome c in the mammalian brain regulates cytochrome c oxidase and caspase-3 activity.

Structural and Antiviral Studies of the Human Norovirus GII.4 Protease.

Norovirus is the leading cause of acute gastroenteritis worldwide with a yearly reported 700 million cases driving a $60 billion global socioeconomic burden. With no United States Food and Drug Administration approved therapeutics and the chance for severe chronic infection and life-threatening complications, researchers have identified the protease as a potential target. However, drug development has focused on the norovirus GI.1 strain despite its accounting for less than 5% of all outbreaks. Our lab aims to change focus for norovirus drug design from GI.1 to the highly infective GII.4, responsible for more than 50% of all outbreaks worldwide. With the first published crystal structure of the norovirus GII.4 protease, we have identified several significant differences in the structure and active site that have hindered development of a potent inhibitor targeting the norovirus GII.4 protease. With these new insights, we have begun designing compounds that demonstrate increased inhibition of the clinically most relevant norovirus GII.4 strain.

Structures of AMP-activated protein kinase bound to novel pharmacological activators in phosphorylated, non-phosphorylated, and nucleotide-free states.

AMP-activated protein kinase (AMPK) is an attractive therapeutic target for managing metabolic diseases. A class of pharmacological activators, including Merck 991, binds the AMPK ADaM site, which forms the interaction surface between the kinase domain (KD) of the α-subunit and the carbohydrate-binding module (CBM) of the ß-subunit. Here, we report the development of two new 991-derivative compounds, R734 and R739, which potently activate AMPK in a variety of cell types, including ß2-specific skeletal muscle cells. Surprisingly, we found that they have only minor effects on direct kinase activity of the recombinant α1ß2γ1 isoform yet robustly enhance protection against activation loop dephosphorylation. This mode of activation is reminiscent of that of ADP, which activates AMPK by binding to the nucleotide-binding sites in the γ-subunit, more than 60 Å away from the ADaM site. To understand the mechanisms of full and partial AMPK activation, we determined the crystal structures of fully active phosphorylated AMPK α1ß1γ1 bound to AMP and R734/R739 as well as partially active nonphosphorylated AMPK bound to R734 and AMP and phosphorylated AMPK bound to R734 in the absence of added nucleotides at <3-Å resolution. These structures and associated analyses identified a novel conformational state of the AMPK autoinhibitory domain associated with partial kinase activity and provide new insights into phosphorylation-dependent activation loop stabilization in AMPK.

A functional proteomics platform to reveal the sequence determinants of lysine methyltransferase substrate selectivity.

Lysine methylation is a key regulator of histone protein function. Beyond histones, few connections have been made to the enzymes responsible for the deposition of these posttranslational modifications. Here, we debut a high-throughput functional proteomics platform that maps the sequence determinants of lysine methyltransferase (KMT) substrate selectivity without a priori knowledge of a substrate or target proteome. We demonstrate the predictive power of this approach for identifying KMT substrates, generating scaffolds for inhibitor design, and predicting the impact of missense mutations on lysine methylation signaling. By comparing KMT selectivity profiles to available lysine methylome datasets, we reveal a disconnect between preferred KMT substrates and the ability to detect these motifs using standard mass spectrometry pipelines. Collectively, our studies validate the use of this platform for guiding the study of lysine methylation signaling and suggest that substantial gaps exist in proteome-wide curation of lysine methylomes.