Biomedical subjects

J C Fontecilla-Camps

Publications and source records attributed to J C Fontecilla-Camps.

At least 19 recordsLinked to original sources

PubMed2002

Fe-only hydrogenases: structure, function and evolution.

Hydrogenases are enzymes capable of catalyzing the oxidation of molecular hydrogen or its production from protons and electrons according to the reversible reaction: H(2)<==>2H(+)+2e(-). Most of these enzymes fall into to major classes: NiFe and Fe-only hydrogenases. Extensive spectroscopic, electrochemical and structural studies have shed appreciable light on the catalytic mechanism of hydrogenases. Although evolutionarily unrelated, NiFe and Fe-hydrogenases share a common, unusual feature: an active site low-spin Fe center with CO and CN coordination. We have recently focused our attention on Fe-hydrogenases because from structural studies by us and others, it appears to be a simpler system than the NiFe counterpart. Thus the primary hydrogen binding site has been identified and plausible, electron, proton and hydrogen pathways from and to the buried active site may be proposed from the structural data. The extensive genome sequencing effort currently under way has shown that eukaryotic organisms contain putatively gene coding sequences that display significant homology to Fe-hydrogenases. Here, we summarize the available evidence concerning the mechanism of these enzymes and carry out a structural comparison between Fe-hydrogenases and related proteins of unknown metal content from yeast, plant, worm, insect and mammals.

Amino Acid Sequence↗

PubMed2001

Crystal structure of the free radical intermediate of pyruvate:ferredoxin oxidoreductase.

In anaerobic organisms, the decarboxylation of pyruvate, a crucial component of intermediary metabolism, is catalyzed by the metalloenzyme pyruvate: ferredoxin oxidoreductase (PFOR) resulting in the generation of low potential electrons and the subsequent acetylation of coenzyme A (CoA). PFOR is the only enzyme for which a stable acetyl thiamine diphosphate (ThDP)-based free radical reaction intermediate has been identified. The 1.87 A-resolution structure of the radical form of PFOR from Desulfovibrio africanus shows that, despite currently accepted ideas, the thiazole ring of the ThDP cofactor is markedly bent, indicating a drastic reduction of its aromaticity. In addition, the bond connecting the acetyl group to ThDP is unusually long, probably of the one-electron type already described for several cation radicals but not yet found in a biological system. Taken together, our data, along with evidence from the literature, suggest that acetyl-CoA synthesis by PFOR proceeds via a condensation mechanism involving acetyl (PFOR-based) and thiyl (CoA-based) radicals.

Acetyl Coenzyme A↗

PubMed2001

Crystallization and preliminary X-ray analysis of the periplasmic nitrate reductase (NapA-NapB complex) from Rhodobacter sphaeroides f. sp. denitrificans.

The periplasmic nitrate reductase of Rhodobacter sphaeroides f. sp. denitrificans is a heterodimer responsible for the first step of reduction in the denitrification process by the conversion of nitrate to nitrite. It consists of a 91 kDa molybdenum-containing catalytic subunit (NapA) and a 17 kDa dihaem cytochrome c (NapB). Crystals of the NapA-NapB complex were obtained by the vapour-diffusion method using ammonium sulfate as precipitant. They belong to the P6(1)22 space group, with unit-cell parameters a = b = 151.9, c = 255.8 A, and contain a single complex in the asymmetric unit. A complete native data set was collected at a synchrotron source to 3.1 A resolution.

2S Albumins, Plant↗

PubMed2001

Crystallographic and FTIR spectroscopic evidence of changes in Fe coordination upon reduction of the active site of the Fe-only hydrogenase from Desulfovibrio desulfuricans.

Fe-only hydrogenases, as well as their NiFe counterparts, display unusual intrinsic high-frequency IR bands that have been assigned to CO and CN(-) ligation to iron in their active sites. FTIR experiments performed on the Fe-only hydrogenase from Desulfovibrio desulfuricans indicate that upon reduction of the active oxidized form, there is a major shift of one of these bands that is provoked, most likely, by the change of a CO ligand from a bridging position to a terminal one. Indeed, the crystal structure of the reduced active site of this enzyme shows that the previously bridging CO is now terminally bound to the iron ion that most likely corresponds to the primary hydrogen binding site (Fe2). The CO binding change may result from changes in the coordination sphere of Fe2 or its reduction. Superposition of this reduced active site with the equivalent region of a NiFe hydrogenase shows a remarkable coincidence between the coordination of Fe2 and that of the Fe ion in the NiFe cluster. Both stereochemical and mechanistic considerations suggest that the small organic molecule found at the Fe-only hydrogenase active site and previously modeled as 1,3-propanedithiolate may, in fact, be di-(thiomethyl)-amine.

Binding Sites↗

PubMed2001

Structural biology of C1: dissection of a complex molecular machinery.

The classical pathway of complement is initiated by the C1 complex, a multimolecular protease comprising a recognition subunit (C1q) and two modular serine proteases (C1r and C1s) associated as a Ca2+-dependent tetramer (C1s-C1r-C1r-C1s). Early studies have allowed identification of specialized functional domains in these proteins and have led to low-resolution models of the C1 complex. The objective of current studies is to gain deeper insights into the structure of C1, and the strategy used for this purpose mainly consists of dissecting the C1 components into modular fragments, in order to solve their three-dimensional structure and establish the structural correlates of their function. The aim of this article is to provide an overview of the structural and functional information generated by this approach, with particular emphasis on the domains involved in the assembly, the recognition function, and the highly specific proteolytic properties of C1.

Animals↗

PubMed2001

Crystallization and 2.2 A resolution structure of R-phycoerythrin from Gracilaria chilensis: a case of perfect hemihedral twinning.

R-phycoerythrin, a light-harvesting component from the red algae Gracilaria chilensis, was crystallized by vapour diffusion using ammonium sulfate as precipitant agent. Red crystals grew after one week at 293 K and diffracted to 2.70 A resolution. Three serial macroseeding assays were necessary to grow a second larger crystal to dimensions of 0.68 x 0.16 x 0.16 mm. This crystal diffracted to 2.24 A resolution using synchrotron radiation at beamline BM14 of the European Synchrotron Radiation Facility (ESRF) at Grenoble, France and was used for structure determination. Data were collected at 100 K to a completeness of 98.6%. The crystal was trigonal, space group R3, with unit-cell parameters a = b = 187.3, c = 59.1 A, alpha = beta = 90, gamma = 120 degrees. Data treatment using the CCP4 suite of programs indicated that the crystal was twinned ((I(2))/(I)(2) = 1.41). Molecular replacement was performed with AMoRe using the R-phycoerythrin from Polysiphonia urceolata [Chang et al. (1996), J. Mol. Biol. 249, 424-440] as a search model. In order to overcome the twinning problem, SHELX97 was used for the crystallographic refinement. The twin fraction was 0.48, indicating a nearly perfect hemihedrally twinned crystal. The final R(work) and R(free) factors are 0.16 and 0.25, respectively. All the residues and chromophores of the alpha- and beta-chains are well defined in the electron-density maps. Some residues belonging to the gamma-linker are also recognizable.

Amino Acid Sequence↗

PubMed2000

Structural model of the Fe-hydrogenase/cytochrome c553 complex combining transverse relaxation-optimized spectroscopy experiments and soft docking calculations.

Fe-hydrogenase is a 54-kDa iron-sulfur enzyme essential for hydrogen cycling in sulfate-reducing bacteria. The x-ray structure of Desulfovibrio desulfuricans Fe-hydrogenase has recently been solved, but structural information on the recognition of its redox partners is essential to understand the structure-function relationships of the enzyme. In the present work, we have obtained a structural model of the complex of Fe-hydrogenase with its redox partner, the cytochrome c(553), combining docking calculations and NMR experiments. The putative models of the complex demonstrate that the small subunit of the hydrogenase has an important role in the complex formation with the redox partner; 50% of the interacting site on the hydrogenase involves the small subunit. The closest contact between the redox centers is observed between Cys-38, a ligand of the distal cluster of the hydrogenase and Cys-10, a ligand of the heme in the cytochrome. The electron pathway from the distal cluster of the Fe-hydrogenase to the heme of cytochrome c(553) was investigated using the software Greenpath and indicates that the observed cysteine/cysteine contact has an essential role. The spatial arrangement of the residues on the interface of the complex is very similar to that already described in the ferredoxin-cytochrome c(553) complex, which therefore, is a very good model for the interacting domain of the Fe-hydrogenase-cytochrome c(553).

Cytochrome c Group↗

PubMed2000

Four crystal structures of the 60 kDa flavoprotein monomer of the sulfite reductase indicate a disordered flavodoxin-like module.

Escherichia coli NADPH-sulfite reductase (SiR) is a 780 kDa multimeric hemoflavoprotein composed of eight alpha-subunits (SiR-FP) and four beta-subunits (SiR-HP) that catalyses the six electron reduction of sulfite to sulfide. Each beta-subunit contains a Fe4S4 cluster and a siroheme, and each alpha-subunit binds one FAD and one FMN as prosthetic groups. The FAD gets electrons from NADPH, and the FMN transfers the electrons to the metal centers of the beta-subunit for sulfite reduction. We report here the 1.94 A X-ray structure of SiR-FP60, a recombinant monomeric fragment of SiR-FP that binds both FAD and FMN and retains the catalytic properties of the native protein. The structure can be divided into three domains. The carboxy-terminal part of the enzyme is composed of an antiparallel beta-barrel which binds the FAD, and a variant of the classical pyridine dinucleotide binding fold which binds NADPH. These two domains form the canonic FNR-like module, typical of the ferredoxin NADP+ reductase family. By analogy with the structure of the cytochrome P450 reductase, the third domain, composed of seven alpha-helices, is supposed to connect the FNR-like module to the N-terminal flavodoxine-like module. In four different crystal forms, the FMN-binding module is absent from electron density maps, although mass spectroscopy, amino acid sequencing and activity experiments carried out on dissolved crystals indicate that a functional module is present in the protein. Our results clearly indicate that the interaction between the FNR-like and the FMN-like modules displays lower affinity than in the case of cytochrome P450 reductase. The flexibility of the FMN-binding domain may be related, as observed in the case of cytochrome bc1, to a domain reorganisation in the course of electron transfer. Thus, a movement of the FMN-binding domain relative to the rest of the enzyme may be a requirement for its optimal positioning relative to both the FNR-like module and the beta-subunit.

Amino Acid Sequence↗

PubMed2000

Crystal structure of the catalytic domain of human complement c1s: a serine protease with a handle.

C1s is the highly specific modular serine protease that mediates the proteolytic activity of the C1 complex and thereby triggers activation of the complement cascade. The crystal structure of a catalytic fragment from human C1s comprising the second complement control protein (CCP2) module and the chymotrypsin-like serine protease (SP) domain has been determined and refined to 1.7 A resolution. In the areas surrounding the active site, the SP structure reveals a restricted access to subsidiary substrate binding sites that could be responsible for the narrow specificity of C1s. The ellipsoidal CCP2 module is oriented perpendicularly to the surface of the SP domain. This arrangement is maintained through a rigid module-domain interface involving intertwined proline- and tyrosine-rich polypeptide segments. The relative orientation of SP and CCP2 is consistent with the fact that the latter provides additional substrate recognition sites for the C4 substrate. This structure provides a first example of a CCP-SP assembly that is conserved in diverse extracellular proteins. Its implications in the activation mechanism of C1 are discussed.

Amino Acid Sequence↗

PubMed2000

Critical role of micelles in pancreatic lipase activation revealed by small angle neutron scattering.

In the duodenum, pancreatic lipase (PL) develops its activity on triglycerides by binding to the bile-emulsified oil droplets in the presence of its protein cofactor pancreatic colipase (PC). The neutron crystal structure of a PC-PL-micelle complex (Hermoso, J., Pignol, D., Penel, S., Roth, M., Chapus, C., and Fontecilla-Camps, J. C. (1997) EMBO J. 16, 5531-5536) has suggested that the stabilization of the enzyme in its active conformation and its adsorption to the emulsified oil droplets are mediated by a preformed lipase-colipase-micelle complex. Here, we correlate the ability of different amphypathic compounds to activate PL, with their association with PC-PL in solution. The method of small angle neutron scattering with D(2)O/H(2)O contrast variation was used to characterize a solution containing PC-PL complex and taurodeoxycholate micelles. The resulting radius of gyration (56 A) and the match point of the solution indicate the formation of a ternary complex that is similar to the one observed in the neutron crystal structure. In addition, we show that either bile salts, lysophospholipids, or nonionic detergents that form micelles with radii of gyration ranging from 13 to 26 A are able to bind to the PC-PL complex, whereas smaller micelles or nonmicellar compounds are not. This further supports the notion of a micelle size-dependent affinity process for lipase activation in vivo.

Animals↗

PubMed2000

Crystal structure of Escherichia coli UvrB C-terminal domain, and a model for UvrB-uvrC interaction.

A crystal structure of the C-terminal domain of Escherichia coli UvrB (UvrB') has been solved to 3.0 A resolution. The domain adopts a helix-loop-helix fold which is stabilised by the packing of hydrophobic side-chains between helices. From the UvrB' fold, a model for a domain of UvrC (UvrC') that has high sequence homology with UvrB' has been made. In the crystal, a dimerisation of UvrB domains is seen involving specific hydrophobic and salt bridge interactions between residues in and close to the loop region of the domain. It is proposed that a homologous mode of interaction may occur between UvrB and UvrC. This interaction is likely to be flexible, potentially spanning > 50 A.

Amino Acid Sequence↗

PubMed2000

Mechanism of calcite crystal growth inhibition by the N-terminal undecapeptide of lithostathine.

Pancreatic juice is supersaturated with calcium carbonate. Calcite crystals therefore may occur, obstruct pancreatic ducts, and finally cause a lithiasis. Human lithostathine, a protein synthesized by the pancreas, inhibits the growth of calcite crystals by inducing a habit modification: the rhombohedral (10 14) usual habit is transformed into a needle-like habit through the (11 0) crystal form. A similar observation was made with the N-terminal undecapeptide (pE(1)R(11)) of lithostathine. We therefore aimed at discovering how peptides inhibit calcium salt crystal growth. We solved the complete x-ray structure of lithostathine, including the flexible N-terminal domain, at 1.3 A. Docking studies of pE(1)R(11) with the (10 14) and (11 0) faces through molecular dynamics simulation resulted in three successive steps. First, the undecapeptide progressively unfolded as it approached the calcite surface. Second, mobile lateral chains of amino acids made hydrogen bonds with the calcite surface. Last, electrostatic bonds between calcium ions and peptide bonds stabilized and anchored pE(1)R(11) on the crystal surface. pE(1)R(11)-calcite interaction was stronger with the (11 0) face than with the (10 14) face, confirming earlier experimental observations. Energy contributions showed that the peptide backbone governed the binding more than did the lateral chains. The ability of peptides to inhibit crystal growth is therefore essentially based on backbone flexibility.

Amino Acid Sequence↗

PubMed2000

A novel FeS cluster in Fe-only hydrogenases.

Many microorganisms can use molecular hydrogen as a source of electrons or generate it by reducing protons. These reactions are catalysed by metalloenzymes of two types: NiFe and Fe-only hydrogenases. Here, we review recent structural results concerning the latter, putting special emphasis on the characteristics of the active site.

Amino Acid Sequence↗

PubMed2000

Crystal structure of a T cell receptor bound to an allogeneic MHC molecule.

Many T cell receptors (TCRs) that are selected to respond to foreign peptide antigens bound to self major histocompatibility complex (MHC) molecules are also reactive with allelic variants of self-MHC molecules. This property, termed alloreactivity, causes graft rejection and graft-versus-host disease. The structural features of alloreactivity have yet to be defined. We now present a basis for this cross-reactivity, elucidated by the crystal structure of a complex involving the BM3.3 TCR and a naturally processed octapeptide bound to the H-2Kb allogeneic MHC class I molecule. A distinguishing feature of this complex is that the eleven-residue-long complementarity-determining region 3 (CDR3) found in the BM3.3 TCR alpha chain folds away from the peptide binding groove and makes no contact with the bound peptide, the latter being exclusively contacted by the BM3.3 CDR3 beta. Our results formally establish that peptide-specific, alloreactive TCRs interact with allo-MHC in a register similar to the one they use to contact self-MHC molecules.

Animals↗

PubMed2000

Towards the charge-density study of proteins: a room-temperature scorpion-toxin structure at 0.96 A resolution as a first test case.

The number of protein structures refined at a resolution higher than 1.0 A is continuously increasing. Subatomic structures may deserve a more sophisticated model than the spherical atomic electron density. In very high resolution structural studies (d < 0.5 A) of small peptides, a multipolar atom model is used to describe the valence electron density. This allows a much more accurate determination of the anisotropic thermal displacement parameters and the estimate of atomic charges. This information is of paramount importance in the understanding of biological processes involving enzymes and metalloproteins. The structure of the scorpion Androctonus australis Hector toxin II has been refined at 0.96 A resolution using synchrotron diffraction data collected at room temperature. Refinement with a multipolar electron-density model in which the multipole populations are transferred from previous peptide studies led to the observation of valence electrons on covalent bonds of the most ordered residues. The refined net charges of the peptide-bond atoms were of the correct sign but were underestimated. Such protein-structure refinements against higher resolution data collected at cryogenic temperature will enable the calculation of experimental atomic charges and properties such as electrostatic potentials.

Crystallography, X-Ray↗

PubMed1999

Structural features of the interaction between an anti-clonotypic antibody and its cognate T-cell antigen receptor.

The crystal structure of the complex between a single chain Fv fragment of the KB5-C20 T-cell antigen receptor (TCR) and the specific anti-clonotypic antibody (Ab) Désiré-1 provides the first description of the interface between a clonotype and an anti-clonotype. In the four idiotype/anti-idiotype complexes of known three-dimensional structures, the interacting Fv fragments associate largely through their complementarity-determining regions (CDRs). In marked contrast, Désiré-1 binds to a face of the KB5-C20 TCR that is almost perpendicular to the TCR antigen binding site, and recognizes discontinuous stretches of TCR Valpha and Vbeta residues that belong to both the CDRs and the framework. Despite this peculiar mode of interaction, Désiré-1 constitutes a genuine anti-clonotypic Ab. Moreover, in spite of the fact that the Désiré-1 contact residues do not constitute a molecular mimic of the physiological ligand normally recognized by the KB5-C20 TCR, the bivalent Désiré-1 Ab is capable of efficiently activating T-cells expressing the KB5-C20 TCR.

Animals↗

PubMed1999

Desulfovibrio desulfuricans iron hydrogenase: the structure shows unusual coordination to an active site Fe binuclear center.

BACKGROUND: Many microorganisms have the ability to either oxidize molecular hydrogen to generate reducing power or to produce hydrogen in order to remove low-potential electrons. These reactions are catalyzed by two unrelated enzymes: the Ni-Fe hydrogenases and the Fe-only hydrogenases. RESULTS: We report here the structure of the heterodimeric Fe-only hydrogenase from Desulfovibrio desulfuricans - the first for this class of enzymes. With the exception of a ferredoxin-like domain, the structure represents a novel protein fold. The so-called H cluster of the enzyme is composed of a typical [4Fe-4S] cubane bridged to a binuclear active site Fe center containing putative CO and CN ligands and one bridging 1, 3-propanedithiol molecule. The conformation of the subunits can be explained by the evolutionary changes that have transformed monomeric cytoplasmic enzymes into dimeric periplasmic enzymes. Plausible electron- and proton-transfer pathways and a putative channel for the access of hydrogen to the active site have been identified. CONCLUSIONS: The unrelated active sites of Ni-Fe and Fe-only hydrogenases have several common features: coordination of diatomic ligands to an Fe ion; a vacant coordination site on one of the metal ions representing a possible substrate-binding site; a thiolate-bridged binuclear center; and plausible proton- and electron-transfer pathways and substrate channels. The diatomic coordination to Fe ions makes them low spin and favors low redox states, which may be required for catalysis. Complex electron paramagnetic resonance signals typical of Fe-only hydrogenases arise from magnetic interactions between the [4Fe-4S] cluster and the active site binuclear center. The paucity of protein ligands to this center suggests that it was imported from the inorganic world as an already functional unit.

Amino Acid Sequence↗

PubMed1999

Crystallization and preliminary crystallographic analysis of the pyruvate-ferredoxin oxidoreductase from Desulfovibrio africanus.

For the first time, crystals of a pyruvate-ferredoxin oxidoreductase (PFOR) suitable for X-ray analysis have been obtained. This enzyme catalyzes, in anaerobic organisms, the crucial energy-yielding reaction of pyruvate decarboxylation to acetylCoA. Polyethylene glycol and divalent metal cations have been used to crystallize the PFOR from the sulfate-reducing bacterium Desulfovibrio africanus. Two different orthorhombic (P212121 ) crystal forms have been grown with unit-cell dimensions a = 86.1, b = 146.7, c = 212.5 A and a = 84.8, b = 144.9, c = 203.0 A. Both crystals diffract to 2.3 A resolution using synchrotron radiation.

Crystallization↗