Functionally Null RAD51D Missense Mutation Associates Strongly with Ovarian Carcinoma.

RAD51D is a key player in DNA repair by homologous recombination (HR), and RAD51D truncating variant carriers have an increased risk for ovarian cancer. However, the contribution of nontruncating RAD51D variants to cancer predisposition remains uncertain. Using deep sequencing and case-control genotyping studies, we show that in French Canadians, the missense RAD51D variant c.620C>T;p.S207L is highly prevalent and is associated with a significantly increased risk for ovarian high-grade serous carcinoma (HGSC; 3.8% cases vs. 0.2% controls). The frequency of the p.S207L variant did not significantly differ from that of controls in breast, endometrial, pancreas, or colorectal adenocarcinomas. Functionally, we show that this mutation impairs HR by disrupting the RAD51D-XRCC2 interaction and confers PARP inhibitor sensitivity. These results highlight the importance of a functional RAD51D-XRCC2 interaction to promote HR and prevent the development of HGSC. This study identifies c.620C>T;p.S207L as the first bona fide pathogenic RAD51D missense cancer susceptibility allele and supports the use of targeted PARP-inhibitor therapies in ovarian cancer patients carrying deleterious missense RAD51D variants. Cancer Res; 77(16); 4517-29. ©2017 AACR.

Structural Basis for Kinase-Mediated Macrolide Antibiotic Resistance.

The macrolides are a class of antibiotic, characterized by a large macrocyclic lactone ring that can be inactivated by macrolide phosphotransferase enzymes. We present structures for MPH(2')-I and MPH(2')-II in the apo state, and in complex with GTP analogs and six different macrolides. These represent the first structures from the two main classes of macrolide phosphotransferases. The structures show that the enzymes are related to the aminoglycoside phosphotransferases, but are distinguished from them by the presence of a large interdomain linker that contributes to an expanded antibiotic binding pocket. This pocket is largely hydrophobic, with a negatively charged patch located at a conserved aspartate residue, rationalizing the broad-spectrum resistance conferred by the enzymes. Complementary mutation studies provide insights into factors governing substrate specificity. A comparison with macrolides bound to their natural target, the 50S ribosome, suggests avenues for next-generation antibiotic development.

Comprehensive characterization of ligand-induced plasticity changes in a dimeric enzyme.

UNLABELLED: An enzyme's inherent structural plasticity is frequently associated with substrate binding, yet detailed structural characterization of flexible proteins remains challenging. This study employs complementary biophysical methods to characterize the partially unfolded structure of substrate-free AAC(6')-Ii, an N-acetyltransferase of the GCN5-related N-acetyltransferase (GNAT) superfamily implicated in conferring broad-spectrum aminoglycoside resistance on Enterococcus faecium. The X-ray crystal structure of AAC(6')-Ii is analyzed to identify relative motions of the structural elements that constitute the dimeric enzyme. Comparison with the previously elucidated crystal structure of AAC(6')-Ii with acetyl coenzyme A (AcCoA) reveals conformational changes that occur upon substrate binding. Our understanding of the enzyme's structural plasticity is further refined with small-angle X-ray scattering and circular dichroism analyses, which together reveal how flexible structural elements impact dimerization and substrate binding. These results clarify the extent of unfolding that AAC(6')-Ii undergoes in the absence of AcCoA and provide a structural connection to previously observed allosteric cooperativity of this enzyme. DATABASE: Structural data are available in the PDB database under the accession number 5E96.

Germline and somatic FGFR1 abnormalities in dysembryoplastic neuroepithelial tumors.

Dysembryoplastic neuroepithelial tumor (DNET) is a benign brain tumor associated with intractable drug-resistant epilepsy. In order to identify underlying genetic alterations and molecular mechanisms, we examined three family members affected by multinodular DNETs as well as 100 sporadic tumors from 96 patients, which had been referred to us as DNETs. We performed whole-exome sequencing on 46 tumors and targeted sequencing for hotspot FGFR1 mutations and BRAF p.V600E was used on the remaining samples. FISH, copy number variation assays and Sanger sequencing were used to validate the findings. By whole-exome sequencing of the familial cases, we identified a novel germline FGFR1 mutation, p.R661P. Somatic activating FGFR1 mutations (p.N546K or p.K656E) were observed in the tumor samples and further evidence for functional relevance was obtained by in silico modeling. The FGFR1 p.K656E mutation was confirmed to be in cis with the germline p.R661P variant. In 43 sporadic cases, in which the diagnosis of DNET could be confirmed on central blinded neuropathology review, FGFR1 alterations were also frequent and mainly comprised intragenic tyrosine kinase FGFR1 duplication and multiple mutants in cis (25/43; 58.1 %) while BRAF p.V600E alterations were absent (0/43). In contrast, in 53 cases, in which the diagnosis of DNET was not confirmed, FGFR1 alterations were less common (10/53; 19 %; p < 0.0001) and hotspot BRAF p.V600E (12/53; 22.6 %) (p < 0.001) prevailed. We observed overexpression of phospho-ERK in FGFR1 p.R661P and p.N546K mutant expressing HEK293 cells as well as FGFR1 mutated tumor samples, supporting enhanced MAP kinase pathway activation under these conditions. In conclusion, constitutional and somatic FGFR1 alterations and MAP kinase pathway activation are key events in the pathogenesis of DNET. These findings point the way towards existing targeted therapies.

Inhibition of outer membrane proteases of the omptin family by aprotinin.

Bacterial proteases are important virulence factors that inactivate host defense proteins and contribute to tissue destruction and bacterial dissemination. Outer membrane proteases of the omptin family, exemplified by Escherichia coli OmpT, are found in some Gram-negative bacteria. Omptins cleave a variety of substrates at the host-pathogen interface, including plasminogen and antimicrobial peptides. Multiple omptin substrates relevant to infection have been identified; nonetheless, an effective omptin inhibitor remains to be found. Here, we purified native CroP, the OmpT ortholog in the murine pathogen Citrobacter rodentium. Purified CroP was found to readily cleave both a synthetic fluorescence resonance energy transfer substrate and the murine cathelicidin-related antimicrobial peptide. In contrast, CroP was found to poorly activate plasminogen into active plasmin. Although classical protease inhibitors were ineffective against CroP activity, we found that the serine protease inhibitor aprotinin displays inhibitory potency in the micromolar range. Aprotinin was shown to act as a competitive inhibitor of CroP activity and to interfere with the cleavage of the murine cathelicidin-related antimicrobial peptide. Importantly, aprotinin was able to inhibit not only CroP but also Yersinia pestis Pla and, to a lesser extent, E. coli OmpT. We propose a structural model of the aprotinin-omptin complex in which Lys15 of aprotinin forms salt bridges with conserved negatively charged residues of the omptin active site.

A recurrent PDGFRB mutation causes familial infantile myofibromatosis.

Infantile myofibromatosis (IM) is the most common benign fibrous tumor of soft tissues affecting young children. By using whole-exome sequencing, RNA sequencing, and targeted sequencing, we investigated germline and tumor DNA in individuals from four distinct families with the familial form of IM and in five simplex IM cases with no previous family history of this disease. We identified a germline mutation c.1681C>T (p.Arg561Cys) in platelet-derived growth factor receptor ß (PDGFRB) in all 11 affected individuals with familial IM, although none of the five individuals with nonfamilial IM had mutations in this gene. We further identified a second heterozygous mutation in PDGFRB in two myofibromas from one of the affected familial cases, indicative of a potential second hit in this gene in the tumor. PDGFR-ß promotes growth of mesenchymal cells, including blood vessels and smooth muscles, which are affected in IM. Our findings indicate p.Arg561Cys substitution in PDGFR-ß as a cause of the dominant form of this disease. They provide a rationale for further investigations of this specific mutation and gene to assess the benefits of targeted therapies against PDGFR-ß in aggressive life-threatening familial forms of the disease.

Structural analysis of a novel cyclohexylamine oxidase from Brevibacterium oxydans IH-35A.

Cyclohexylamine oxidase (CHAO) is a flavoprotein first described in Brevibacterium oxydans strain IH-35A that carries out the initial step of the degradation of the industrial chemical cyclohexylamine to cyclohexanone. We have cloned and expressed in Escherichia coli the CHAO-encoding gene (chaA) from B. oxydans, purified CHAO and determined the structures of both the holoenzyme form of the enzyme and a product complex with cyclohexanone. CHAO is a 50 kDa monomer with a PHBH fold topology. It belongs to the flavin monooxygenase family of enzymes and exhibits high substrate specificity for alicyclic amines and sec-alkylamines. The overall structure is similar to that of other members of the flavin monooxygenase family, but lacks either of the C- or N-terminal extensions observed in these enzymes. Active site features of the flavin monooxygenase family are conserved in CHAO, including the characteristic aromatic cage. Differences in the orientations of residues of the CHAO aromatic cage result in a substrate-binding site that is more open than those of its structural relatives. Since CHAO has a buried hydrophobic active site with no obvious route for substrates and products, a random acceleration molecular dynamics simulation has been used to identify a potential egress route. The path identified includes an intermediate cavity and requires transient conformation changes in a shielding loop and a residue at the border of the substrate-binding cavity. These results provide a foundation for further studies with CHAO aimed at identifying features determining substrate specificity and for developing the biocatalytic potential of this enzyme.

BSSF: a fingerprint based ultrafast binding site similarity search and function analysis server.

BACKGROUND: Genome sequencing and post-genomics projects such as structural genomics are extending the frontier of the study of sequence-structure-function relationship of genes and their products. Although many sequence/structure-based methods have been devised with the aim of deciphering this delicate relationship, there still remain large gaps in this fundamental problem, which continuously drives researchers to develop novel methods to extract relevant information from sequences and structures and to infer the functions of newly identified genes by genomics technology. RESULTS: Here we present an ultrafast method, named BSSF(Binding Site Similarity & Function), which enables researchers to conduct similarity searches in a comprehensive three-dimensional binding site database extracted from PDB structures. This method utilizes a fingerprint representation of the binding site and a validated statistical Z-score function scheme to judge the similarity between the query and database items, even if their similarities are only constrained in a sub-pocket. This fingerprint based similarity measurement was also validated on a known binding site dataset by comparing with geometric hashing, which is a standard 3D similarity method. The comparison clearly demonstrated the utility of this ultrafast method. After conducting the database searching, the hit list is further analyzed to provide basic statistical information about the occurrences of Gene Ontology terms and Enzyme Commission numbers, which may benefit researchers by helping them to design further experiments to study the query proteins. CONCLUSIONS: This ultrafast web-based system will not only help researchers interested in drug design and structural genomics to identify similar binding sites, but also assist them by providing further analysis of hit list from database searching.

DrugViz: a Cytoscape plugin for visualizing and analyzing small molecule drugs in biological networks.

SUMMARY: DrugViz is a Cytoscape plugin that is designed to visualize and analyze small molecules within the framework of the interactome. DrugViz can import drug-target network information in an extended SIF file format to Cytoscape and display the two-dimensional (2D) structures of small molecule nodes in a unified visualization environment. It also can identify small molecule nodes by means of three different 2D structure searching methods, namely isomorphism, substructure and fingerprint-based similarity searches. After selections, users can furthermore conduct a two-side clustering analysis on drugs and targets, which allows for a detailed analysis of the active compounds in the network, and elucidate relationships between these drugs and targets. DrugViz represents a new tool for the analysis of data from chemogenomics, metabolomics and systems biology. AVAILABILITY: DrugViz and data set used in Application are freely available for download at http://202.127.30.184:8080/software.html.

The type IA topoisomerase catalytic cycle: A normal mode analysis and molecular dynamics simulation.

Type IA topoisomerases alter the topological state of DNA to relax the supercoils introduced during the DNA replication and transcription process, giving them critical roles in many cellular functions. To manipulate the DNA, type IA topoisomerases first cleave one DNA strand and form a covalent linkage between a catalytic tyrosine residue and the 5'-phosphoryl of the DNA. This is followed by a movement of domain III of the topoisomerase to accommodate the second DNA strand in the center channel of the topoisomerase. Domain III is then closed for religation of the cleaved DNA and subsequently reopened to release the passing strand. Although numerous biophysical and biochemical studies have examined this catalytic cycle, fundamental questions remain such as how domain III opens and closes during this process. We have used computational simulation methods, namely normal mode analysis and molecular dynamics, to investigate the catalytic cycle of Escherichia coli topoisomerase III as a representative of the type IA topoisomerases. It was found that domain II is intrinsically flexible and may empower the enzyme to perform its function by triggering domain III opening and closing. A molecular dynamics simulation and MM-PBSA analysis shows that topoisomerase III alone cannot overcome the large energy barrier of the conformational transition. A detailed examination of the DNA binding sites suggests that the processing DNA cooperates with the topoisomerase to accomplish this dramatic conformational change. These findings will guide future mutagenesis studies of type IA topoisomerases aimed at dissecting the driving forces and conformations in the catalytic cycle.

Structures of aminoglycoside acetyltransferase AAC(6')-Ii in a novel crystal form: structural and normal-mode analyses.

The aminoglycoside-modifying enzyme aminoglycoside 6'-N-acetyltransferase type Ii [AAC(6')-Ii] has been crystallized with its cofactor coenzyme A in space group C222(1), with unit-cell parameters a = 71.5, b = 127.4, c = 76.9 A and one physiologically relevant dimer species per asymmetric unit. The space group previously observed for this complex was P2(1)2(1)2(1), with two dimers per asymmetric unit. By comparing the six available protomer structures of the AAC(6')-Ii-CoA complex, it has been possible to identify regions of plasticity within the protein. Normal-mode analysis of this complex suggests that this plasticity is not an artefact of crystal-packing forces, but that the region of the protomer that displays multiple conformations is intrinsically flexible. It is conjectured that the flexibility is relevant for the cooperative activity observed for the enzyme.

Roles of the equatorial tyrosyl iron ligand of protocatechuate 3,4-dioxygenase in catalysis.

The active site Fe(III) of protocatechuate 3,4-dioxygenase (3,4-PCD) from Pseudomonas putida is ligated axially by Tyr447 and His462 and equatorially by Tyr408, His460, and OH(-). Tyr447 and OH(-) are displaced as protocatechuate (3,4-dihydroxybenzoate, PCA) chelates the iron and appear to serve as in situ bases to promote this process. The role(s) of Tyr408 is (are) explored here using mutant enzymes that exhibit less than 0.1% wild-type activity. The X-ray crystal structures of the mutants and their PCA complexes show that the new shorter residues in the 408 position cannot ligate the iron and instead interact with the iron through solvents. Moreover, PCA binds as a monodentate rather than a bidentate ligand, and Tyr447 fails to dissociate. Although the new residues at position 408 do not directly bind to the iron, large changes in the spectroscopic and catalytic properties are noted among the mutant enzymes. Resonance Raman features show that the Fe-O bond of the monodentate 4-hydroxybenzoate (4HB) inhibitor complex is significantly stronger in the mutants than in wild-type 3,4-PCD. Transient kinetic studies show that PCA and 4HB bind to 3,4-PCD in a fast, reversible step followed by a step in which coordination to the metal occurs; the latter process is at least 50-fold slower in the mutant enzymes. It is proposed that, in wild-type 3,4-PCD, the Lewis base strength of Tyr408 lowers the Lewis acidity of the iron to foster the rapid exchange of anionic ligands during the catalytic cycle. Accordingly, the increase in Lewis acidity of the iron caused by substitution of this residue by solvent tends to make the iron substitution inert. Tyr447 cannot be released to allow formation of the usual dianionic PCA chelate complex with the active site iron, and the rate of electrophilic attack by O(2) becomes rate limiting overall. The structures of the PCA complexes of these mutant enzymes show that hydrogen-bonding interactions between the new solvent ligand and the new second-sphere residue in position 408 allow this residue to significantly influence the spectroscopic and kinetic properties of the enzymes.

X-ray structure of the AAC(6')-Ii antibiotic resistance enzyme at 1.8 A resolution; examination of oligomeric arrangements in GNAT superfamily members.

The rise of antibiotic resistance as a public health concern has led to increased interest in studying the ways in which bacteria avoid the effects of antibiotics. Enzymatic inactivation by several families of enzymes has been observed to be the predominant mechanism of resistance to aminoglycoside antibiotics such as kanamycin and gentamicin. Despite the importance of acetyltransferases in bacterial resistance to aminoglycoside antibiotics, relatively little is known about their structure and mechanism. Here we report the three-dimensional atomic structure of the aminoglycoside acetyltransferase AAC(6')-Ii in complex with coenzyme A (CoA). This structure unambiguously identifies the physiologically relevant AAC(6')-Ii dimer species, and reveals that the enzyme structure is similar in the AcCoA and CoA bound forms. AAC(6')-Ii is a member of the GCN5-related N-acetyltransferase (GNAT) superfamily of acetyltransferases, a diverse group of enzymes that possess a conserved structural motif, despite low sequence homology. AAC(6')-Ii is also a member of a subset of enzymes in the GNAT superfamily that form multimeric complexes. The dimer arrangements within the multimeric GNAT superfamily members are compared, revealing that AAC(6')-Ii forms a dimer assembly that is different from that observed in the other multimeric GNAT superfamily members. This different assembly may provide insight into the evolutionary processes governing dimer formation.

Protein kinase inhibitors and antibiotic resistance.

While antibiotics revolutionized the treatment of infectious disease in the 20th century, bacterial resistance now threatens to render many of them ineffective. Aminoglycosides are a class of clinically important antibiotics used in the treatment of infections caused by Gram-positive and -negative organisms. They are bactericidal, targeting the bacterial ribosome, where they bind to the A-site and disrupt protein synthesis. Clinical resistance to these drugs occurs mainly via enzymatic inactivation by aminoglycoside-modifying enzymes that phosphorylate, adenylate, or acetylate the aminoglycoside. Those that phosphorylate (i.e., aminoglycoside kinases) have been shown to be structurally related to eukaryotic protein kinases. This was surprising, given the low degree of sequence similarity between the groups of enzymes. The nucleotide-binding site, specifically, is very similar in structure, suggesting that the two classes of enzymes share a common mechanism of phosphoryl transfer. Three strategies can be envisaged for combating aminoglycoside kinase-mediated bacterial resistance. The first involves compounds that target the antibiotic binding region. Secondly, protein kinase inhibitors have been identified that disable aminoglycoside-modifying enzymes by targeting the ATP-binding site. Lastly, compounds are being developed that exploit the bridged nature of the active site, incorporating nucleotide and substrate motifs. A strategy using bifunctional aminoglycoside dimers has also been pursued, yielding molecules that bind to the target site on the bacterial ribosome, while serving as poor substrates for modifying enzymes. This work holds out the promise that effective inhibitors of aminoglycoside-modifying enzymes may eventually restore the usefulness of aminoglycoside antibiotics.