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.

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.

The crystal structure of nsp10-nsp16 heterodimer from SARS-CoV-2 in complex with S-adenosylmethionine.

SARS-CoV-2 is a member of the coronaviridae family and is the etiological agent of the respiratory Coronavirus Disease 2019. The virus has spread rapidly around the world resulting in over two million cases and nearly 150,000 deaths as of April 17, 2020. Since no treatments or vaccines are available to treat COVID-19 and SARS-CoV-2, respiratory complications derived from the infections have overwhelmed healthcare systems around the world. This virus is related to SARS-CoV-1, the virus that caused the 2002-2004 outbreak of Severe Acute Respiratory Syndrome. In January 2020, the Center for Structural Genomics of Infectious Diseases implemented a structural genomics pipeline to solve the structures of proteins essential for coronavirus replication-transcription. Here we show the first structure of the SARS-CoV-2 nsp10-nsp16 2'-O-methyltransferase complex with S-adenosylmethionine at a resolution of 1.80 Å. This heterodimer complex is essential for capping viral mRNA transcripts for efficient translation and to evade immune surveillance.

A comparative genomics approach identifies contact-dependent growth inhibition as a virulence determinant.

Emerging evidence suggests the Pseudomonas aeruginosa accessory genome is enriched with uncharacterized virulence genes. Identification and characterization of such genes may reveal novel pathogenic mechanisms used by particularly virulent isolates. Here, we utilized a mouse bacteremia model to quantify the virulence of 100 individual P. aeruginosa bloodstream isolates and performed whole-genome sequencing to identify accessory genomic elements correlated with increased bacterial virulence. From this work, we identified a specific contact-dependent growth inhibition (CDI) system enriched among highly virulent P. aeruginosa isolates. CDI systems contain a large exoprotein (CdiA) with a C-terminal toxin (CT) domain that can vary between different isolates within a species. Prior work has revealed that delivery of a CdiA-CT domain upon direct cell-to-cell contact can inhibit replication of a susceptible target bacterium. Aside from mediating interbacterial competition, we observed our virulence-associated CdiA-CT domain to promote toxicity against mammalian cells in culture and lethality during mouse bacteremia. Structural and functional studies revealed this CdiA-CT domain to have in vitro tRNase activity, and mutations that abrogated this tRNAse activity in vitro also attenuated virulence. Furthermore, CdiA contributed to virulence in mice even in the absence of contact-dependent signaling. Overall, our findings indicate that this P. aeruginosa CDI system functions as both an interbacterial inhibition system and a bacterial virulence factor against a mammalian host. These findings provide an impetus for continued studies into the complex role of CDI systems in P. aeruginosa pathogenesis.

Structure of galactarate dehydratase, a new fold in an enolase involved in bacterial fitness after antibiotic treatment.

Galactarate dehydratase (GarD) is the first enzyme in the galactarate/glucarate pathway and catalyzes the dehydration of galactarate to 3-keto-5-dehydroxygalactarate. This protein is known to increase colonization fitness of intestinal pathogens in antibiotic-treated mice and to promote bacterial survival during stress. The galactarate/glucarate pathway is widespread in bacteria, but not in humans, and thus could be a target to develop new inhibitors for use in combination therapy to combat antibiotic resistance. The structure of almost all the enzymes of the galactarate/glucarate pathway were solved previously, except for GarD, for which only the structure of the N-terminal domain was determined previously. Herein, we report the first crystal structure of full-length GarD solved using a seleno-methoionine derivative revealing a new protein fold. The protein consists of three domains, each presenting a novel twist as compared to their distant homologs. GarD in the crystal structure forms dimers and each monomer consists of three domains. The N-terminal domain is comprised of a ß-clip fold, connected to the second domain by a long unstructured linker. The second domain serves as a dimerization interface between two monomers. The C-terminal domain forms an unusual variant of a Rossmann fold with a crossover and is built around a seven-stranded parallel ß-sheet supported by nine α-helices. A metal binding site in the C-terminal domain is occupied by Ca2+ . The activity of GarD was corroborated by the production of 5-keto-4-deoxy-D-glucarate under reducing conditions and in the presence of iron. Thus, GarD is an unusual enolase with a novel protein fold never previously seen in this class of enzymes.

Structural comparison of p-hydroxybenzoate hydroxylase (PobA) from Pseudomonas putida with PobA from other Pseudomonas spp. and other monooxygenases.

The crystal structure is reported of p-hydroxybenzoate hydroxylase (PobA) from Pseudomonas putida, a possible drug target to combat tetracycline resistance, in complex with flavin adenine dinucleotide (FAD). The structure was refined at 2.2 Šresolution with four polypeptide chains in the asymmetric unit. Based on the results of pairwise structure alignments, PobA from P. putida is structurally very similar to PobA from P. fluorescens and from P. aeruginosa. Key residues in the FAD-binding and substrate-binding sites of PobA are highly conserved spatially across the proteins from all three species. Additionally, the structure was compared with two enzymes from the broader class of oxygenases: 2-hydroxybiphenyl 3-monooxygenase (HbpA) from P. nitroreducens and 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO) from Mesorhizobium japonicum. Despite having only 14% similarity in their primary sequences, pairwise structure alignments of PobA from P. putida with HbpA from P. nitroreducens and MHPCO from M. japonicum revealed local similarities between these structures. Key secondary-structure elements important for catalysis, such as the ßαß fold, ß-sheet wall and α12 helix, are conserved across this expanded class of oxygenases.

Analysis of crystalline and solution states of ligand-free spermidine N-acetyltransferase (SpeG) from Escherichia coli.

Spermidine N-acetyltransferase (SpeG) transfers an acetyl group from acetyl-coenzyme A to an N-terminal amino group of intracellular spermidine. This acetylation inactivates spermidine, reducing the polyamine toxicity that tends to occur under certain chemical and physical stresses. The structure of the SpeG protein from Vibrio cholerae has been characterized: while the monomer possesses a structural fold similar to those of other Gcn5-related N-acetyltransferase superfamily members, its dodecameric structure remains exceptional. In this paper, structural analyses of SpeG isolated from Escherichia coli are described. Like V. cholerae SpeG, E. coli SpeG forms dodecamers, as revealed by two crystal structures of the ligand-free E. coli SpeG dodecamer determined at 1.75 and 2.9 Šresolution. Although both V. cholerae SpeG and E. coli SpeG can adopt an asymmetric open dodecameric state, solution analysis showed that the oligomeric composition of ligand-free E. coli SpeG differs from that of ligand-free V. cholerae SpeG. Based on these data, it is proposed that the equilibrium balance of SpeG oligomers in the absence of ligands differs from one species to another and thus might be important for SpeG function.

The spermidine acetyltransferase SpeG regulates transcription of the small RNA rprA.

Spermidine N-acetyltransferase (SpeG) acetylates and thus neutralizes toxic polyamines. Studies indicate that SpeG plays an important role in virulence and pathogenicity of many bacteria, which have evolved SpeG-dependent strategies to control polyamine concentrations and survive in their hosts. In Escherichia coli, the two-component response regulator RcsB is reported to be subject to Nε-acetylation on several lysine residues, resulting in reduced DNA binding affinity and reduced transcription of the small RNA rprA; however, the physiological acetylation mechanism responsible for this behavior has not been fully determined. Here, we performed an acetyltransferase screen and found that SpeG inhibits rprA promoter activity in an acetylation-independent manner. Surface plasmon resonance analysis revealed that SpeG can physically interact with the DNA-binding carboxyl domain of RcsB. We hypothesize that SpeG interacts with the DNA-binding domain of RcsB and that this interaction might be responsible for SpeG-dependent inhibition of RcsB-dependent rprA transcription. This work provides a model for SpeG as a modulator of E. coli transcription through its ability to interact with the transcription factor RcsB. This is the first study to provide evidence that an enzyme involved in polyamine metabolism can influence the function of the global regulator RcsB, which integrates information concerning envelope stresses and central metabolic status to regulate diverse behaviors.

Crystal structures of the transpeptidase domain of the Mycobacterium tuberculosis penicillin-binding protein PonA1 reveal potential mechanisms of antibiotic resistance.

UNLABELLED: Mycobacterium tuberculosis is a human respiratory pathogen that causes the deadly disease tuberculosis. The rapid global spread of antibiotic-resistant M. tuberculosis makes tuberculosis infections difficult to treat. To overcome this problem new effective antimicrobial strategies are urgently needed. One promising target for new therapeutic approaches is PonA1, a class A penicillin-binding protein, which is required for maintaining physiological cell wall synthesis and cell shape during growth in mycobacteria. Here, crystal structures of the transpeptidase domain, the enzymatic domain responsible for penicillin binding, of PonA1 from M. tuberculosis in the inhibitor-free form and in complex with penicillin V are reported. We used site-directed mutagenesis, antibiotic profiling experiments, and fluorescence thermal shift assays to measure PonA1's sensitivity to different classes of ß-lactams. Structural comparison of the PonA1 apo-form and the antibiotic-bound form shows that binding of penicillin V induces conformational changes in the position of the loop ß4'-α3 surrounding the penicillin-binding site. We have also found that binding of different antibiotics including penicillin V positively impacts protein stability, while other tested ß-lactams such as clavulanate or meropenem resulted in destabilization of PonA1. Our antibiotic profiling experiments indicate that the transpeptidase activity of PonA1 in both M. tuberculosis and M. smegmatis mediates tolerance to specific cell wall-targeting antibiotics, particularly to penicillin V and meropenem. Because M. tuberculosis is an important human pathogen, these structural data provide a template to design novel transpeptidase inhibitors to treat tuberculosis infections. DATABASE: Structural data are available in the PDB database under the accession numbers 5CRF and 5CXW.

Small angle X-ray scattering data and structure factor fitting for the study of the quaternary structure of the spermidine N-acetyltransferase SpeG.

Here we describe the treatment of the small-angle X-ray Scattering (SAXS) data used during SpeG quaternary structure study as part of the research article "Substrate induced allosteric change in the quaternary structure of the spermidine N-acetyltransferase SpeG" published in Journal of Molecular Biology [1]. These data were collected on two separate area detectors as separate dilution series of the SpeG and the SpeG with spermine samples along with data from their companion buffers. The data were radially integrated, corrected for incident beam variation, and scaled to absolute units. After subtraction of volume-fraction scaled buffer scattering and division by the SpeG concentration, multiple scattering curves free of an inter-molecular structure factor were derived from the dilution series. Rather than extrapolating to infinite dilution, the structure factor contribution was estimated by fitting to the full set of data provided by dividing the scattering curves of a dilution series by the curve from the most dilute sample in that series.

Substrate-Induced Allosteric Change in the Quaternary Structure of the Spermidine N-Acetyltransferase SpeG.

The spermidine N-acetyltransferase SpeG is a dodecameric enzyme that catalyzes the transfer of an acetyl group from acetyl coenzyme A to polyamines such as spermidine and spermine. SpeG has an allosteric polyamine-binding site and acetylating polyamines regulate their intracellular concentrations. The structures of SpeG from Vibrio cholerae in complexes with polyamines and cofactor have been characterized earlier. Here, we present the dodecameric structure of SpeG from V. cholerae in a ligand-free form in three different conformational states: open, intermediate and closed. All structures were crystallized in C2 space group symmetry and contain six monomers in the asymmetric unit cell. Two hexamers related by crystallographic 2-fold symmetry form the SpeG dodecamer. The open and intermediate states have a unique open dodecameric ring. This SpeG dodecamer is asymmetric except for the one 2-fold axis and is unlike any known dodecameric structure. Using a fluorescence thermal shift assay, size-exclusion chromatography with multi-angle light scattering, small-angle X-ray scattering analysis, negative-stain electron microscopy and structural analysis, we demonstrate that this unique open dodecameric state exists in solution. Our combined results indicate that polyamines trigger conformational changes and induce the symmetric closed dodecameric state of the protein when they bind to their allosteric sites.

A novel polyamine allosteric site of SpeG from Vibrio cholerae is revealed by its dodecameric structure.

Spermidine N-acetyltransferase, encoded by the gene speG, catalyzes the initial step in the degradation of polyamines and is a critical enzyme for determining the polyamine concentrations in bacteria. In Escherichia coli, studies have shown that SpeG is the enzyme responsible for acetylating spermidine under stress conditions and for preventing spermidine toxicity. Not all bacteria contain speG, and many bacterial pathogens have developed strategies to either acquire or silence it for pathogenesis. Here, we present thorough kinetic analyses combined with structural characterization of the VCA0947 SpeG enzyme from the important human pathogen Vibrio cholerae. Our studies revealed the unexpected presence of a previously unknown allosteric site and an unusual dodecameric structure for a member of the Gcn5-related N-acetyltransferase superfamily. We show that SpeG forms dodecamers in solution and in crystals and describe its three-dimensional structure in several ligand-free and liganded structures. Importantly, these structural data define the first view of a polyamine bound in an allosteric site of an N-acetyltransferase. Kinetic characterization of SpeG from V. cholerae showed that it acetylates spermidine and spermine. The behavior of this enzyme is complex and exhibits sigmoidal curves and substrate inhibition. We performed a detailed non-linear regression kinetic analysis to simultaneously fit families of substrate saturation curves to uncover a simple kinetic mechanism that explains the apparent complexity of this enzyme. Our results provide a fundamental understanding of the bacterial SpeG enzyme, which will be key toward understanding the regulation of polyamine levels in bacteria during pathogenesis.

Structural characterization of a hypothetical protein: a potential agent involved in trimethylamine metabolism in Catenulispora acidiphila.

Catenulispora acidiphila is a newly identified lineage of actinomycetes that produces antimicrobial activities and represents a promising source of novel antibiotics and secondary metabolites. Among the discovered protein coding genes, 68 % were assigned a putative function, while the remaining 32 % are genes encoding "hypothetical" proteins. Caci_0382 is one of the "hypothetical" proteins that has very few homologs. Sequence analysis shows that the protein belongs to the NTF2-like protein family. The structure of Caci_0382 demonstrates that it shares the same fold and has a similar active site as limonene-1,2-epoxide hydrolase, which suggests that it may have a related function. Using a fluorescence thermal shift assay, we identified stabilizing compounds that suggest potential natural ligands of Caci_0382. Using this information, we determined the crystal structure in complex with trimethylamine to provide a better understanding of the function of this uncharacterized protein.

Characterization of the deoxynucleotide triphosphate triphosphohydrolase (dNTPase) activity of the EF1143 protein from Enterococcus faecalis and crystal structure of the activator-substrate complex.

The EF1143 protein from Enterococcus faecalis is a distant homolog of deoxynucleotide triphosphate triphosphohydrolases (dNTPases) from Escherichia coli and Thermus thermophilus. These dNTPases are important components in the regulation of the dNTP pool in bacteria. Biochemical assays of the EF1143 dNTPase activity demonstrated nonspecific hydrolysis of all canonical dNTPs in the presence of Mn(2+). In contrast, with Mg(2+) hydrolysis required the presence of dGTP as an effector, activating the degradation of dATP and dCTP with dGTP also being consumed in the reaction with dATP. The crystal structure of EF1143 and dynamic light scattering measurements in solution revealed a tetrameric oligomer as the most probable biologically active unit. The tetramer contains four dGTP specific allosteric regulatory sites and four active sites. Examination of the active site with the dATP substrate suggests an in-line nucleophilic attack on the α-phosphate center as a possible mechanism of the hydrolysis and two highly conserved residues, His-129 and Glu-122, as an acid-base catalytic dyad. Structural differences between EF1143 apo and holo forms revealed mobility of the α3 helix that can regulate the size of the active site binding pocket and could be stabilized in the open conformation upon formation of the tetramer and dGTP effector binding.

Crystal structure of an apo form of Shigella flexneri ArsH protein with an NADPH-dependent FMN reductase activity.

The arsH gene or its homologs are a frequent part of the arsenic resistance system in bacteria and eukaryotes. Although a specific biological function of the gene product is unknown, the ArsH protein was annotated as a member of the NADPH-dependent FMN reductase family based on a conserved (T/S)XRXXSX(T/S) fingerprint motif common for FMN binding proteins. Presented here are the first crystal structure of an ArsH protein from Shigella flexneri refined at 1.7 A resolution and results of enzymatic activity assays that revealed a strong NADPH-dependent FMN reductase and low azoreductase activities. The ArsH apo protein has an alpha/beta/alpha-fold typical for FMN binding proteins. The asymmetric unit consists of four monomers, which form a tetramer. Buried surface analysis suggests that this tetramer is likely to be the relevant biological assembly. Dynamic light scattering experiments are consistent with this hypothesis and show that ArsH in solution at room temperature does exist predominantly in the tetrameric form.

Cyclic AMP and AKAP-mediated targeting of protein kinase A regulates lactate dehydrogenase subunit A mRNA stability.

Expression of the lactate dehydrogenase A subunit (ldh-A) gene is controlled through transcriptional as well as post-transcriptional mechanisms. Both mechanisms involve activation of protein kinase A (PKA) into its subunits and subsequent phosphorylation and activation of several key regulatory factors. In rat C6 glioma cells, post-transcriptional gene regulation occurs through PKA-mediated stabilization of LDH-A mRNA and subsequent increase of intracellular LDH-A mRNA levels. Previous studies have demonstrated a cAMP-stabilizing region (CSR) located in the LDH-A 3'-untranslated region which, in combination with several phosphorylated CSR-binding proteins (CSR-BP), regulates the PKA-mediated stabilization of LDH-A mRNA. However, the mechanistic details of interaction of CSR with proteins as they pertain to mRNA stabilization by PKA are so far largely unknown. In this study we tested the hypothesis that ribosomal protein extracts (RSW) from glioma cells contain PKA regulatory (RII) and catalytic (C) subunits that, in combination with a protein kinase A anchoring protein (AKAP 95) and CSR-BPs participate in forming CSR-protein complexes that are responsible for mRNA stability regulation. To demonstrate the importance of CSR-protein complex formation, the PKA subunits and AKAP 95 were removed from the RSW by immunoprecipitation, and the antigen-deleted RSW were subjected to CSR binding analysis using gel mobility shift and UV cross-linking. It was shown that AKAP 95 as well as RII formed a direct linkage with CSR during CSR-protein complex formation. In contrast, the catalytic subunit formed part of the CSR-protein complex but did not bind to CSR directly in a covalent linkage. To determine whether formation of CSR complexes that included C, RII, and AKAP 95 constituted a functional event and was necessary for mRNA stabilization, cell-free decay reactions were carried out with RSW extracts, and the kinetics of decay of LDH-A mRNA was determined. Depletion of PKA subunits and AKAP 95 from RSW extracts by immunoprecipitation resulted in a marked loss of mRNA stabilization activity indicating that the presence of the PKA regulatory and catalytic subunits as well as AKAP 95 in the CSR-protein complexes was absolutely necessary to achieve LDH-A mRNA stabilization.