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Barry L Stoddard

Publications and source records attributed to Barry L Stoddard.

33 records · Page 2Linked to original sources
PubMed2004

Disruption of protein-membrane binding and identification of small-molecule inhibitors of coagulation factor VIII.

Factor VIII is a critical member of the blood coagulation cascade. It binds to the membrane surfaces of activated platelets at the site of vascular injury via a highly specific interaction between factor VIII's carboxy-terminal C2 domain and their phosphatidylserine-rich lipid bilayer. We have identified small-molecule inhibitors of factor VIII's membrane binding activity that have IC50 values as low as 2.5 microM. This interaction is approximately 10(3)-fold tighter than that of free o-phospho-L-serine. These compounds also inhibit factor VIII-dependent activation of factor X, indicating that disruption of membrane lipid binding leads to inhibition of the intrinsic coagulation pathway. The tightest binding inhibitor is specific and does not prevent membrane binding by the closely related coagulation factor V. These results indicate that this and related compounds may be used as leads to develop novel antithrombotic agents.

Blood Coagulation Factor Inhibitors↗
PubMed2004

Homology modeling and mutational analysis of Ho endonuclease of yeast.

Ho endonuclease is a LAGLIDADG homing endonuclease that initiates mating-type interconversion in yeast. Ho is encoded by a free-standing gene but shows 50% primary sequence similarity to the intein (protein-intron encoded) PI-SceI. Ho is unique among LAGLIDADG endonucleases in having a 120-residue C-terminal putative zinc finger domain. The crystal structure of PI-SceI revealed a bipartite enzyme with a protein-splicing domain (Hint) and intervening endonuclease domain. We made a homology model for Ho on the basis of the PI-SceI structure and performed mutational analysis of putative critical residues, using a mating-type switch as a bioassay for activity and GFP-fusion proteins to detect nuclear localization. We found that residues of the N-terminal sequence of the Hint domain are important for Ho activity, in particular the DNA recognition region. C-terminal residues of the Hint domain are dispensable for Ho activity; however, the C-terminal putative zinc finger domain is essential. Mutational analysis indicated that residues in Ho that are conserved relative to catalytic, active-site residues in PI-SceI and other related homing endonucleases are essential for Ho activity. Our results indicate that in addition to the conserved catalytic residues, Hint domain residues and the zinc finger domain have evolved a critical role in Ho activity.

Amino Acid Sequence↗
PubMed2003

Structural and biochemical analyses of DNA and RNA binding by a bifunctional homing endonuclease and group I intron splicing factor.

We determined the crystal structure of a bifunctional group I intron splicing factor and homing endonuclease, termed the I-AniI maturase, in complex with its DNA target at 2.6 A resolution. The structure demonstrates the remarkable structural conservation of the beta-sheet DNA-binding motif between highly divergent enzyme subfamilies. DNA recognition by I-AniI was further studied using nucleoside deletion and DMS modification interference analyses. Correlation of these results with the crystal structure provides information on the relative importance of individual nucleotide contacts for DNA recognition. Alignment and modeling of two homologous maturases reveals conserved basic surface residues, distant from the DNA-binding surface, that might be involved in RNA binding. A point mutation that introduces a single negative charge in this region uncouples the maturase and endonuclease functions of the protein, inhibiting RNA binding and splicing while maintaining DNA binding and cleavage.

Amino Acid Sequence↗
PubMed2003

Design of a novel globular protein fold with atomic-level accuracy.

A major challenge of computational protein design is the creation of novel proteins with arbitrarily chosen three-dimensional structures. Here, we used a general computational strategy that iterates between sequence design and structure prediction to design a 93-residue alpha/beta protein called Top7 with a novel sequence and topology. Top7 was found experimentally to be folded and extremely stable, and the x-ray crystal structure of Top7 is similar (root mean square deviation equals 1.2 angstroms) to the design model. The ability to design a new protein fold makes possible the exploration of the large regions of the protein universe not yet observed in nature.

Algorithms↗
PubMed2003

The crystal structure and mutational analysis of human NUDT9.

Human ADP-ribose pyrophosphatase NUDT9 belongs to a superfamily of Nudix hydrolases that catabolize potentially toxic compounds in the cell. The enzyme hydrolyzes ADP-ribose (ADPR) to AMP and ribose 5'-phosphate. NUDT9 shares 39% sequence identity with the C-terminal cytoplasmic domain of the ADPR-gated calcium channel TRPM2, which exhibits low but specific enzyme activity. We determined crystal structures of NUDT9 in the presence and in the absence of the reaction product ribose 5'-phosphate. On the basis of these structures and comparison with a bacterial homologue, a model of the substrate complex was built. The structure and activity of a double point mutant (R(229)E(230)F(231) to R(229)I(230)L(231)), which mimics the Nudix signature of the ion channel domain, was determined. Finally, the activities of a pair of additional mutated constructs were compared to the wild-type enzyme. The first corresponds to a minimal Nudix domain missing an N-terminal domain and C-terminal tail; the second disrupts two potential general bases in the active site. NUDT9 contains an N-terminal domain with a novel fold and a catalytic C-terminal Nudix domain. Unlike its closest functional homologue (homodimeric Escherichia coli ADPRase), it is active as a monomer, and the substrate is bound in a cleft between the domains. The structure of the RIL mutant provides structural basis for the reduced activity of the TRPM2 ion channel. The conformation and binding interactions of ADPR substrate are predicted to differ from those observed for E.coli ADPRase; mutation of structurally aligned acidic residues in their active sites produce significantly different effects on catalytic efficiency, indicating that their reaction pathways and mechanisms may have diverged.

Amino Acid Sequence↗
PubMed2003

Flexible DNA target site recognition by divergent homing endonuclease isoschizomers I-CreI and I-MsoI.

Homing endonucleases are highly specific catalysts of DNA strand breaks that induce the transposition of mobile intervening sequences containing the endonuclease open reading frame. These enzymes recognize long DNA targets while tolerating individual sequence polymorphisms within those sites. Sequences of the homing endonucleases themselves diversify to a great extent after founding intron invasion events, generating highly divergent enzymes that recognize similar target sequences. Here, we visualize the mechanism of flexible DNA recognition and the pattern of structural divergence displayed by two homing endonuclease isoschizomers. We determined structures of I-CreI bound to two DNA target sites that differ at eight of 22 base-pairs, and the structure of an isoschizomer, I-MsoI, bound to a nearly identical DNA target site. This study illustrates several principles governing promiscuous base-pair recognition by DNA-binding proteins, and demonstrates that the isoschizomers display strikingly different protein/DNA contacts. The structures allow us to determine the information content at individual positions in the binding site as a function of the distribution of direct and water-mediated contacts to nucleotide bases, and provide an evolutionary snapshot of endonucleases at an early stage of divergence in their target specificity.

Amino Acid Sequence↗
PubMed2003

The 1.14 A crystal structure of yeast cytosine deaminase: evolution of nucleotide salvage enzymes and implications for genetic chemotherapy.

Cytosine deaminase (CD) catalyzes the deamination of cytosine and is only present in prokaryotes and fungi, where it is a member of the pyrimidine salvage pathway. The enzyme is of interest both for antimicrobial drug design and gene therapy applications against tumors. The structure of Saccharomyces cerevisiae CD has been determined in the presence and absence of a mechanism-based inhibitor, at 1.14 and 1.43 A resolution, respectively. The enzyme forms an alpha/beta fold similar to bacterial cytidine deaminase, but with no similarity to the alpha/beta barrel fold used by bacterial cytosine deaminase or mammalian adenosine deaminase. The structures observed for bacterial, fungal, and mammalian nucleic acid deaminases represent an example of the parallel evolution of two unique protein folds to carry out the same reaction on a diverse array of substrates.

Amino Acid Motifs↗
PubMed2002

Catalytic mechanisms of restriction and homing endonucleases.

The catalytic mechanisms of type II restriction endonucleases and homing endonucleases are discussed and compared. Brief reviews of the chemistry of phosphoryl transfers and canonical one-metal and two-metal endonucleolytic mechanisms are provided along with possible future directions in the study of endonuclease active sites. The discussion of type II restriction endonucleases is comprised of a description of the general architecture of the canonical active site structural motif followed by more in-depth examples of one- and two-metal mechanisms. The homing endonuclease section is comprised of four sections describing what is known regarding the cleavage mechanisms of the four group I intron homing endonuclease families: LAGLIDADG, His-Cys box, H-N-H, and GIY-YIG.

Amino Acid Sequence↗
PubMed2002

NUDT9, a member of the Nudix hydrolase family, is an evolutionarily conserved mitochondrial ADP-ribose pyrophosphatase.

We have recently characterized the protein product of the human NUDT9 gene as a highly specific ADP-ribose (ADPR) pyrophosphatase. We now report an analysis of the human NUDT9 gene and its potential alternative transcripts along with detailed studies of the enzymatic properties and cell biological behavior of human NUDT9 protein. Our analysis of the human NUDT9 gene and twenty-two distinct cloned NUDT9 transcripts indicates that the full-length NUDT9 alpha transcript is the dominant form, and suggests that an alternative NUDT9 beta transcript occurs as the result of a potentially aberrant splice from a cryptic donor site within the first exon to the splice acceptor site of exon 2. Computer analysis of the predicted protein of the NUDT9 alpha transcript identified an N-terminal signal peptide or subcellular targeting sequence. Using green fluorescence protein tagging, we demonstrate that the predicted human NUDT9 alpha protein is targeted highly specifically to mitochondria, whereas the predicted protein of the NUDT9 beta transcript, which is missing this sequence, exhibits no clear subcellular localization. Investigation of the physical and enzymatic properties of NUDT9 indicates that it is functional as a monomer, optimally active at near neutral pH, and that it requires divalent metal ions and an intact Nudix motif for enzymatic activity. Furthermore, partial proteolysis of NUDT9 suggests that NUDT9 enzymes consist of two distinct domains: a proteolytically resistant C-terminal domain retaining essentially full specific ADPR pyrophosphatase activity and a proteolytically labile N-terminal portion that functions to enhance the affinity of the C-terminal domain for ADPR.

Amino Acid Sequence↗
PubMed2002

The structure of Escherichia coli cytosine deaminase.

Cytosine deaminase (CD) catalyzes the deamination of cytosine, producing uracil. This enzyme is present in prokaryotes and fungi (but not multicellular eukaryotes) and is an important member of the pyrimidine salvage pathway in those organisms. The same enzyme also catalyzes the conversion of 5-fluorocytosine to 5-fluorouracil; this activity allows the formation of a cytotoxic chemotherapeutic agent from a non-cytotoxic precursor. The enzyme is of widespread interest both for antimicrobial drug design and for gene therapy applications against tumors. The structure of Escherichia coli CD has been determined in the presence and absence of a bound mechanism-based inhibitor. The enzyme forms an (alphabeta)(8) barrel structure with structural similarity to adenosine deaminase, a relationship that is undetectable at the sequence level, and no similarity to bacterial cytidine deaminase. The enzyme is packed into a hexameric assembly stabilized by a unique domain-swapping interaction between enzyme subunits. The active site is located in the mouth of the enzyme barrel and contains a bound iron ion that coordinates a hydroxyl nucleophile. Substrate binding involves a significant conformational change that sequesters the reaction complex from solvent.

Adenosine Deaminase↗
PubMed2002

Structure of a tRNA repair enzyme and molecular biology workhorse: T4 polynucleotide kinase.

T4 phage polynucleotide kinase (PNK) was identified over 35 years ago and has become a staple reagent for molecular biologists. The enzyme displays 5'-hydroxyl kinase, 3'-phosphatase, and 2',3'-cyclic phosphodiesterase activities against a wide range of substrates. These activities modify the ends of nicked tRNA generated by a bacterial response to infection and facilitate repair by T4 RNA ligase. DNA repair enzymes that share conserved motifs with PNK have been identified in eukaryotes. PNK contains two functionally distinct structural domains and forms a homotetramer. The C-terminal phosphatase domain is homologous to the L-2-haloacid dehalogenase family and the N-terminal kinase domain is homologous to adenylate kinase. The active sites have been characterized through structural homology analyses and visualization of bound substrate.

Bacteriophage T4↗
PubMed2002

Nuclease enzymes.

Explore the source record for details and available documents.

Endonucleases↗
PubMed2002

Design, activity, and structure of a highly specific artificial endonuclease.

We have generated an artificial highly specific endonuclease by fusing domains of homing endonucleases I-DmoI and I-CreI and creating a new 1400 A(2) protein interface between these domains. Protein engineering was accomplished by combining computational redesign and an in vivo protein-folding screen. The resulting enzyme, E-DreI (Engineered I-DmoI/I-CreI), binds a long chimeric DNA target site with nanomolar affinity, cleaving it precisely at a rate equivalent to its natural parents. The structure of an E-DreI/DNA complex demonstrates the accuracy of the protein interface redesign algorithm and reveals how catalytic function is maintained during the creation of the new endonuclease. These results indicate that it may be possible to generate novel highly specific DNA binding proteins from homing endonucleases.

Algorithms↗