Biomedical subjects

Mark Bartlam

Publications and source records attributed to Mark Bartlam.

At least 19 recordsLinked to original sources

PubMed2006

Preliminary crystallographic analysis of avian infectious bronchitis virus main protease.

Infectious bronchitis virus (IBV) is the prototype of the genus Coronavirus. It causes a highly contagious disease which affects the respiratory, reproductive, neurological and renal systems of chickens, resulting great economic losses in the poultry industry worldwide. The coronavirus (CoV) main protease (M(pro)), which plays a pivotal role in viral gene expression and replication through a highly complex cascade involving the proteolytic processing of replicase polyproteins, is an attractive target for antiviral drug design. In this study, IBV M(pro) was overexpressed in Escherichia coli. Crystals suitable for X-ray crystallography have been obtained using microseeding techniques and belong to space group P6(1)22. X-ray diffraction data were collected in-house to 2.7 A resolution from a single crystal. The unit-cell parameters were a = b = 119.1, c = 270.7 A, alpha = beta = 90, gamma = 120 degrees. Three molecules were predicted to be present in the asymmetric unit from a calculated self-rotation function.

Animals↗

PubMed2006

Production of authentic SARS-CoV M(pro) with enhanced activity: application as a novel tag-cleavage endopeptidase for protein overproduction.

The viral proteases have proven to be the most selective and useful for removing the fusion tags in fusion protein expression systems. As a key enzyme in the viral life-cycle, the main protease (M(pro)) is most attractive for drug design targeting the SARS coronavirus (SARS-CoV), the etiological agent responsible for the outbreak of severe acute respiratory syndrome (SARS) in 2003. In this study, SARS-CoV M(pro) was used to specifically remove the GST tag in a new fusion protein expression system. We report a new method to produce wild-type (WT) SARS-CoV M(pro) with authentic N and C termini, and compare the activity of WT protease with those of three different types of SARS-CoV M(pro) with additional residues at the N or C terminus. Our results show that additional residues at the N terminus, but not at the C terminus, of M(pro) are detrimental to enzyme activity. To explain this, the crystal structures of WT SARS-CoV M(pro) and its complex with a Michael acceptor inhibitor were determined to 1.6 Angstroms and 1.95 Angstroms resolution respectively. These crystal structures reveal that the first residue of this protease is important for sustaining the substrate-binding pocket and inhibitor binding. This study suggests that SARS-CoV M(pro) could serve as a new tag-cleavage endopeptidase for protein overproduction, and the WT SARS-CoV M(pro) is more appropriate for mechanistic characterization and inhibitor design.

Calbindins↗

PubMed2006

An insight into the mechanism of human cysteine dioxygenase. Key roles of the thioether-bonded tyrosine-cysteine cofactor.

Cysteine dioxygenase is a non-heme mononuclear iron metalloenzyme that catalyzes the oxidation of cysteine to cysteine sulfinic acid with addition of molecular dioxygen. This irreversible oxidative catabolism of cysteine initiates several important metabolic pathways related to diverse sulfurate compounds. Cysteine dioxygenase is therefore very important for maintaining the proper hepatic concentration of intracellular free cysteine. Mechanisms for mouse and rat cysteine dioxygenases have recently been reported based on their crystal structures in the absence of substrates, although there is still a lack of direct evidence. Here we report the first crystal structure of human cysteine dioxygenase in complex with its substrate L-cysteine to 2.7A, together with enzymatic activity and metal content assays of several single point mutants. Our results provide an insight into a new mechanism of cysteine thiol dioxygenation catalyzed by cysteine dioxygenase, which is tightly associated with a thioether-bonded tyrosine-cysteine cofactor involving Tyr-157 and Cys-93. This cross-linked protein-derived cofactor plays several key roles different from those in galactose oxidase. This report provides a new potential target for therapy of diseases related to human cysteine dioxygenase, including neurodegenerative and autoimmune diseases.

Amino Acid Sequence↗

PubMed2006

Structure of human spindlin1. Tandem tudor-like domains for cell cycle regulation.

Spindlin1, a meiotic spindle-binding protein that is highly expressed in ovarian cancer cells, was first identified as a gene involved in gametogenesis. It appeared to be a target for cell cycle-dependent phosphorylation and was demonstrated to disturb the cell cycle. Here we report the crystal structure of human spindlin1 to 2.2A of resolution, representing the first three-dimensional structure from the spin/ssty (Y-linked spermiogenesis-specific transcript) gene family. The refined structure, containing three repeats of five/four anti-parallel beta-strands, exhibits a novel arrangement of tandem Tudor-like domains. Two phosphate ions, chelated by Thr-95 and other residues, appear to stabilize the long loop between domains I and II, which might mediate the cell cycle regulation activity of spindlin1. Flow cytometry experiments indicate that cells expressing spindlin1 display a different cell cycle distribution in mitosis, whereas those expressing a T95A mutant, which had a great decrease in phosphorous content, have little effect on the cell cycle. We further identified associations of spindlin1 with nucleic acid to provide a biochemical basis for its cell cycle regulation and other functions.

Amino Acid Sequence↗

PubMed2006

Structural basis of Robo proline-rich motif recognition by the srGAP1 Src homology 3 domain in the Slit-Robo signaling pathway.

The Slit-Robo (sr) GTPase-activating protein (GAPs) are important components in the intracellular pathway mediating Slit-Robo signaling in axon guidance and cell migration. We report the first crystal structure of the srGAP1 SH3 domain at 1.8-A resolution. The unusual side chain conformation of the conserved Phe-13 in the P1 pocket renders the ligand binding pocket shallow and narrow, which contributes toward the low binding affinity. Moreover, the opposing electrostatic charge and the hydrophobic properties of the P3 specificity pocket are consistent with the observed binding characteristics of the srGAP1 SH3 domain to its ligand. Surface plasmon resonance experiments indicate that the srGAP1 SH3 domain interacts with its natural ligand inaCtoN orientation. The srGAP1 SH3 domain can bind to both the CC2 and CC3 motifs in vitro. The N-terminal two acidic residues in the CC3 motif recognition site are necessary for srGAP1 SH3 domain binding. A longer CC3 peptide (CC3-FL) binds with greater affinity than its shorter counterpart, suggesting that the residues surrounding the proline-rich core are important for protein-peptide interactions. Our study reveals previously unknown properties of the srGAP-Robo interaction. Our data provide a structural basis for the srGAP-Robo interaction, consistent with the role of the Robo intracellular domain in interacting with other downstream signaling molecules and mediating versatile and dynamic responses to axon guidance and cell migration cues.

Amino Acid Motifs↗

PubMed2006

Expression, crystallization and preliminary X-ray studies of the immunoglobulin-like domain 3 of human palladin.

Palladin is a member of the recently discovered palladin/myotilin/myopalladin family, the members of which associate with alpha-actinin. Palladin may play important roles in actin stress-fibre formation, cell adhesion and migration. The immunoglobulin-like domain 3 of human palladin has been overexpressed in Escherichia coli and crystallized suitable for X-ray crystallographic study. Crystals have been obtained using the vapour-diffusion method and belong to space group P2(1). X-ray diffraction data were collected in-house to 1.8 A resolution from a single crystal. The unit-cell parameters are a = 40.9, b = 33.3, c = 34.8 A, beta = 90.3 degrees . One molecule was predicted to be present in the asymmetric unit.

Cloning, Molecular↗

PubMed2006

Crystal structure of human pyrroline-5-carboxylate reductase.

Pyrroline-5-carboxylate reductase (P5CR) is a universal housekeeping enzyme that catalyzes the reduction of Delta(1)-pyrroline-5-carboxylate (P5C) to proline using NAD(P)H as the cofactor. The enzymatic cycle between P5C and proline is very important for the regulation of amino acid metabolism, intracellular redox potential, and apoptosis. Here, we present the 2.8 Angstroms resolution structure of the P5CR apo enzyme, its 3.1 Angstroms resolution ternary complex with NAD(P)H and substrate-analog. The refined structures demonstrate a decameric architecture with five homodimer subunits and ten catalytic sites arranged around a peripheral circular groove. Mutagenesis and kinetic studies reveal the pivotal roles of the dinucleotide-binding Rossmann motif and residue Glu221 in the human enzyme. Human P5CR is thermostable and the crystals were grown at 37 degrees C. The enzyme is implicated in oxidation of the anti-tumor drug thioproline.

Amino Acid Sequence↗

PubMed2006

Purification, characterization, and crystallization of human pyrroline-5-carboxylate reductase.

Pyrroline-5-carboxylate reductase (P5CR) catalyzes the reduction of Delta1-pyrroline-5-carboxylate (P5C) to proline with concomitant oxidation of NAD(P)H to NAD(P)(+). The enzymatic cycle between P5C and proline is very important in many physiological and pathological processes. Human P5CR was over-expressed in Escherichia coli and purified to homogeneity by chromatography. Enzymatic assays of the wild-type protein were carried out using 3,4-dehydro-L-proline as substrate and NAD(+) as cofactor. The homopolymer was characterized by cross-linking and size exclusion gel filtration chromatography. Human P5CR was crystallized by the hanging-drop vapor-diffusion method at 37 degrees C. Diffraction data were obtained to a resolution of 2.8A and were suitable for high resolution X-ray structure determination.

Crystallization↗

PubMed2006

Crystal structures of human glycerol 3-phosphate dehydrogenase 1 (GPD1).

Homo sapiens L-alpha-glycerol-3-phosphate dehydrogenase 1 (GPD1) catalyzes the reversible biological conversion of dihydroxyacetone (DHAP) to glycerol-3-phosphate. The GPD1 protein was expressed in Escherichia coli, and purified as a fusion protein with glutathione S-transferase. Here we report the apoenzyme structure of GPD1 determined by multiwavelength anomalous diffraction phasing, and other complex structures with small molecules (NAD+ and DHAP) by the molecular replacement method. This enzyme structure is organized into two distinct domains, the N-terminal eight-stranded beta-sheet sandwich domain and the C-terminal helical substrate-binding domain. An electrophilic catalytic mechanism by the epsilon-NH3+ group of Lys204 is proposed on the basis of the structural analyses. In addition, the inhibitory effects of zinc and sulfate on GPDHs are assayed and discussed.

Catalysis↗

PubMed2006

Crystal structures of Nipah and Hendra virus fusion core proteins.

The Nipah and Hendra viruses are highly pathogenic paramyxoviruses that recently emerged from flying foxes to cause serious disease outbreaks in humans and livestock in Australia, Malaysia, Singapore and Bangladesh. Their unique genetic constitution, high virulence and wide host range set them apart from other paramyxoviruses. These characteristics have led to their classification into the new genus Henpavirus within the family Paramyxoviridae and to their designation as Biosafety Level 4 pathogens. The fusion protein, an enveloped glycoprotein essential for viral entry, belongs to the family of class I fusion proteins and is characterized by the presence of two heptad repeat (HR) regions, HR1 and HR2. These two regions associate to form a fusion-active hairpin conformation that juxtaposes the viral and cellular membranes to facilitate membrane fusion and enable subsequent viral entry. The Hendra and Nipah virus fusion core proteins were crystallized and their structures determined to 2.2 A resolution. The Nipah and Hendra fusion core structures are six-helix bundles with three HR2 helices packed against the hydrophobic grooves on the surface of a central coiled coil formed by three parallel HR1 helices in an oblique antiparallel manner. Because of the high level of conservation in core regions, it is proposed that the Nipah and Hendra virus fusion cores can provide a model for membrane fusion in all paramyxoviruses. The relatively deep grooves on the surface of the central coiled coil represent a good target site for drug discovery strategies aimed at inhibiting viral entry by blocking hairpin formation.

Amino Acid Sequence↗

PubMed2006

Dodecamer structure of severe acute respiratory syndrome coronavirus nonstructural protein nsp10.

The severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural proteins nsp1 to nsp16 have been implicated by genetic analysis in the assembly of a functional replication/transcription complex. We report the crystal structure of nsp10 from SARS-CoV at 2.1-A resolution. The nsp10 structure has a novel fold, and 12 identical subunits assemble to form a unique spherical dodecameric architecture. Two zinc fingers have been identified from the nsp10 monomer structure with the sequence motifs C-(X)2-C-(X)5-H-(X)6-C and C-(X)2-C-(X)7-C-(X)-C. The nsp10 crystal structure is the first of a new class of zinc finger protein three-dimensional structures to be revealed experimentally. The zinc finger sequence motifs are conserved among all three coronavirus antigenic groups, implicating an essential function for nsp10 in all coronaviruses. Based on the structure, we propose that nsp10 is a transcription factor for coronavirus replication/transcription.

Amino Acid Sequence↗

PubMed2006

New antiviral target revealed by the hexameric structure of mouse hepatitis virus nonstructural protein nsp15.

The unique coronavirus transcription/replication machinery comprised of multiple virus-encoded nonstructural proteins (nsp) plays a vital role during initial and intermediate phases of the viral life cycle. The crystal structure of mouse hepatitis virus strain A59 (MHV-A59) nsp15 is reported at 2.15-A resolution. nsp15 is an XendoU endoribonuclease and is the first one from this family to have its structure unveiled. The MHV-A59 nsp15 monomer structure has a novel protein fold. Two nsp15 trimers form a back-to-back hexamer that is believed to be the functional unit. The structure reveals the catalytic site including the highly conserved residues His262, His277, and Lys317, which is supported by mutagenesis analysis. Gel filtration and enzyme activity assays confirmed that the hexamer is the active form for nsp15 and demonstrate the specificity of nsp15 for uridylate. The high sequence conservation of nsp15 in coronaviruses, including that of severe acute respiratory syndrome, suggests that this protein may provide a new target for the design of antiviral therapeutics.

Amino Acid Sequence↗

PubMed2006

Crystallization and preliminary crystallographic analysis of allograft inflammatory factor 1.

Allograft inflammatory factor-1 (AIF-1) is a 17-kDa IFN-gamma inducible Ca2+-binding EF-hand protein involved in immune dysfunction and smooth muscle cell activation. AIF-1 was solubly expressed in E. coli and purified. Crystals of AIF-1 were grown at 291 K using PEG-8000 as precipitant. Diffraction by the AIF-1 crystal extends to 3.3 A resolution, and the crystal belongs to the space group P4(3) with unit cell parameters a=b=73.4, c=49.1 A.

Calcium-Binding Proteins↗

PubMed2006

Drug design targeting the main protease, the Achilles' heel of coronaviruses.

Coronaviruses (CoVs), a genus containing about 26 known species to date, cause highly prevalent diseases and are often severe or fatal in humans and animals. In 2003, a previously unknown coronavirus was identified to be the etiological agent of a global outbreak of a form of life-threatening pneumonia called severe acute respiratory syndrome (SARS). No efficacious therapy is currently available, and vaccines and drugs are under development to prevent SARS-CoV infection in many countries. The CoV main protease (M(pro)), which plays a pivotal role in viral gene expression and replication through a highly complex cascade involving the proteolytic processing of replicase polyproteins, is an attractive target for drug design. This review summarizes the recent advances in biological and structural studies, together with development of inhibitors targeting CoV M(pro)s. It is expected that inhibitors targeting CoV M(pro)s could be developed into wide-spectrum antiviral drugs against existing and possible future emerging CoV-associated diseases.

Amino Acid Sequence↗

PubMed2005

Crystal structure of the N-terminal SH3 domain of mouse betaPIX, p21-activated kinase-interacting exchange factor.

The mouse betaPIX-SH3 domain, residues 8-63 of P21-activated kinase interacting exchange factor, has been characterized by X-ray diffraction. Crystals belonging to space group P3(2)21 diffracted to 2.0 A and the structure was phased by the single-wavelength anomalous diffraction method. The domain is a compact beta-barrel with an overall conformation similar to the general SH3 structure. The X-ray structure shows mouse betaPIX-SH3 domain binding the way in which the betaPIX characteristic amino acids do so for an unconventional ligand binding surface. This arrangement provides a rationale for the unusual ligand recognition motif exhibited by mouse betaPIX-SH3 domain. Comparison with another SH3/peptide complex shows that the recognition mode of the mouse betaPIX-SH3 domain should be very similar to the RXXK ligand binding mode. The unique large and planar hydrophobic pocket may contribute to the promiscuity of betaPIX-SH3 domain resulting in its multiple biological functions.

Amino Acid Sequence↗

PubMed2005

Structural insights into SARS coronavirus proteins.

The SARS coronavirus was identified as the pathogen of a global outbreak of SARS (severe acute respiratory syndrome) in 2003. Its large RNA genome encodes four structural proteins, sixteen non-structural proteins and eight accessory proteins. The availability of structures of SARS coronavirus macromolecules has enabled the elucidation of their important functions, such as mediating the fusion of viral and host cellular membranes, and in replication and transcription. In particular, the spike protein fusion core and the main protease have been the most extensively studied, with the aim of designing anti-SARS therapeutics. Attention is now being focused on replicase proteins, which should enhance our understanding of the replication and transcription machinery. The structures and functions of most SARS proteins remain unknown, and further structural studies will be important for revealing their functions and for designing potential anti-SARS therapeutics.

Membrane Proteins↗

PubMed2005

Crystal structure of methyl parathion hydrolase from Pseudomonas sp. WBC-3.

Methyl parathion hydrolase (MPH, E.C.3.1.8.1), isolated from the soil-dwelling bacterium Pseudomonas sp. WBC-3, is a Zn(II)-containing enzyme that catalyzes the degradation of the organophosphate pesticide methyl parathion. We have determined the structure of MPH from Pseudomonas sp. WBC-3 to 2.4 angstroms resolution. The enzyme is dimeric and each subunit contains a mixed hybrid binuclear zinc center, in which one of the zinc ions is replaced by cadmium. In both subunits, the more solvent-exposed beta-metal ion is substituted for Cd2+ due to high cadmium concentration in the crystallization condition. Both ions are surrounded by ligands in an octahedral arrangement. The ions are separated by 3.5 angstroms and are coordinated by the amino acid residues His147, His149, Asp151, His152, His234 and His302 and a water molecule. Asp255 and a water molecule serve to bridge the zinc ions together. MPH is homologous with other metallo-beta-lactamases but does not show any similarity to phosphotriesterase that can also catalyze the degradation of methyl parathion with lower rate, despite the lack of sequence homology. Trp179, Phe196 and Phe119 form an aromatic cluster at the entrance of the catalytic center. Replacement of these three amino acids by alanine resulted in a significant increase of K(m) and loss of catalytic activity, indicating that the aromatic cluster has an important role to facilitate affinity of enzyme to the methyl parathion substrates.

Catalytic Domain↗

PubMed2005

Design of wide-spectrum inhibitors targeting coronavirus main proteases.

The genus Coronavirus contains about 25 species of coronaviruses (CoVs), which are important pathogens causing highly prevalent diseases and often severe or fatal in humans and animals. No licensed specific drugs are available to prevent their infection. Different host receptors for cellular entry, poorly conserved structural proteins (antigens), and the high mutation and recombination rates of CoVs pose a significant problem in the development of wide-spectrum anti-CoV drugs and vaccines. CoV main proteases (M(pro)s), which are key enzymes in viral gene expression and replication, were revealed to share a highly conservative substrate-recognition pocket by comparison of four crystal structures and a homology model representing all three genetic clusters of the genus Coronavirus. This conclusion was further supported by enzyme activity assays. Mechanism-based irreversible inhibitors were designed, based on this conserved structural region, and a uniform inhibition mechanism was elucidated from the structures of Mpro-inhibitor complexes from severe acute respiratory syndrome-CoV and porcine transmissible gastroenteritis virus. A structure-assisted optimization program has yielded compounds with fast in vitro inactivation of multiple CoV M(pro)s, potent antiviral activity, and extremely low cellular toxicity in cell-based assays. Further modification could rapidly lead to the discovery of a single agent with clinical potential against existing and possible future emerging CoV-related diseases.

Animals↗