Biomedical subjects

Howard Robinson

Publications and source records attributed to Howard Robinson.

At least 37 records · Page 2Linked to original sources

PubMed2005

The 1.9 A structure of a proteasome-11S activator complex and implications for proteasome-PAN/PA700 interactions.

Proteasomes are cylindrical structures that function in multiple cellular processes by degrading a wide variety of cytosolic and nuclear proteins. Substrate access and product release from the enclosed catalytic chamber occurs through axial pores that are opened by activator complexes. Here, we report high-resolution structures of wild-type and mutant archaeal proteasomes bound to the activator PA26. These structures support the proposal that an ordered open conformation is required for proteolysis and that its formation can be triggered by outward displacement of surrounding residues. The structures and associated biochemical assays reveal the mechanism of binding, which involves an interaction between the PA26 C terminus and a conserved lysine. Surprisingly, biochemical observations implicate an equivalent interaction for the unrelated ATP-dependent activators PAN and PA700.

Amino Acid Sequence↗

PubMed2005

Crystal structures of Aedes aegypti kynurenine aminotransferase.

Aedes aegypti kynurenine aminotransferase (AeKAT) catalyzes the irreversible transamination of kynurenine to kynurenic acid, the natural antagonist of NMDA and 7-nicotinic acetycholine receptors. Here, we report the crystal structure of AeKAT in its PMP and PLP forms at 1.90 and 1.55 A, respectively. The structure was solved by a combination of single-wavelength anomalous dispersion and molecular replacement approaches. The initial search model in the molecular replacement method was built with the result of single-wavelength anomalous dispersion data from the Br-AeKAT crystal in combination with homology modeling. The solved structure shows that the enzyme is a homodimer, and that the two subunits are stabilized by a number of hydrogen bonds, salts bridges, and hydrophobic interactions. Each subunit is divided into an N-terminal arm and small and large domains. Based on its folding, the enzyme belongs to the prototypical fold type, aminotransferase subgroup I. The three-dimensional structure shows a strictly conserved 'PLP-phosphate binding cup' featuring PLP-dependent enzymes. The interaction between Cys284 (A) and Cys284 (B) is unique in AeKAT, which might explain the cysteine effect of AeKAT activity. Further mutation experiments of this residue are needed to eventually understand the mechanism of the enzyme modulation by cysteine.

Aedes↗

PubMed2005

Crystal structures of the BlaI repressor from Staphylococcus aureus and its complex with DNA: insights into transcriptional regulation of the bla and mec operons.

The 14-kDa BlaI protein represses the transcription of blaZ, the gene encoding beta-lactamase. It is homologous to MecI, which regulates the expression of mecA, the gene encoding the penicillin binding protein PBP2a. These genes mediate resistance to beta-lactam antibiotics in staphylococci. Both repressors can bind either bla or mec DNA promoter-operator sequences. Regulated resistance genes are activated via receptor-mediated cleavage of the repressors. Cleavage is induced when beta-lactam antibiotics bind the extramembrane sensor of the sensor-transducer signaling molecules, BlaR1 or MecR1. The crystal structures of BlaI from Staphylococcus aureus, both in free form and in complex with 32 bp of DNA of the mec operator, have been determined to 2.0- and 2.7-A resolutions, respectively. The structure of MecI, also in free form and in complex with the bla operator, has been previously reported. Both repressors form homodimers, with each monomer composed of an N-terminal DNA binding domain of winged helix-turn-helix topology and a C-terminal dimerization domain. The structure of BlaI in complex with the mec operator shows a protein-DNA interface that is conserved between both mec and bla targets. The recognition helix alpha3 interacts specifically with the conserved TACA/TGTA DNA binding motif. BlaI and, probably, MecI dimers bind to opposite faces of the mec DNA double helix in an up-and-down arrangement, whereas MecI and, probably, BlaI dimers bind to the same DNA face of bla promoter-operator DNA. This is due to the different spacing of mec and bla DNA binding sites. Furthermore, the flexibility of the dimeric proteins may make the C-terminal proteolytic cleavage site more accessible when the repressors are bound to DNA than when they are in solution, suggesting that the induction cascade involves bound rather than free repressor.

Amino Acid Sequence↗

PubMed2004

Structural analysis of Pseudomonas 1-aminocyclopropane-1-carboxylate deaminase complexes: insight into the mechanism of a unique pyridoxal-5'-phosphate dependent cyclopropane ring-opening reaction.

1-Aminocyclopropane-1-carboxylate (ACC) deaminase is a pyridoxal 5'-phosphate (PLP) dependent enzyme catalyzing the opening of the cyclopropane ring of ACC to give alpha-ketobutyric acid and ammonia as the products. This ring cleavage reaction is unusual because the substrate, ACC, contains no abstractable alpha-proton and the carboxyl group is retained in the product. How the reaction is initiated to generate an alpha-carbanionic intermediate, which is the common entry for most PLP-dependent reactions, is not obvious. To gain insight into this unusual ring-opening reaction, we have solved the crystal structures of ACC deaminase from Pseudomonas sp. ACP in complex with substrate ACC, an inhibitor, 1-aminocyclopropane-1-phosphonate (ACP), the product alpha-ketobutyrate, and two d-amino acids. Several notable observations of these structural studies include the following: (1) a typically elusive gem-diamine intermediate is trapped in the enzyme complex with ACC or ACP; (2) Tyr294 is in close proximity (3.0 A) to the pro-S methylene carbon of ACC in the gem-diamine complexes, implicating a direct role of this residue in the ring-opening reaction; (3) Tyr294 may also be responsible for the abstraction of the alpha-proton from d-amino acids, a prelude to the subsequent deamination reaction; (4) the steric hindrance precludes accessibility of active site functional groups to the l-amino acid substrates and may account for the stereospecificity of this enzyme toward d-amino acids. These structural data provide evidence favoring a mechanism in which the ring cleavage is induced by a nucleophilic attack at the pro-S beta-methylene carbon of ACC, with Tyr294 as the nucleophile. However, these observations are also consistent with an alternative mechanistic possibility in which the ring opening is acid-catalyzed and may be facilitated by charge relay through PLP, where Tyr294 functions as a general acid. The results of mutagenesis studies corroborated the assigned critical role for Tyr294 in the catalysis.

Binding Sites↗

PubMed2004

Structure of 2C-methyl-D-erythritol-2,4-cyclodiphosphate synthase from Shewanella oneidensis at 1.6 A: identification of farnesyl pyrophosphate trapped in a hydrophobic cavity.

Isopentenyl pyrophosphate (IPP) is a universal building block for the ubiquitous isoprenoids that are essential to all organisms. The enzymes of the non-mevalonate pathway for IPP synthesis, which is unique to many pathogenic bacteria, have recently been explored as targets for antibiotic development. Several crystal structures of 2C-methyl-D-erythritol-2,4-cyclophosphate (MECDP) synthase, the fifth of seven enzymes involved in the non-mevalonate pathway for synthesis of IPP, have been reported; however, the composition of metal ions in the active site and the presence of a hydrophobic cavity along the non-crystallographic threefold symmetry axis has varied between the reported structures. Here, the structure of MEDCP from Shewanella oneidensis MR1 (SO3437) was determined to 1.6 A resolution in the absence of substrate. The presence of a zinc ion in the active-site cleft, tetrahedrally coordinated by two histidine side chains, an aspartic acid side chain and an ambiguous fourth ligand, was confirmed by zinc anomalous diffraction. Based on analysis of anomalous diffraction data and typical metal-to-ligand bond lengths, it was concluded that an octahedral sodium ion was 3.94 A from the zinc ion. A hydrophobic cavity was observed along the threefold non-crystallographic symmetry axis, filled by a well defined non-protein electron density that could be modeled as farnesyl pyrophosphate (FPP), a downstream product of IPP, suggesting a possible feedback mechanism for enzyme regulation. The high-resolution data clarified the FPP-binding mode compared with previously reported structures. Multiple sequence alignment indicated that the residues critical to the formation of the hydrophobic cavity and for coordinating the pyrophosphate group of FPP are present in the majority of MEDCP synthase enzymes, supporting the idea of a specialized biological function related to FPP binding in a subfamily of MEDCP synthase homologs.

Aldose-Ketose Isomerases↗

PubMed2004

Crystallization and preliminary X-ray diffraction study of the farnesyl diphosphate synthase from Trypanosoma brucei.

Farnesyl diphosphate synthase (FPPS) catalyses the formation of farnesyl diphosphate from dimethylallyl diphosphate and isopentenyl diphosphate and is an RNAi-validated drug target in Trypanosoma brucei, the causative agent of African sleeping sickness. A T. brucei FPPS (390 amino acids) has been expressed in Escherichia coli and the recombinant protein has been crystallized in the absence and presence of the bisphosphonate inhibitor minodronate. Diffraction data were collected at 100 K using synchrotron radiation from both crystal types. Crystals obtained in the absence of minodronate belong to space group I222, with unit-cell parameters a = 61.43, b = 118.12, c = 120.04 A, while crystals grown in the presence of minodronate belong to space group C2, with unit-cell parameters a = 131.98, b = 118.10, c = 63.25 A, beta = 112.48 degrees. An initial model of the drug-free protein has been built using a homology model with the molecular-replacement method and refined to 3.3 A resolution. It shows mostly helical structure and resembles the structure of avian farnesyl diphosphate synthase, but with the addition of two loop regions.

Alkyl and Aryl Transferases↗

PubMed2004

A dimerized coiled-coil domain and an adjoining part of geminin interact with two sites on Cdt1 for replication inhibition.

Geminin is a cellular protein that associates with Cdt1 and inhibits Mcm2-7 loading during S phase. It prevents multiple cycles of replication per cell cycle and prevents episome replication. It also directly inhibits the HoxA11 transcription factor. Here we report that geminin forms a parallel coiled-coil homodimer with atypical residues in the dimer interface. Point mutations that disrupt the dimerization abolish interaction with Cdt1 and inhibition of replication. An array of glutamic acid residues on the coiled-coil domain surface interacts with positive charges in the middle of Cdt1. An adjoining region interacts independently with the N-terminal 100 residues of Cdt1. Both interactions are essential for replication inhibition. The negative residues on the coiled-coil domain and a different part of geminin are also required for interaction with HoxA11. Therefore a rigid cylinder with negative surface charges is a critical component of a bipartite interaction interface between geminin and its cellular targets.

Amino Acid Sequence↗

PubMed2004

Crystal structure of phosphodiesterase 9 shows orientation variation of inhibitor 3-isobutyl-1-methylxanthine binding.

Cyclic nucleotide phosphodiesterases (PDEs) are enzymes controlling cellular concentrations of the second messengers cAMP and cGMP. The crystal structure of the catalytic domain of PDE9A2, a member of a PDE family specifically hydrolyzing cGMP, has been determined at 2.23-A resolution. The PDE9A2 catalytic domain closely resembles the cAMP-specific PDE4D2 but is significantly different from the cGMP-specific PDE5A1, implying that each individual PDE family has its own characteristic substrate recognition mechanism. The different conformations of the H and M loops between PDE9A2 and PDE5A1 imply their less critical roles in nucleotide recognition. The nonselective inhibitor 3-isobutyl-1-methylxanthine (IBMX) binds to a similar subpocket in the active sites of PDE4, PDE5, and PDE9 and has a common pattern of the binding. However, significantly different orientations and interactions of IBMXs are observed among the three PDE families and also between two monomers of the PDE9A2 dimer. The kinetic properties of the PDE9A2 catalytic domain similar to those of full-length PDE9A imply that the N-terminal regulatory domain does not significantly alter the catalytic activity and the IBMX inhibition.

1-Methyl-3-isobutylxanthine↗

PubMed2004

Crystal structure of the oxygen-dependant coproporphyrinogen oxidase (Hem13p) of Saccharomyces cerevisiae.

Coproporphyrinogen oxidase (CPO) is an essential enzyme that catalyzes the sixth step of the heme biosynthetic pathway. Unusually for heme biosynthetic enzymes, CPO exists in two evolutionarily and mechanistically distinct families, with eukaryotes and some prokaryotes employing members of the highly conserved oxygen-dependent CPO family. Here, we report the crystal structure of the oxygen-dependent CPO from Saccharomyces cerevisiae (Hem13p), which was determined by optimized sulfur anomalous scattering and refined to a resolution of 2.0 A. The protein adopts a novel structure that is quite different from predicted models and features a central flat seven-stranded anti-parallel sheet that is flanked by helices. The dimeric assembly, which is seen in different crystal forms, is formed by packing of helices and a short isolated strand that forms a beta-ladder with its counterpart in the partner subunit. The deep active-site cleft is lined by conserved residues and has been captured in open and closed conformations in two different crystal forms. A substratesized cavity is completely buried in the closed conformation by the approximately 8-A movement of a helix that forms a lid over the active site. The structure therefore suggests residues that likely play critical roles in catalysis and explains the deleterious effect of many of the mutations associated with the disease hereditary coproporphyria.

Amino Acid Sequence↗

PubMed2004

Structural mechanism for inactivation and activation of CAD/DFF40 in the apoptotic pathway.

CAD/DFF40 is responsible for the degradation of chromosomal DNA into nucleosomal fragments and subsequent chromatin condensation during apoptosis. It exists as an inactive complex with its inhibitor ICAD/DFF45 in proliferating cells but becomes activated upon cleavage of ICAD/DFF45 into three domains by caspases in dying cells. The molecular mechanism underlying the control and activation of CAD/DFF40 was unknown. Here, the crystal structure of activated CAD/DFF40 reveals that it is a pair of molecular scissors with a deep active-site crevice that appears ideal for distinguishing internucleosomal DNA from nucleosomal DNA. Ensuing studies show that ICAD/DFF45 sequesters the nonfunctional CAD/DFF40 monomer and is also able to disassemble the functional CAD/DFF40 dimer. This capacity requires the involvement of the middle domain of ICAD/DFF45, which by itself cannot remain bound to CAD/DFF40 due to low binding affinity for the enzyme. Thus, the consequence of the caspase-cleavage of ICAD/DFF45 is a self-assembly of CAD/DFF40 into the active dimer.

Amino Acid Sequence↗

PubMed2004

Crystal structure of the [Mg2+-(chromomycin A3)2]-d(TTGGCCAA)2 complex reveals GGCC binding specificity of the drug dimer chelated by a metal ion.

The anticancer antibiotic chromomycin A3 (Chro) is a DNA minor groove binding drug belonging to the aureolic family. Chro likely exerts its activity by interfering with replication and transcription. Chro forms a dimer, mediated by a divalent metal ion, which binds to G/C-rich DNA. Herein we report the first crystal structure of Chro bound to d(TTG GCCAA)2 DNA duplex solved by multiwavelength anomalous diffraction (MAD) based on the chelated Co3+ ion. The structure of the Mg2+ complex was subsequently refined at 2.15 A resolution, which revealed two complexes of metal-coordinated dimers of Chro bound to the octamer DNA duplex in the asymmetric unit. The metal ion is octahedrally coordinated to the O1 and O9 oxygen atoms of the chromophore (CPH), and two water molecules act as the fifth and sixth ligands. The two coordinated water molecules are hydrogen bonded to O2 atoms of C5 and C13 bases. The Chro dimer binds at and significantly widens the minor groove of the GGCC sequence. The long axis of each chromophore lies along and stacks over the sugar-phosphate backbone with the two attached saccharide moieties (rings A/B and C/D/E) wrapping across the minor groove. DNA is kinked by 30 degrees and 36 degrees in the two complexes, respectively. Six G-specific hydrogen bonds between Chro and DNA provide the GGCC sequence specificity. Interestingly, DNA in concert with Chro appears to act as an effective template to catalyze the deamination of Co(NH3)6(3+), as shown by circular dichroism and crystal structure data. Our results present useful structural information for designing new anticancer drug derivatives in the future.

Antibiotics, Antineoplastic↗

PubMed2004

Structure of C73G putidaredoxin from Pseudomonas putida.

The structure of the C73G mutant of putidaredoxin (Pdx), the Fe(2)S(2) ferredoxin that supplies electrons to cytochrome CYP101 (p450cam) for camphor oxidation, is reported at 1.9 A resolution in a C2 crystal form. The structure was solved by single-wavelength iron anomalous diffraction, which yielded electron density above the 2sigma level for over 97% of the non-H atoms in the protein. The final structure with R = 0.19 and R(free) = 0.21 has been deposited in the Protein Data Bank with accession code 1r7s. The C2 crystal contains three Pdx molecules in the asymmetric unit, giving three independent models of the protein that are very similar (r.m.s.d. < 0.3 A for the 106 C(alpha) atoms). The unusually high solvent fraction of 80% results in comparatively few crystal-packing artifacts. The structure is briefly compared with the recently reported crystal structures of the C73S and C73S/C85S mutants. In general, the eight independent molecules in the three crystal structures (three in C73G, three in C73S and two in C73S/C85S) are much more similar to each other than to the previously reported NMR structure of wild-type Pdx in solution. The present findings show a unanimous structure in some regions crucial for electron-transfer interactions, including the cluster-binding loop 39-48 and the cytochrome-interaction region of Asp38 and Trp106. In addition, the Cys45 amide group donates a hydrogen bond to cluster sulfur S1, with Ala46 adopting an Lalpha conformation, in all three molecules in the crystal.

Binding Sites↗

PubMed2004

Structure of the Chlamydia protein CADD reveals a redox enzyme that modulates host cell apoptosis.

The Chlamydia protein CADD (Chlamydia protein associating with death domains) has been implicated in the modulation of host cell apoptosis via binding to the death domains of tumor necrosis factor family receptors. Transfection of CADD into mammalian cells induces apoptosis. Here we present the CADD crystal structure, which reveals a dimer of seven-helix bundles. Each bundle contains a di-iron center adjacent to an internal cavity, forming an active site similar to that of methane mono-oxygenase hydrolase. We further show that CADD mutants lacking critical metal-coordinating residues are substantially less effective in inducing apoptosis but retain their ability to bind to death domains. We conclude that CADD is a novel redox protein toxin unique to Chlamydia species and propose that both its redox activity and death domain binding ability are required for its biological activity.

Amino Acid Sequence↗

PubMed2003

Crystallization and phasing of alanine dehydrogenase from Archaeoglobus fulgidus.

Alanine dehydrogenase (AlaDH) from the hyperthermophilic archaeon Archaeoglobus fulgidus is a dimer of 35 kDa chains. The archaeal enzyme appears to represent a new class of AlaDH that is not homologous to bacterial AlaDH enzymes, but has close evolutionary links to the broad ornithine cyclodeaminase/micro-crystallin family, which includes human thyroid hormone binding protein, which has 30% sequence identity to the A. fulgidus gene. The enzyme has been cloned, shown to catalyze the NAD-dependent interconversion of alanine and pyruvate and crystallized in several forms. Although the purified protein crystallized readily under many conditions, most of the crystals diffracted weakly or not at all. One polymorph growing in space group P2(1)2(1)2(1) has non-crystallographic symmetry that becomes crystallographic, changing the space group to P2(1)2(1)2, upon binding iridium or samarium. Before and after derivatization, these crystals diffracted to 2.5 A using synchrotron radiation. Multiwavelength diffraction data were collected from the non-isomorphous iridium derivative, enabling structure determination.

Alanine Dehydrogenase↗

PubMed2003

A 1.7A structure of Fve, a member of the new fungal immunomodulatory protein family.

Fve, a major fruiting body protein from Flammulina velutipes, a mushroom possessing immunomodulatory activity, stimulates lymphocyte mitogenesis, suppresses systemic anaphylaxis reactions and edema, enhances transcription of IL-2, IFN-gamma and TNF-alpha, and hemagglutinates red blood cells. It appears to be a lectin with specificity for complex cell-surface carbohydrates. Fve is a non-covalently linked homodimer containing no Cys, His or Met residues. It shares sequence similarity only to the other fungal immunomodulatory proteins (FIPs) LZ-8, Gts, Vvo and Vvl, all of unknown structure. The 1.7A structure of Fve solved by single anomalous diffraction of NaBr-soaked crystals is novel: each monomer consists of an N-terminal alpha-helix followed by a fibronectin III (FNIII) fold. The FNIII fold is the first instance of "pseudo-h-type" topology, a transition between the seven beta-stranded s-type and the eight beta-stranded h-type topologies. The structure suggests that dimerization, critical for the activity of FIPs, occurs by 3-D domain swapping of the N-terminal helices and is stabilized predominantly by hydrophobic interactions. The structure of Fve is the first in this lectin family to be reported, and the first of an FNIII domain-containing protein of fungal origin.

Adjuvants, Immunologic↗

PubMed2003

Crystal structure of DJ-1/RS and implication on familial Parkinson's disease.

DJ-1 is a protein involved in multiple physiological processes, including cancer, Parkinson's disease, and male fertility. It is unknown how DJ-1 functions in the apparently different systems. The crystal structure of DJ-1 at 1.6 A resolution shows that DJ-1 is a helix-strand-helix sandwich and forms a dimer. The DJ-1 structure is similar to the members of the intracellular protease PfpI family. However, the catalytic triad of Cys-His-Glu is not strictly conserved in DJ-1, implying that DJ-1 has a different catalytic mechanism if it acts as a protease or DJ-1 serves as a regulatory protein in the physiological processes. The structure shows that Leu166 positions in the middle of a helix and thus predicts that the L166P mutation will bend the helix and impact the dimerization of DJ-1. As a result, the conformational changes may diminish the DJ-1 binding with its partner, leading to the familial Parkinson's disease caused by the single L166P mutation.

Catalytic Domain↗

PubMed2003

Structure and ubiquitin binding of the ubiquitin-interacting motif.

Ubiquitylation is used to target proteins into a large number of different biological processes including proteasomal degradation, endocytosis, virus budding, and vacuolar protein sorting (Vps). Ubiquitylated proteins are typically recognized using one of several different conserved ubiquitin binding modules. Here, we report the crystal structure and ubiquitin binding properties of one such module, the ubiquitin-interacting motif (UIM). We found that UIM peptides from several proteins involved in endocytosis and vacuolar protein sorting including Hrs, Vps27p, Stam1, and Eps15 bound specifically, but with modest affinity (Kd = 0.1-1 mm), to free ubiquitin. Full affinity ubiquitin binding required the presence of conserved acidic patches at the N and C terminus of the UIM, as well as highly conserved central alanine and serine residues. NMR chemical shift perturbation mapping experiments demonstrated that all of these UIM peptides bind to the I44 surface of ubiquitin. The 1.45 A resolution crystal structure of the second yeast Vps27p UIM (Vps27p-2) revealed that the ubiquitin-interacting motif forms an amphipathic helix. Although Vps27p-2 is monomeric in solution, the motif unexpectedly crystallized as an antiparallel four-helix bundle, and the potential biological implications of UIM oligomerization are therefore discussed.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Structure-function relationships in human Class III alcohol dehydrogenase (formaldehyde dehydrogenase).

Human Class III alcohol dehydrogenase (ADH), also known as glutathione-dependent formaldehyde dehydrogenase plays an important role in the formaldehyde detoxification and reduction of the nitric oxide metabolite s-nitrosoglutathione (GSNO). It follows a random bi bi kinetic mechanism and prefers bulkier substrates like long chain primary alcohols and glutathione adducts like s-hydroxymethylglutathione and GSNO over smaller alcohols like ethanol. The structure of the FDH.NAD(H) binary complex reported here, in conjunction with the other complexes of FDH, provide the structural basis of the kinetic observations. These structures show that the apoenzyme has a semi-open domain conformation that permits random random addition of alcohol or NAD(H). Moreover, there is no significant domain movement upon binding of the coenzyme or the substrate, 12-hydroxydodecanoic acid. Interestingly, two active site zinc coordination environments are observed in FDH. In the apoenzyme, the active site zinc is coordinated to Cys44, His66, Cys173 and a water molecule. In the FDH.NAD(H) binary complex reported here, Glu67 is added to the coordination environment of the active site zinc and the distance between the water molecule and zinc is increased. This change in the zinc coordination, brought about by the displacement of zinc of about 2 A towards Glu67 could promote substrate exchange at the active site metal during catalysis.

Aldehyde Oxidoreductases↗