Accurate Representation of Protein-Ligand Structural Diversity in the Protein Data Bank (PDB)

The number of available protein structures in the Protein Data Bank (PDB) has considerably increased in recent years. Thanks to the growth of structures and complexes, numerous large-scale studies have been done in various research areas, e.g., protein–protein, protein–DNA, or in drug discovery. While protein redundancy was only simply managed using simple protein sequence identity threshold, the similarity of protein-ligand complexes should also be considered from a structural perspective. Hence, the protein-ligand duplicates in the PDB are widely known, but were never quantitatively assessed, as they are quite complex to analyze and compare. Here, we present a specific clustering of protein-ligand structures to avoid bias found in different studies. The methodology is based on binding site superposition, and a combination of weighted Root Mean Square Deviation (RMSD) assessment and hierarchical clustering. Repeated structures of proteins of interest are highlighted and only representative conformations were conserved for a non-biased view of protein distribution. Three types of cases are described based on the number of distinct conformations identified for each complex. Defining these categories decreases by 3.84-fold the number of complexes, and offers more refined results compared to a protein sequence-based method. Widely distinct conformations were analyzed using normalized B-factors. Furthermore, a non-redundant dataset was generated for future molecular interactions analysis or virtual screening studies.

606. Identification and Characterization of HMB-2, a Novel Metallo-β-Lactamase in a Pseudomonas aeruginosa Isolate

Background Carbapenemases, a global health threat, are a diverse group of β-lactamases active against cephalosporins and carbapenems, which are often last resort treatments for multidrug-resistant gram-negative infections. The most common carbapenemases reported among Pseudomonas aeruginosa are metallo-β-lactamase (MBLs). We describe a novel MBL (designated HMB-2) identified in a P. aeruginosa isolate from a urine specimen collected in 2015 as part of CDC’s Emerging Infections Program. Methods We performed antimicrobial susceptibility testing (AST) by broth microdilution, real-time PCR to screen for common carbapenemases (IMP, KPC, NDM, VIM, and OXA-48), and modified carbapenem inactivation method (mCIM) to test for carbapenemase production. The isolate underwent whole-genome sequencing (WGS) using Illumina MiSeq and PacBio RS II (Pacific Biosciences) platforms. Long read sequences were polished using Quiver and corrected by Pilon utilizing Illumina reads. We further characterized a putative novel MBL identified in WGS data by amplifying and cloning the gene into the pCR2.1-TOPO II vector (Invitrogen), which was then sub-cloned into a pET21 expression vector (Sigma–Aldrich). The resulting hmb2+ pET21 plasmid was transformed into a susceptible Escherichia coli for AST, including the imipenem-EDTA method to confirm MBL activity. Results The isolate displayed resistance to carbapenems and demonstrated phenotypic carbapenemase activity (mCIM positive), but was negative for carbapenemase genes by PCR. WGS analyses identified a putative MBL gene located on the chromosome. The gene shared 98% DNA and protein sequence identity with an MBL reported in 2016 in a P. aeruginosa isolate from Germany (HMB-1) and thus was named hmb-2. The cloned hmb-2 gene conferred resistance to carbapenems (meropenem and ertapenem) and third-generation cephalosporins (cefotaxime and ceftazidime) in transformed E. coli. The Minimum Inhibitory Concentrationratio for the imipenem-EDTA method was ≥4. Conclusion A putative, novel β-lactamase gene, blaHMB-2, was identified and cloned. The imipenem-EDTA results indicated that HMB-2 is an MBL. This discovery underscores the important role WGS plays in identifying new mechanisms of antimicrobial resistance. Disclosures All authors: No reported disclosures.

Structural analysis ofClostridium acetobutylicumATCC 824 glycoside hydrolase from CAZy family GH105

Clostridium acetobutylicumATCC 824 gene CA_C0359 encodes a putative unsaturated rhamnogalacturonyl hydrolase (URH) with distant amino-acid sequence homology to YteR ofBacillus subtilisstrain 168. YteR, like other URHs, has core structural homology to unsaturated glucuronyl hydrolases, but hydrolyzes the unsaturated disaccharide derivative of rhamnogalacturonan I. The crystal structure of the recombinant CA_C0359 protein was solved to 1.6 Å resolution by molecular replacement using the phase information of the previously reported structure of YteR (PDB entry 1nc5) fromBacillus subtilisstrain 168. The YteR-like protein is a six-α-hairpin barrel with two β-sheet strands and a small helix overlaying the end of the hairpins next to the active site. The protein has low primary protein sequence identity to YteR but is structurally similar. The two tertiary structures align with a root-mean-square deviation of 1.4 Å and contain a highly conserved active pocket. There is a conserved aspartic acid residue in both structures, which has been shown to be important for hydration of the C=C bond during the release of unsaturated galacturonic acid by YteR. A surface electrostatic potential comparison of CA_C0359 and proteins from CAZy families GH88 and GH105 reveals the make-up of the active site to be a combination of the unsaturated rhamnogalacturonyl hydrolase and the unsaturated glucuronyl hydrolase fromBacillus subtilisstrain 168. Structural and electrostatic comparisons suggests that the protein may have a slightly different substrate specificity from that of YteR.

Crystal structure and functional characterization of a glucosamine-6-phosphate N-acetyltransferase from Arabidopsis thaliana

GlcNAc (N-acetylglucosamine) is an essential part of the glycan chain in N-linked glycoproteins. It is a building block for polysaccharides such as chitin, and several glucosaminoglycans and proteins can be O-GlcNAcylated. The deacetylated form, glucosamine, is an integral part of GPI (glycosylphosphatidylinositol) anchors. Both are incorporated into polymers by glycosyltransferases that utilize UDP-GlcNAc. This UDP-sugar is synthesized in a short pathway comprising four steps starting from fructose 6-phosphate. GNA (glucosamine-6-phosphate N-acetyltransferase) catalyses the second of these four reactions in the de novo synthesis in eukaryotes. A phylogenetic analysis revealed that only one GNA isoform can be found in most of the species investigated and that the most likely Arabidopsis candidate is encoded by the gene At5g15770 (AtGNA). qPCR (quantitative PCR) revealed the ubiquitous expression of AtGNA in all organs of Arabidopsis plants. Heterologous expression of AtGNA showed that it is highly active between pH 7 and 8 and at temperatures of 30–40°C. It showed Km values of 231 μM for glucosamine 6-phosphate and 33 μM for acetyl-CoA respectively and a catalytic efficiency comparable with that of other GNAs characterized. The solved crystal structure of AtGNA at a resolution of 1.5 Å (1 Å=0.1 nm) revealed a very high structural similarity to crystallized GNA proteins from Homo sapiens and Saccharomyces cerevisiae despite less well conserved protein sequence identity.

Surprising Results from a Search for Effective Disinfectants for Tobacco mosaic virus–Contaminated Tools

Over 100 years after its discovery, Tobacco mosaic virus (TMV) remains an economically important pathogen for producers of many vegetatively propagated crops including petunias (Petunia × hybrida). To directly address this concern, we have developed a robust system to determine efficacy of disinfectants for treating TMV-contaminated cutting tools using a combination of preliminary screens and replicated trials. Contrary to widely held beliefs, wild-type (wt) TMV and four additional tobamovirus species infected four petunia cultivars without producing obvious viral symptoms. In contrast, a petunia isolate of TMV with 99.0% (nucleotide) and 99.4% (amino acid) coat protein sequence identity to wt TMV produced symptoms on all but one tested cultivar. We also show that TMV transmission can occur up to the twentieth petunia plant cut following a single cutting event on a TMV-infected plant. Although many new products are now available, treatment of TMV-contaminated tools with a 20% (wt/vol) solution of nonfat dry milk (NFDM) plus 0.1% Tween 20 or a 1:10 dilution of household bleach (0.6% sodium hypochlorite), two “old standbys”, completely eliminated TMV transmission to petunias. Treatment of contaminated tools with 1% (wt/vol) Virkon S or 20% NFDM also significantly reduced the incidence of infected petunias. Other treatments identified in the preliminary screens are candidates for the second phase of screening that simulates contamination during the process of taking cuttings.

A Highly Divergent Picornavirus in a Marine Mammal

ABSTRACT Nucleic acids from an unidentified virus from ringed seals (Phoca hispida) were amplified using sequence-independent PCR, subcloned, and then sequenced. The full genome of a novel RNA virus was derived, identifying the first sequence-confirmed picornavirus in a marine mammal. The phylogenetic position of the tentatively named seal picornavirus 1 (SePV-1) as an outlier to the grouping of parechoviruses was found consistently in alignable regions of the genome. A mean protein sequence identity of only 19.3 to 30.0% was found between the 3D polymerase gene sequence of SePV-1 and those of other picornaviruses. The predicted secondary structure of the short 506-base 5′-untranslated region showed some attributes of a type IVB internal ribosome entry site, and the polyprotein lacked an apparent L peptide, both properties associated with the Parechovirus genus. The presence of two SePV-1 2A genes and of the canonical sequence required for cotranslational cleavage resembled the genetic organization of Ljungan virus. Minor genetic variants were detected in culture supernatants derived from 8 of 108 (7.4%) seals collected in 2000 to 2002, indicating a high prevalence of SePV-1 in this hunted seal population. The high level of genetic divergence of SePV-1 compared to other picornaviruses and its mix of characteristics relative to its closest relatives support the provisional classification of SePV-1 as the prototype for a new genus in the family Picornaviridae.

Evaluation of Two Homologous Proline-Rich Proteins of Coccidioides posadasii as Candidate Vaccines against Coccidioidomycosis

ABSTRACT Evaluation of the protective efficacy of recombinant T-cell-reactive proteins of Coccidioides posadasii in a murine model of coccidioidomycosis has led to the discovery of potential vaccines against this respiratory disease. A recombinant proline-rich antigen (rAg2/Pra) has been reported to be a leading vaccine candidate. However, contradictory results exist on the protection afforded by this antigen. Subcutaneous vaccination of either C57BL/6 or BALB/c mice with rAg2/Pra plus adjuvant followed by intraperitoneal challenge with C. posadasii resulted in a significant reduction of the fungal burden at 12 to 14 days postchallenge compared to that in nonvaccinated animals. Use of the same vaccination protocol followed by intranasal (i.n.) challenge of C57BL/6 mice with an equal number of organisms culminated in chronic pulmonary infection or death over a 90-day period. Early studies of Ag2/Pra suggested that it is a component of an immunogenic complex. We reveal in this study that C. posadasii produces a homolog of the reported proline-rich antigen, designated Prp2, which shows 69% protein sequence identity and 86% similarity to Ag2/Pra. Protection against i.n. challenge of C57BL/6 mice was evaluated by vaccination with the single bacterially expressed homolog, rAg2/Pra, or rPrp2 in combination with rAg2/Pra, each in the presence of the same adjuvant. The combined vaccine provided significantly better protection than either of the single recombinant protein vaccines. Results of enzyme-linked immunospot assays of the immunized mice revealed that the two proline-rich homologs contain unique T-cell epitopes. In combination, the recombinant proteins stimulate a more heterogeneous and protective T-cell repertoire than the monovalent vaccines.

Comparative genomics and genome evolution in yeasts

Yeasts provide a powerful model system for comparative genomics research. The availability of multiple complete genome sequences from different fungal groups—currently 18 hemiascomycetes, 8 euascomycetes and 4 basidiomycetes—enables us to gain a broad perspective on genome evolution. The sequenced genomes span a continuum of divergence levels ranging from multiple individuals within a species to species pairs with low levels of protein sequence identity and no conservation of gene order. One of the most interesting emerging areas is the growing number of events such as gene losses, gene displacements and gene relocations that can be attributed to the action of natural selection.

Comparative Sequence Analysis of IS50/Tn5 Transposase

ABSTRACT Comparative sequence analysis of IS50 transposase-related protein sequences in conjunction with known structural, biochemical, and genetic data was used to determine domains and residues that play key roles in IS50 transposase function. BLAST and ClustalW analyses have been used to find and analyze six complete protein sequences that are related to the IS50 transposase. The protein sequence identity of these six homologs ranged from 25 to 55% in comparison to the IS50 transposase. Homologous motifs were found associated with each of the three catalytic residues. Residues that play roles in transposase-DNA binding, protein autoregulation, and DNA hairpin formation were also found to be conserved in addition to other residues of unknown function. On the other hand, some homologous sequences did not appear to be competent to encode the inhibitor regulatory protein. The results were also used to compare the IS50 transposase with the more distantly related transposase encoded by IS10.