Biomedical subjects

Christian Siebold

Publications and source records attributed to Christian Siebold.

14 recordsLinked to original sources

PubMed2007

Mutations in alpha-tubulin cause abnormal neuronal migration in mice and lissencephaly in humans.

The development of the mammalian brain is dependent on extensive neuronal migration. Mutations in mice and humans that affect neuronal migration result in abnormal lamination of brain structures with associated behavioral deficits. Here, we report the identification of a hyperactive N-ethyl-N-nitrosourea (ENU)-induced mouse mutant with abnormalities in the laminar architecture of the hippocampus and cortex, accompanied by impaired neuronal migration. We show that the causative mutation lies in the guanosine triphosphate (GTP) binding pocket of alpha-1 tubulin (Tuba1) and affects tubulin heterodimer formation. Phenotypic similarity with existing mouse models of lissencephaly led us to screen a cohort of patients with developmental brain anomalies. We identified two patients with de novo mutations in TUBA3, the human homolog of Tuba1. This study demonstrates the utility of ENU mutagenesis in the mouse as a means to discover the basis of human neurodevelopmental disorders.

Amino Acid Sequence↗

PubMed2006

Molecular analysis of receptor protein tyrosine phosphatase mu-mediated cell adhesion.

Type IIB receptor protein tyrosine phosphatases (RPTPs) are bi-functional cell surface molecules. Their ectodomains mediate stable, homophilic, cell-adhesive interactions, whereas the intracellular catalytic regions can modulate the phosphorylation state of cadherin/catenin complexes. We describe a systematic investigation of the cell-adhesive properties of the extracellular region of RPTPmu, a prototypical type IIB RPTP. The crystal structure of a construct comprising its N-terminal MAM (meprin/A5/mu) and Ig domains was determined at 2.7 A resolution; this assigns the MAM fold to the jelly-roll family and reveals extensive interactions between the two domains, which form a rigid structural unit. Structure-based site-directed mutagenesis, serial domain deletions and cell-adhesion assays allowed us to identify the four N-terminal domains (MAM, Ig, fibronectin type III (FNIII)-1 and FNIII-2) as a minimal functional unit. Biophysical characterization revealed at least two independent types of homophilic interaction which, taken together, suggest that there is the potential for formation of a complex and possibly ordered array of receptor molecules at cell contact sites.

Catalytic Domain↗

PubMed2006

MHC class II proteins and disease: a structural perspective.

MHC class II molecules on the surface of antigen-presenting cells display a range of peptides for recognition by the T-cell receptors of CD4+ T helper cells. Therefore, MHC class II molecules are central to effective adaptive immune responses, but conversely, genetic and epidemiological data have implicated these molecules in the pathogenesis of autoimmune diseases. Indeed, the strength of the associations between particular MHC class II alleles and disease render them the main genetic risk factors for autoimmune disorders such as type 1 diabetes. Here, we discuss the insights that the crystal structures of MHC class II molecules provide into the molecular mechanisms by which sequence polymorphisms might contribute to disease susceptibility.

Autoimmune Diseases↗

PubMed2005

High-resolution structure of the catalytic region of MICAL (molecule interacting with CasL), a multidomain flavoenzyme-signaling molecule.

Semaphorins are extracellular cell guidance cues that govern cytoskeletal dynamics during neuronal and vascular development. MICAL (molecule interacting with CasL) is a multidomain cytosolic protein with a putative flavoprotein monooxygenase (MO) region required for semaphorin-plexin repulsive axon guidance. Here, we report the 1.45-A resolution crystal structure of the FAD-containing MO domain of mouse MICAL-1 (residues 1-489). The topology most closely resembles that of the NADPH-dependent flavoenzyme p-hydroxybenzoate hydroxylase (PHBH). Comparison of structures before and after reaction with NADPH reveals that, as in PHBH, the flavin ring can switch between two discrete positions. In contrast with other MOs, this conformational switch is coupled with the opening of a channel to the active site, suggestive of a protein substrate. In support of this hypothesis, distinctive structural features highlight putative protein-binding sites in suitable proximity to the active site entrance. The unusual juxtaposition of this N-terminal MO (hydroxylase) activity with the characteristics of a multiprotein-binding scaffold exhibited by the C-terminal portion of the MICALs represents a unique combination of functionality to mediate signaling.

Animals↗

PubMed2005

A procedure for setting up high-throughput nanolitre crystallization experiments. Crystallization workflow for initial screening, automated storage, imaging and optimization.

Crystallization trials at the Division of Structural Biology in Oxford are now almost exclusively carried out using a high-throughput workflow implemented in the Oxford Protein Production Facility. Initial crystallization screening is based on nanolitre-scale sitting-drop vapour-diffusion experiments (typically 100 nl of protein plus 100 nl of reservoir solution per droplet) which use standard crystallization screening kits and 96-well crystallization plates. For 294 K crystallization trials the barcoded crystallization plates are entered into an automated storage system with a fully integrated imaging system. These plates are imaged in accordance with a pre-programmed schedule and the resulting digital data for each droplet are harvested into a laboratory information-management system (LIMS), scored by crystal recognition software and displayed for user analysis via a web-based interface. Currently, storage for trials at 277 K is not automated and for imaging the crystallization plates are fed by hand into an imaging system from which the data enter the LIMS. The workflow includes two procedures for nanolitre-scale optimization of crystallization conditions: (i) a protocol for variation of pH, reservoir dilution and protein:reservoir ratio and (ii) an additive screen. Experience based on 592 crystallization projects is reported.

Animals↗

PubMed2005

Multiple sclerosis: MHC associations and therapeutic implications.

Multiple sclerosis (MS) is an autoimmune disease with an important genetic component. The strongest genetic association is with the major histocompatibility complex (MHC) region. Several MHC alleles predispose to the disease, the most prominent of which are certain alleles in the HLA-DR2 haplotype. Functional and structural studies have helped to explain the molecular basis of these associations. Although there is currently no curative treatment for MS, an increased understanding of the disease has aided the design of immunotherapies that act on the immune system more specifically than the longstanding drugs. Many of these therapies work at the antigen-specific level, disrupting the interaction between T-cell receptors and MHC molecules that leads to disease.

Animals↗

PubMed2005

The structure and function of the outer coat protein VP9 of Banna virus.

Banna virus (BAV: genus Seadornavirus, family Reoviridae) has a double-shelled morphology similar to rotavirus and bluetongue virus. The structure of BAV outer-capsid protein VP9 was determined by X-ray crystallography at 2.6 A resolution, revealing a trimeric molecule, held together by an N-terminal helical bundle, reminiscent of coiled-coil structures found in fusion-active proteins such as HIV gp41. The major domain of VP9 contains stacked beta sheets with marked structural similarities to the receptor binding protein VP8 of rotavirus. Anti-VP9 antibodies neutralize viral infectivity, and, remarkably, pretreatment of cells with trimeric VP9 increased viral infectivity, indicating that VP9 is involved in virus attachment to cell surface and subsequent internalization. Sequence similarities were also detected between BAV VP10 and VP5 portion of rotavirus VP4, suggesting that the receptor binding and internalization apparatus, which is a single gene product activated by proteoloysis in rotavirus, is the product of two separate genome segments in BAV.

Amino Acid Sequence↗

PubMed2004

Crystal structure of the phosphoenolpyruvate-binding enzyme I-domain from the Thermoanaerobacter tengcongensis PEP: sugar phosphotransferase system (PTS).

Enzyme I (EI), the first component of the phosphoenolpyruvate (PEP):sugar phosphotransferase system (PTS), consists of an N-terminal protein-binding domain (EIN) and a C-terminal PEP-binding domain (EIC). EI transfers phosphate from PEP by double displacement via a histidine residue on EIN to the general phosphoryl carrier protein HPr. Here, we report the 1.82A crystal structure of the homodimeric EIC domain from Thermoanaerobacter tengcongensis, a saccharolytic eubacterium that grows optimally at 75 degrees C. EIC folds into a (betaalpha)(8) barrel with three large helical insertions between beta2/alpha2, beta3/alpha3 and beta6/alpha6. The large amphipathic dimer interface buries 3750A(2) of accessible surface area per monomer. A comparison with pyruvate phosphate dikinase (PPDK) reveals that the active-site residues in the empty PEP-binding site of EIC and in the liganded PEP-binding site of PPDK have almost identical conformations, pointing to a rigid structure of the active site. In silico models of EIC in complex with the Z and E-isomers of chloro-PEP provide a rational explanation for their difference as substrates and inhibitors of EI. The EIC domain exhibits 54% amino acid sequence identity with Escherichia coli and 60% with Bacillus subtilis EIC, has the same amino acid composition but contains additional salt-bridges and a more complex salt-bridge network than the homology model of E.coli EIC. The easy crystallization of EIC suggests that T.tengcongensis can serve as source for stable homologs of mesophilic proteins that are too labile for crystallization.

Binding Sites↗

PubMed2004

Phosphoenolpyruvate- and ATP-dependent dihydroxyacetone kinases: covalent substrate-binding and kinetic mechanism.

Dihydroxyacetone (Dha) kinases are a sequence-conserved family of enzymes, which utilize two different phosphoryldonors, ATP in animals, plants, and some bacteria, and a multiphosphoprotein of the phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) in most bacteria. Here, we compare the PTS-dependent kinase of Escherichia coli and the ATP-dependent kinase of Citrobacter freundii. They display 30% sequence identity. The binding constants of the E. coli kinase for eleven short-chain carbonyl compounds were determined by acetone precipitation of the enzyme-substrate complexes. They are 3.4 microM for Dha, 780 microM for Dha-phosphate (DhaP), 50 microM for D,L-glyceraldehyde (GA), and 90 microM for D,L-glyceraldehyde-3-phosphate. The k(cat) for Dha of the PTS-dependent kinase is 290 min(-1), and that of the ATP-dependent kinase is 1050 min(-1). The Km for Dha of both kinases is <6 microM. The X-ray structures of the enzyme-GA and the enzyme-DhaP complex show that substrates as well as products are bound in hemiaminal linkage to an active-site histidine. Quantum-mechanical calculations offer no indication for activation of the reacting hydroxyl group by the formation of the hemiaminal. However, the formation of the hemiaminal bond allows selection for short-chain carbonyl compounds and discrimination against structurally similar polyols. The Dha kinase remains fully active in the presence of 2 M glycerol, and phosphorylates trace impurities of carbonyl compounds present in glycerol.

Acetone↗

PubMed2004

Crystal structure of HLA-DQ0602 that protects against type 1 diabetes and confers strong susceptibility to narcolepsy.

The MHC class II molecule DQ0602 confers strong susceptibility to narcolepsy but dominant protection against type 1 diabetes. The crystal structure of DQ0602 reveals the molecular features underlying these contrasting genetic properties. Structural comparisons to homologous DQ molecules with differential disease associations highlight a previously unrecognized interplay between the volume of the P6 pocket and the specificity of the P9 pocket, which implies that presentation of an expanded peptide repertoire is critical for dominant protection against type 1 diabetes. In narcolepsy, the volume of the P4 pocket appears central to the susceptibility, suggesting that the presentation of a specific peptide population plays a major role.

Alleles↗

PubMed2003

Crystal structure of the Citrobacter freundii dihydroxyacetone kinase reveals an eight-stranded alpha-helical barrel ATP-binding domain.

Dihydroxyacetone kinases are a sequence-conserved family of enzymes, which utilize two different phosphoryldonors, ATP in animals, plants and some bacteria, and a multiphosphoprotein of the phosphoenolpyruvate carbohydrate phosphotransferase system in bacteria. Here we report the 2.5-A crystal structure of the homodimeric Citrobacter freundii dihydroxyacetone kinase complex with an ATP analogue and dihydroxyacetone. The N-terminal domain consists of two alpha/beta-folds with a molecule of dihydroxyacetone covalently bound in hemiaminal linkage to the N epsilon 2 of His-220. The C-terminal domain consists of a regular eight-helix alpha-barrel. The eight helices form a deep pocket, which includes a tightly bound phospholipid. Only the lipid headgroup protrudes from the surface. The nucleotide is bound on the top of the barrel across from the entrance to the lipid pocket. The phosphate groups are coordinated by two Mg2+ ions to gamma-carboxyl groups of aspartyl residues. The ATP binding site does not contain positively charged or aromatic groups. Paralogues of dihydroxyacetone kinase also occur in association with transcription regulators and proteins of unknown function pointing to biological roles beyond triose metabolism.

Adenosine Triphosphate↗

PubMed2003

A mechanism of covalent substrate binding in the x-ray structure of subunit K of the Escherichia coli dihydroxyacetone kinase.

Dihydroxyacetone (Dha) kinases are homologous proteins that use different phosphoryl donors, a multiphosphoryl protein of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system in bacteria, ATP in animals, plants, and some bacteria. The Dha kinase of Escherichia coli consists of three subunits, DhaK and DhaL, which are colinear to the ATP-dependent Dha kinases of eukaryotes, and the multiphosphoryl protein DhaM. Here we show the crystal structure of the DhaK subunit in complex with Dha at 1.75 A resolution. DhaK is a homodimer with a fold consisting of two six-stranded mixed beta-sheets surrounded by nine alpha-helices and a beta-ribbon covering the exposed edge strand of one sheet. The core of the N-terminal domain has an alpha/beta fold common to subunits of carbohydrate transporters and transcription regulators of the phosphoenolpyruvate-dependent carbohydrate:phosphotransferase system. The core of the C-terminal domain has a fold similar to the C-terminal domain of the cell-division protein FtsZ. A molecule of Dha is covalently bound in hemiaminal linkage to the N epsilon 2 of His-230. The hemiaminal does not participate in covalent catalysis but is the chemical basis for discrimination between short-chain carbonyl compounds and polyols. Paralogs of Dha kinases occur in association with transcription regulators of the TetR/QacR and the SorC families, pointing to their biological role as sensors in signaling.

Amino Acid Sequence↗

PubMed2002

Intein-mediated cyclization of a soluble and a membrane protein in vivo: function and stability.

Cyclized subunits of the E. coli glucose transporter were produced in vivo by intein mediated trans-splicing. IIA(Glc) is a beta-sandwich protein, IICB(Glc) spans the membrane eight times. Genes encoding the circularly permuted precursors U(Cdelta)-IIA(Glc)-U(Ndelta) and U(Cdelta)-IICB(Glc)-U(Ndelta) were assembled from DNA fragments encoding the 3' and 5' segments of the recA intein of M. tuberculosis and crr and ptsG of E. coli, respectively. A 20-residues long, Ala-Pro rich linker peptide and/or a histidine tag were used to join the native N- and C-termini in the cyclized proteins. The cyclized proteins complemented growth of glucose auxotrophic strains. Purified, cyclized IIA(Glc) and IICB(Glc) had 100 and 25%, respectively, of wild-type glucose phosphotransferase activity. They had an increased electrophoretic mobility, which decreased upon linearization of the proteins with chymotrypsin. Cyclized IIA(Glc) displayed increased stability against temperature and GuHCl-induced unfolding (75 vs. 70 degrees C; 1.52 vs. 1.05 M).

Cyclization↗

PubMed2001

Mechanism-based inhibition of enzyme I of the Escherichia coli phosphotransferase system. Cysteine 502 is an essential residue.

Four phosphoenolpyruvate (PEP) derivatives, carrying reactive or activable chemical functions in each of the three chemical regions of PEP, were assayed as alternative substrates of enzyme I (EI) of the Escherichia coli PEP:glucose phosphotransferase system. The Z- and E-isomers of 3-chlorophosphoenolpyruvate (3-Cl-PEP) were substrates, presenting K(m) values of 0.08 and 0.12 mm, respectively, very similar to the K(m) of 0.14 mm measured for PEP, and k(cat) of 40 and 4 min(-1), compared with 2,200 min(-1), for PEP. The low catalytic efficiency of these substrates permits the study of activity at in vivo EI concentrations. Z-Cl-PEP was a competitive inhibitor of PEP with a K(I) of 0.4 mm. E-Cl-PEP was not an inhibitor. Compounds 3 and 4, obtained by modification of the carboxylic and phosphate groups of PEP, were neither substrates nor inhibitors of EI, highlighting the importance of these functionalities for recognition by EI. Z-Cl-PEP is a suicide inhibitor. About 10-50 turnovers sufficed to inactivate EI completely. Such a property can be exploited to reveal and quantitate phosphoryl transfer from EI to other proteins at in vivo concentrations. Inactivation was saturatable in Z-Cl-PEP, with an apparent K(m)(inact) of 0.2-0.4 mm. The rate of inactivation increased with the concentration of EI, indicating a preferential or exclusive reaction with the dimeric form of EI. E-Cl-PEP inactivates EI much more slowly, and unlike PEP, it did not protect against inactivation by Z-Cl-PEP. This and the ineffectiveness of E-Cl-PEP as a competitive inhibitor have been related to the presence of two EI active species. Cys-502 of EI was identified by mass spectrometry as the reacting residue. The C502A EI mutant showed less than 0.06% wild-type activity. Sequence alignments and comparisons of x-ray structures of different PEP-utilizing enzymes indicate that Cys-502 might serve as a proton donor during catalysis.

Amino Acid Sequence↗