Biomedical subjects

Duncan E McRee

Publications and source records attributed to Duncan E McRee.

At least 19 recordsLinked to original sources

PubMed2006

Structural basis for the inhibition of Aurora A kinase by a novel class of high affinity disubstituted pyrimidine inhibitors.

The 2.25 A crystal structure of a complex of Aurora A kinase (AIKA) with cyclopropanecarboxylic acid-(3-(4-(3-trifluoromethyl-phenylamino)-pyrimidin-2-ylamino)-phenyl)-amide 1 is described here. The inhibitor binding mode is novel, with the cyclopropanecarboxylic acid moiety directed towards the solvent exposed region of the ATP-binding pocket, and several induced structural changes in the active-site compared with other published AIK structures. This structure provides context for the available SAR data on this compound class, and could be exploited for the design of analogs with increased affinity and selectivity for AIK.

Animals↗

PubMed2006

Two conformations of a crystalline human tRNA synthetase-tRNA complex: implications for protein synthesis.

Aminoacylation of tRNA is the first step of protein synthesis. Here, we report the co-crystal structure of human tryptophanyl-tRNA synthetase and tRNATrp. This enzyme is reported to interact directly with elongation factor 1alpha, which carries charged tRNA to the ribosome. Crystals were generated from a 50/50% mixture of charged and uncharged tRNATrp. These crystals captured two conformations of the complex, which are nearly identical with respect to the protein and a bound tryptophan. They are distinguished by the way tRNA is bound. In one, uncharged tRNA is bound across the dimer, with anticodon and acceptor stem interacting with separate subunits. In this cross-dimer tRNA complex, the class I enzyme has a class II-like tRNA binding mode. This structure accounts for biochemical investigations of human TrpRS, including species-specific charging. In the other conformation, presumptive aminoacylated tRNA is bound only by the anticodon, the acceptor stem being free and having space to interact precisely with EF-1alpha, suggesting that the product of aminoacylation can be directly handed off to EF-1alpha for the next step of protein synthesis.

Amino Acid Sequence↗

PubMed2004

Novel anion-independent iron coordination by members of a third class of bacterial periplasmic ferric ion-binding proteins.

The uptake of the element iron is vital for the survival of most organisms. Numerous pathogenic Gram-negative bacteria utilize a periplasm-to-cytosol ATP-binding cassette transport pathway to transport this essential atom in to the cell. In this study, we investigated the Yersinia enterocolitica (YfuA) and Serratia marcescens (SfuA) iron-binding periplasmic proteins. We have determined the 1.8-angstroms structures of iron-loaded (YfuA) and iron-free (SfuA) forms of this class of proteins. Although the sequence of these proteins varies considerably from the other members of the transferrin structural superfamily, they adopt the same three-dimensional fold. The iron-loaded YfuA structure illustrates the unique nature of this new class of proteins in that they are able to octahedrally coordinate the ferric ion in the absence of a bound anion. The iron-free SfuA structure contains a bound citrate anion in the iron-binding cleft that tethers the N- and C-terminal domains of the apo protein and stabilizes the partially open structure.

Amino Acid Sequence↗

PubMed2004

Differential evolution for protein crystallographic optimizations.

Genetic algorithms are powerful optimizers that have been underutilized in protein crystallography, given that many crystallographic problems have characteristics that would benefit from these algorithms: non-linearity, interdependent parameters and a complex function landscape. These functions have been implemented for real-space optimizations in a new fitting program, MIfit, for real-space refinement of protein models and heavy-atom searches. Some programming tips and examples will be presented here to aid others who might want to use genetic algorithms in their own work.

Algorithms↗

PubMed2004

Alanyl-tRNA synthetase crystal structure and design for acceptor-stem recognition.

Early work on aminoacylation of alanine-specific tRNA (tRNA(Ala)) by alanyl-tRNA synthetase (AlaRS) gave rise to the concept of an early "second genetic code" imbedded in the acceptor stems of tRNAs. A single conserved and position-specific G:U base pair in the tRNA acceptor stem is the key identity determinant. Further understanding has been limited due to lack of a crystal structure of the enzyme. We determined a 2.14 A crystal structure of the 453 amino acid catalytic fragment of Aquifex aeolicus AlaRS. It contains the catalytic domain characteristic of class II synthetases, a helical domain with a hairpin motif critical for acceptor-stem recognition, and a C-terminal domain of a mixed alpha/beta fold. Docking of tRNA(Ala) on AlaRS shows critical contacts with the three domains, consistent with previous mutagenesis and functional data. It also suggests conformational flexibility within the C domain, which might allow for the positional variation of the key G:U base pair seen in some tRNA(Ala)s.

Alanine-tRNA Ligase↗

PubMed2004

Structural snapshots of human HDAC8 provide insights into the class I histone deacetylases.

Modulation of the acetylation state of histones plays a pivotal role in the regulation of gene expression. Histone deacetylases (HDACs) catalyze the removal of acetyl groups from lysines near the N termini of histones. This reaction promotes the condensation of chromatin, leading to repression of transcription. HDAC deregulation has been linked to several types of cancer, suggesting a potential use for HDAC inhibitors in oncology. Here we describe the first crystal structures of a human HDAC: the structures of human HDAC8 complexed with four structurally diverse hydroxamate inhibitors. This work sheds light on the catalytic mechanism of the HDACs, and on differences in substrate specificity across the HDAC family. The structure also suggests how phosphorylation of Ser39 affects HDAC8 activity.

Amino Acid Sequence↗

PubMed2004

Structural basis for iron binding and release by a novel class of periplasmic iron-binding proteins found in gram-negative pathogens.

We have determined the 1.35- and 1.45-A structures, respectively, of closed and open iron-loaded forms of Mannheimia haemolytica ferric ion-binding protein A. M. haemolytica is the causative agent in the economically important and fatal disease of cattle termed shipping fever. The periplasmic iron-binding protein of this gram-negative bacterium, which has homologous counterparts in many other pathogenic species, performs a key role in iron acquisition from mammalian host serum iron transport proteins and is essential for the survival of the pathogen within the host. The ferric (Fe(3+)) ion in the closed structure is bound by a novel asymmetric constellation of four ligands, including a synergistic carbonate anion. The open structure is ligated by three tyrosyl residues and a dynamically disordered solvent-exposed anion. Our results clearly implicate the synergistic anion as the primary mediator of global protein conformation and provide detailed insights into the molecular mechanisms of iron binding and release in the periplasm.

Animals↗

PubMed2003

Crystal structures that suggest late development of genetic code components for differentiating aromatic side chains.

Early forms of the genetic code likely generated "statistical" proteins, with similar side chains occupying the same sequence positions at different ratios. In this scenario, groups of related side chains were treated by aminoacyl-tRNA synthetases as a single molecular species until a discrimination mechanism developed that could separate them. The aromatic amino acids tryptophan, tyrosine, and phenylalanine likely constituted one of these groups. A crystal structure of human tryptophanyl-tRNA synthetase was solved at 2.1 A with a tryptophanyl-adenylate bound at the active site. A cocrystal structure of an active fragment of human tyrosyl-tRNA synthetase with its cognate amino acid analog was also solved at 1.6 A. The two structures enabled active site identifications and provided the information for structure-based sequence alignments of approximately 45 orthologs of each enzyme. Two critical positions shared by all tyrosyl-tRNA synthetases and tryptophanyl-tRNA synthetases for amino acid discrimination were identified. The variations at these two positions and phylogenetic analyses based on the structural information suggest that, in contrast to many other amino acids, discrimination of tyrosine from tryptophan occurred late in the development of the genetic code.

Adenosine Monophosphate↗

PubMed2003

Presence of ferric hydroxide clusters in mutants of Haemophilus influenzae ferric ion-binding protein A.

The periplasmic iron binding protein plays an essential role in the iron uptake pathway of Gram-negative pathogenic bacteria from the Pasteurellaceae and Neisseriaceae families and is critical for survival of these pathogens within the host. In this study, we report the crystal structures of two mutant forms of ferric ion-binding protein A (FbpA) from Haemophilus influenzae with bound multinuclear oxo-metal clusters. Crystals of site-directed mutants in the metal or anion binding ligands contain protein in the open conformation, and two mutant FbpAs, H9A and N175L, contain different cluster arrangements in the iron-binding pocket. The iron clusters are anchored by binding to the two tyrosine ligands (Tyr195 and Tyr196) positioned at the vertex of the iron-binding pocket but are not coordinated by the other metal binding ligands. Our results suggest that the metal clusters may have formed in situ, suggesting that the mutant FbpAs may serve as a simple model for protein-mediated mineralization.

Alanine↗

PubMed2003

Crystal structure of Pasteurella haemolytica ferric ion-binding protein A reveals a novel class of bacterial iron-binding proteins.

Pasteurellosis caused by the Gram-negative pathogen Pasteurella haemolytica is a serious disease leading to death in cattle. To scavenge growth-limiting iron from the host, the pathogen utilizes the periplasmic ferric ion-binding protein A (PhFbpA) as a component of an ATP-binding cassette transport pathway. We report the 1.2-A structure of the iron-free (apo) form of PhFbpA, which is a member of the transferrin structural superfamily. The protein structure adopts a closed conformation, allowing us to reliably assign putative iron-coordinating residues. Based on our analysis, PhFbpA utilizes a unique constellation of binding site residues and anions to octahedrally coordinate an iron atom. A surprising finding in the structure is the presence of two formate anions on opposite sides of the iron-binding pocket. The formate ions tether the N- and C-terminal domains of the protein and stabilize the closed structure, also providing clues as to probable candidates for synergistic anions in the iron-loaded state. PhFbpA represents a new class of bacterial iron-binding proteins.

Bacterial Outer Membrane Proteins↗

PubMed2003

High-resolution structure of the soluble, respiratory-type Rieske protein from Thermus thermophilus: analysis and comparison.

The structure of the soluble Rieske protein from Thermus thermophilus has been determined at a resolution of 1.3 A at pH 8.5 using multiwavelength anomalous dispersion (MAD) techniques. This is the first report of a Rieske protein from a menaquinone-utilizing organism. The structure shows an overall fold similar to previously reported Rieske proteins. A novel feature of this crystal form appears to be a shared hydrogen between the His-134 imidazole ring ligated to Fe2 of the [2Fe-2S] cluster and its symmetry partner, His-134', one being formally an imidazolate anion, Fe2-(His-134)N(epsilon)(-)...H-N(epsilon')(His-134')-Fe2', in which crystallographic C(2) axes pass equidistant between N(epsilon)...N(epsilon') and normal to the line defined by N(epsilon)...N(epsilon'). This provides evidence for a stable, oxidized cluster with a His(-) ligand and lends support to a previously proposed mechanism of coupled proton and electron transfer. A detailed comparison of the Thermus Rieske protein with six other Rieske and Rieske-type proteins indicates: (a) The cluster binding domain is tightly conserved. (b) The 3-D structure of the 10 beta-strand fold is conserved, even among the most divergent proteins. (c) There is an approximately linear relation between acid-pH redox potential and number of H-bonds to the cluster. (d) These proteins have two faces, one points into the larger complex (bc(1), b(6)f, or other), is involved in the proton coupled electron transfer function, and is highly conserved. The second is oriented toward the solvent and shows wide variation in charge, sequence, length, hydrophobicity, and secondary elements in the loops that connect the beta-sheets.

Amino Acid Sequence↗

PubMed2003

Structure of a c-kit product complex reveals the basis for kinase transactivation.

The c-Kit proto-oncogene is a receptor protein-tyrosine kinase associated with several highly malignant human cancers. Upon binding its ligand, stem cell factor (SCF), c-Kit forms an active dimer that autophosphorylates itself and activates a signaling cascade that induces cell growth. Disease-causing human mutations that activate SCF-independent constitutive expression of c-Kit are found in acute myelogenous leukemia, human mast cell disease, and gastrointestinal stromal tumors. We report on the phosphorylation state and crystal structure of a c-Kit product complex. The c-Kit structure is in a fully active form, with ordered kinase activation and phosphate-binding loops. These results provide key insights into the molecular basis for c-Kit kinase transactivation to assist in the design of new competitive inhibitors targeting activated mutant forms of c-Kit that are resistant to current chemotherapy regimes.

Chromatography, Liquid↗

PubMed2003

High-resolution crystal structures and spectroscopy of native and compound I cytochrome c peroxidase.

Cytochrome c peroxidase (CCP) is a 32.5 kDa mitochondrial intermembrane space heme peroxidase from Saccharomyces cerevisiae that reduces H(2)O(2) to 2H(2)O by oxidizing two molecules of cytochrome c (cyt c). Here we compare the 1.2 A native structure (CCP) with the 1.3 A structure of its stable oxidized reaction intermediate, Compound I (CCP1). In addition, crystals were analyzed by UV-vis absorption and electron paramagnetic resonance spectroscopies before and after data collection to determine the state of the Fe(IV) center and the cationic Trp191 radical formed in Compound I. The results show that X-ray exposure does not lead to reduction of Fe(IV) and only partial reduction of the Trp radical. A comparison of the two structures reveals subtle but important conformational changes that aid in the stabilization of the Trp191 cationic radical in Compound I. The higher-resolution data also enable a more accurate determination of changes in heme parameters. Most importantly, when one goes from resting state Fe(III) to Compound I, the His-Fe bond distance increases, the iron moves into the porphyrin plane leading to shorter pyrrole N-Fe bonds, and the Fe(IV)-O bond distance is 1.87 A, suggesting a single Fe(IV)-O bond and not the generally accepted double bond.

Crystallography, X-Ray↗

PubMed2003

High resolution structure of an alternate form of the ferric ion binding protein from Haemophilus influenzae.

The periplasmic iron binding protein of pathogenic Gram-negative bacteria performs an essential role in iron acquisition from transferrin and other iron sources. Structural analysis of this protein from Haemophilus influenzae identified four amino acids that ligand the bound iron: His(9), Glu(57), Tyr(195), and Tyr(196). A phosphate provides an additional ligand, and the presence of a water molecule is required to complete the octahedral geometry for stable iron binding. We report the 1.14-A resolution crystal structure of the iron-loaded form of the H. influenzae periplasmic ferric ion binding protein (FbpA) mutant H9Q. This protein was produced in the periplasm of Escherichia coli and, after purification and conversion to the apo form, was iron-loaded. H9Q is able to bind ferric iron in an open conformation. A surprising finding in the present high resolution structure is the presence of EDTA located at the previously determined anion ternary binding site, where phosphate is located in the wild type holo and apo structures. EDTA contributes four of the six coordinating ligands for iron, with two Tyr residues, 195 and 196, completing the coordination. This is the first example of a metal binding protein with a bound metal.EDTA complex. The results suggest that FbpA may have the ability to bind and transport iron bound to biological chelators, in addition to bare ferric iron.

Bacterial Proteins↗

PubMed2003

A fully integrated protein crystallization platform for small-molecule drug discovery.

Structure-based drug discovery in the pharmaceutical industry benefits from cost-efficient methodologies that quickly assess the feasibility of specific, often refractory, protein targets to form well-diffracting crystals. By tightly coupling construct and purification diversity with nanovolume crystallization, the Structural Biology Group at Syrrx has developed such a platform to support its small-molecule drug-discovery program. During the past 18 months of operation at Syrrx, the Structural Biology Group has executed several million crystallization and imaging trials on over 400 unique drug-discovery targets. Here, key components of the platform, as well as an analysis of some experimental results that allowed for platform optimization, will be described.

Cloning, Molecular↗

PubMed2003

The crystal structure of shikimate dehydrogenase (AroE) reveals a unique NADPH binding mode.

Shikimate dehydrogenase catalyzes the NADPH-dependent reversible reduction of 3-dehydroshikimate to shikimate. We report the first X-ray structure of shikimate dehydrogenase from Haemophilus influenzae to 2.4-A resolution and its complex with NADPH to 1.95-A resolution. The molecule contains two domains, a catalytic domain with a novel open twisted alpha/beta motif and an NADPH binding domain with a typical Rossmann fold. The enzyme contains a unique glycine-rich P-loop with a conserved sequence motif, GAGGXX, that results in NADPH adopting a nonstandard binding mode with the nicotinamide and ribose moieties disordered in the binary complex. A deep pocket with a narrow entrance between the two domains, containing strictly conserved residues primarily contributed by the catalytic domain, is identified as a potential 3-dehydroshikimate binding pocket. The flexibility of the nicotinamide mononucleotide portion of NADPH may be necessary for the substrate 3-dehydroshikimate to enter the pocket and for the release of the product shikimate.

Alcohol Oxidoreductases↗

PubMed2003

Crystal structures of active fully assembled substrate- and product-bound complexes of UDP-N-acetylmuramic acid:L-alanine ligase (MurC) from Haemophilus influenzae.

UDP-N-acetylmuramic acid:L-alanine ligase (MurC) catalyzes the addition of the first amino acid to the cytoplasmic precursor of the bacterial cell wall peptidoglycan. The crystal structures of Haemophilus influenzae MurC in complex with its substrate UDP-N-acetylmuramic acid (UNAM) and Mg(2+) and of a fully assembled MurC complex with its product UDP-N-acetylmuramoyl-L-alanine (UMA), the nonhydrolyzable ATP analogue AMPPNP, and Mn(2+) have been determined to 1.85- and 1.7-A resolution, respectively. These structures reveal a conserved, three-domain architecture with the binding sites for UNAM and ATP formed at the domain interfaces: the N-terminal domain binds the UDP portion of UNAM, and the central and C-terminal domains form the ATP-binding site, while the C-terminal domain also positions the alanine. An active enzyme structure is thus assembled at the common domain interfaces when all three substrates are bound. The MurC active site clearly shows that the gamma-phosphate of AMPPNP is positioned between two bound metal ions, one of which also binds the reactive UNAM carboxylate, and that the alanine is oriented by interactions with the positively charged side chains of two MurC arginine residues and the negatively charged alanine carboxyl group. These results indicate that significant diversity exists in binding of the UDP moiety of the substrate by MurC and the subsequent ligases in the bacterial cell wall biosynthesis pathway and that alterations in the domain packing and tertiary structure allow the Mur ligases to bind sequentially larger UNAM peptide substrates.

Adenosine Triphosphate↗

PubMed2002

Crystal structure of a human aminoacyl-tRNA synthetase cytokine.

The 20 aminoacyl-tRNA synthetases catalyze the first step of protein synthesis and establish the rules of the genetic code through aminoacylation reactions. Biological fragments of two human enzymes, tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase, connect protein synthesis to cell-signaling pathways including angiogenesis. Alternative splicing or proteolysis produces these fragments. The proangiogenic N-terminal fragment mini-TyrRS has IL-8-like cytokine activity that, like other CXC cytokines, depends on a Glu-Leu-Arg motif. Point mutations in this motif abolish cytokine activity. The full-length native TyrRS lacks cytokine activity. No structure has been available for any mammalian tRNA synthetase that, in turn, might give insight into why mini-TyrRS and not TyrRS has cytokine activities. Here, the structure of human mini-TyrRS, which contains both the catalytic and the anticodon recognition domain, is reported to a resolution of 1.18 A. The critical Glu-Leu-Arg motif is located on an internal alpha-helix of the catalytic domain, where the guanidino side chain of R is part of a hydrogen-bonding network tethering the anticodon-recognition domain back to the catalytic site. Whereas the catalytic domains of the human and bacterial enzymes superimpose, the spatial disposition of the anticodon recognition domain relative to the catalytic domain is unique in mini-TyrRS relative to the bacterial orthologs. This unique orientation of the anticodon-recognition domain can explain why the fragment mini-TyrRS, and not full-length native TyrRS, is active in cytokine-signaling pathways.

Alternative Splicing↗