Biomedical subjects

C Cambillau

Publications and source records attributed to C Cambillau.

At least 91 records · Page 5Linked to original sources

PubMed1994

The 2.6-A refined structure of the Escherichia coli recombinant Saccharomyces cerevisiae flavocytochrome b2-sulfite complex.

Flavocytochrome b2 from Saccharomyces cerevisiae catalyzes the oxidation of L-lactate to pyruvate and the electron transfer to cytochrome c in the mitochondrial intermembrane space. It is a homotetramer with a molecular weight of 4 x 58 kDa, each monomer of which is composed of 2 distinct domains, the one carrying FMN and the other, a "b5-like" heme. The native structure has been described at a resolution of 2.4 A (Xia ZX, Mathews FS, 1990, J Mol Biol 212:837-863). The heme domains protrude from the central body of the tetramer consisting of the 4 FMN binding domains. Because only 2 heme domains are visible in the electron density map, the other 2 are probably disordered. We crystallized the Escherichia coli recombinant flavocytochrome b2 from S. cerevisiae inhibited by sulfite. Although the crystals were obtained under very different conditions from those of the pyruvate-containing native enzyme, they were found to be isostructural (P 3(2) 2 1, a = b = 164.5 A, c = 114.0 A). The 2.6-A X-ray structure was extensively refined with X-PLOR (R = 17.3%), which made it possible to describe in detail the recombinant flavocytochrome b2 molecular structure. There exist few differences between the native and recombinant structures, in line with the fact that they show similar kinetic behavior, and they further confirm the intrinsic mobility of the heme domain (Labeyrie F, Beloil JC, Thomas MA, 1988, Biochim Biophys Acta 953:134-141). This structure will be used as a starting model in the structural resolution of flavocytochrome b2 point mutants.

Binding Sites↗

PubMed1994

Structural studies on recombinant and point mutants of flavocytochrome b2.

Flavocytochrome b2 from S cerevisiae is a homotetramer with a molecular mass of 4 x 58 kDa. It catalyses the oxidation of L-lactate into pyruvate and the electron transfer to cytochrome c in the mitochondrial intermembrane space. Each monomer is composed of a flavinmononucleotide (FMN) carrying domain and a 'b5-like' heme domain. The wild type structure has been described at a resolution of 2.4 A. We report here on the refined structure of the E. coli native recombinant flavocytochrome b2 from S cerevisiae inhibited by sulphite and that of two point mutants, Y143F and Y254F, in which pyruvate is bound to the active site. The crystals, obtained under very different conditions from those of the native enzyme, are isostructural (P 3(2) 2 1, a=b=164.5 A, c=114.0 A). In line with the similarities found to exist in the kinetic behaviour of the native and recombinant protein, few structural differences were observed here, and the crystallographic data further confirm the intrinsic mobility of the heme domain. The superimposable position of the aromatic rings of Phe 143 in the mutant Y143F and Tyr 143 in the native protein makes it seem unlikely that the aromatic ring may be directly involved in the intramolecular electron transfer. The fact that a very restricted number of domain interactions was observed in Y143F shows that Tyr 143 is one of the amino acids essential to the formation of the productive complex. In the Y143F mutant, the number of catalytically efficient complexes is probably drastically decreased, which will severely limit the rate of intramolecular election transfer. The structure of Y254F shows a reorientation of the substrate at the active site. Together with the kinetic results, this finding definitely excludes the possibility that Tyr 254 may act as general base and that the substrate may interact directly with Phe 254 in the mutant. The model between flavocytochrome b2 and cytochrome c will serve as a basis for designing suitable mutants of the amino acids involved either in the interaction or the electron transfer.

Cytochrome c Group↗

PubMed1994

Crosslinking of mammalian lectin (galectin-1) by complex biantennary saccharides.

Galectins are beta-galactoside-binding proteins that occur intra- and extracellularly in many animal tissues. They have been proposed to form networks of glycoconjugates on the cell surface, where they may modulate various cell response pathways such as growth, activation and adhesion. The high resolution X-ray crystallographic analyses of three crystal forms of bovine galectin-1 in complex with biantennary saccharides of N-acetyllactosamine type reveal infinite chains of lectin dimers cross-linked through N-acetyllactosamine units located at the end of the oligosaccharide antenna. The oligosaccharide adopts a different low energy conformation in each of the three crystal forms.

Amino Acid Sequence↗

PubMed1993

Solution structure of P05-NH2, a scorpion toxin analog with high affinity for the apamin-sensitive potassium channel.

The venom of the scorpion Androctonus mauretanicus mauretanicus contains a toxin--P05--which is structurally and functionally similar to scorpion leiurotoxin I (87% sequence identity), a blocker of the apamin-sensitive Ca(2+)-activated K+ channels. P05, a 31-residue polypeptide cross-linked by three disulfide bridges, also possesses binding and physiological properties similar to those of the bee venom toxin apamin (18 residues, two disulfides). However, the amino acid sequences of these two polypeptides are dissimilar, except for a common Arg-Arg-Cys-Gln motif which is located on an alpha-helix. P05-NH2, a synthetic analog of P05, unlike native P05, was found to bind irreversibly to the apamin receptor. The solution structure of P05-NH2 has been solved by conventional two-dimensional NMR techniques followed by distance geometry and energy minimization. The obtained conformation is composed of two and an half turns of alpha-helix (residues 5-14) connected by a tight turn to a two-stranded antiparallel beta-sheet (sequences 17-22 and 25-29). This beta-sheet has a right-handed twist as usual for such secondary structures. The beta-turn connecting the two strands belongs to type II'. This structure is homologous to all scorpion toxin structures known so far as well as to insect defensins. The three arginines known to be involved in the pharmacological activity, i.e., Arg6, Arg7, and Arg13, are all located on the solvent-exposed side of the helix and form a positively charged surface which includes Gln9. The calculated electrostatic potential is highly asymmetric with the greatest positive potential centered on the Arg-rich alpha-helix side.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

A structural domain (the lid) found in pancreatic lipases is absent in the guinea pig (phospho)lipase.

Typically pancreatic lipases are characterized by the following properties: (1) they are activated by lipid/water interfaces (interfacial activation), (2) they are inhibited by bile salts but reactivated by colipase (a small activator protein), and (3) they do not hydrolyze significantly phospholipids. A cDNA clone encoding a guinea pig pancreatic (phospho)lipase (GPL) has been sequenced and expressed. The enzyme (recombinant as well as native) differs from other pancreatic lipases in that (1) it is not interfacially activated, (2) its activity is unaffected by the presence of bile salts and/or colipase using tributyrin as substrate, and (3) it exhibits equally phospholipase A1 and lipase activities. The amino acid sequence of GPL is highly homologous to that of other known pancreatic lipases, with the exception of a deletion in the so-called lid domain that regulates access to the active centers of other lipases. We propose that this deletion is directly responsible for the anomalous behavior of this enzyme. Thus GPL challenges the classical distinction between lipases, esterases, and phospholipases.

Amino Acid Sequence↗

PubMed1993

Interfacial activation of the lipase-procolipase complex by mixed micelles revealed by X-ray crystallography.

The three-dimensional structure of the lipase-procolipase complex, co-crystallized with mixed micelles of phosphatidylcholine and bile salt, has been determined at 3 A resolution by X-ray crystallography. The lid, a surface helix covering the catalytic triad of lipase, adopts a totally different conformation which allows phospholipid to bind to the enzyme's active site. The open lid is an essential component of the active site and interacts with procolipase. Together they form the lipid-water interface binding site. This reorganization of the lid structure provokes a second drastic conformational change in an active site loop, which in its turn creates the oxyanion hole (induced fit).

Binding Sites↗

PubMed1993

Crystallization of pancreatic procolipase and of its complex with pancreatic lipase.

The human pancreatic lipase-porcine procolipase complex has been crystallized in space group P3(2)21 (a = b = 80.3 A and c = 251 A) from a solution containing polyethylene glycol, NaCl and beta-octyl glucoside. The crystals diffract to 2.6 A on a synchrotron beam. The complex in the presence of bile salts and phospholipids crystallizes in a tetragonal space group P4(2)2(1)2 (a = b = 133.4 A, c = 92.6 A). Crystals of procolipase alone were obtained under slightly different experimental conditions (space group I432, a = b = c = 164.3 A).

Animals↗

PubMed1993

A hypothetical complex between crystalline flavocytochrome b2 and cytochrome c.

Flavocytochrome b2 and cytochrome c are physiological electron transfer partners in yeast mitochondria. The formation of a stable complex between them has been demonstrated both in solution and in the crystalline state. On the basis of the three-dimensional structures, using molecular modeling and energy minimization, we have generated a hypothetical model for the interaction of these redox partners in the crystal lattice. General criteria such as good charge and surface complementarity, plausible orientation, and separation distance of the prosthetic groups, as well as more specific criteria such as the stoichiometry determined in the crystal, and the involvement of both domains and of more than one subunit of flavocytochrome b2 led us to discriminate between several possible interaction sites. In the hypothetical model we present, four cytochrome c molecules interact with a tetramer of flavocytochrome b2. The b2 and c hemes are coplanar, with an edge-to-edge distance of 14 A. The contact surface area is ca. 800 A2. Several electrostatic interactions involving the flavin and the heme domains of flavocytochrome b2 stabilize the binding of cytochrome c.

Computer Graphics↗

PubMed1993

Solution structure of human corticotropin releasing factor by 1H NMR and distance geometry with restrained molecular dynamics.

The structure of human corticotropin releasing factor (hCRF) has been determined by proton nuclear magnetic resonance (1H NMR) in a mixed-solvent system of 66% trifluoroethanol/34% H2O at pH 3.8 and 37 degrees C. Nearly complete resonance assignment was achieved by using standard two-dimensional methods. Distance restraints for structure calculations were obtained by qualitative analysis of intra- and inter-residue nuclear Overhauser effects. Structures were obtained from the distance restraints by distance geometry, followed by refinement using molecular dynamics and were completed with amide hydrogen exchange data. The structure of hCRF in this solvent comprises an extended N-terminal tetrapeptide connected to a well-defined alpha-helix between residues 6 and 36. The first half of the alpha-helix (residues 6-20) is clearly amphipathic. The five carboxy-terminal residues are predominantly disordered.

Amino Acid Sequence↗

PubMed1993

Engineering cysteine mutants to obtain crystallographic phases with a cutinase from Fusarium solani pisi.

Cutinases are extracellular enzymes involved in the disruption of cutine, an insoluble polyester which covers the surface of plants. They belong to a class of serine esterases that are able to hydrolyse fatty acid esters and emulsified triglycerides as efficiently as lipases, but without displaying interfacial activation. Classical crystallographic methods for obtaining heavy-atom derivatives failed, so the cutinase structure has been solved exclusively by the multiple isomorphous replacement method using four Hg derivatives obtained from mutants S4C, S92C, S120C and S129C. Two of these derivatives behaved as expected: (i) the cys mutant of the catalytic Ser S120C, located at the surface of the active site pocket, leads to a good derivative; and (ii) the Hg atom of the derivative obtained with the S92C mutant is completely accessible to the solvent and occupies two alternative positions--consequently a poor derivative results. In contrast, two mutants show an unexpected behaviour: (i) the Hg atom in the S129C mutant was completely buried 10 A below the protein surface and yielded the best derivative; and (ii) a poor quality derivative was obtained with the S4C mutant. Cys 4 belongs to the disordered propeptide 1-16. The Cys 4 bound Hg atom is located in front of the Asp58 side chain, but neither Cys4 nor parts of the propeptide are clearly visible in the electron density maps of the derivative structure.

Binding Sites↗

PubMed1993

Interactions of indolo[3,2-b]carbazoles and related polycyclic aromatic hydrocarbons with specific binding sites for 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat liver.

In the present study we have investigated the capacity of various compounds sterically related to indolo[3,2-b]carbazole to inhibit specific 2,3,7,8-tetrachloro[1,6-3H]dibenzo-p-dioxin binding in rat liver cytosol, as analyzed by electrofocusing in polyacrylamide gels. When the two nitrogen atoms of indolo[3,2-b]carbazole (IC50 = 3.6 nM) were replaced with sulfur atoms, the affinity for the specific binding sites (IC50 = 3.3 nM) was similar to that of the parent compound, whereas the affinity decreased when the two nitrogen atoms were replaced with oxygen atoms (IC50 = 29 nM). Substitution with methyl groups at positions 5 and 11 (on the nitrogens) of indolo[3,2-b]carbazole resulted in increased affinity (IC50 = 1.2 nM), compared with that of the parent compound, whereas dimethylation at the 4,10- or 2,8-positions decreased the affinity (IC50 = 19 nM and IC50 > 150 nM, respectively). Substitution at positions 5 and 11 of indolo[3,2-b]carbazole with substituents larger than methyl, as in 5,11-diethylindolo[3,2-b]carbazole (IC50 = 8.9 nM), diacetylindolo[3,2-b]carbazole (IC50 = 11.2 nM), 5,11-dibutylindolo[3,2-b]carbazole (IC50 > 150 nM), and 5,11-di(N,N-dimethylaminoethyl)indolo[3,2-b]carbazole (IC50 > 1500 nM), also decreased the affinity. Introduction of oxygen in, or hydroxylation of, the middle ring of indolo[3,2-b]carbazole, giving indolo[3,2-b]carbazole-6,12-quinone (IC50 > 150 nM) or 6,12-dihydroxyindolo[3,2-b]carbazole (IC50 > 1500 nM), respectively, also lowered the affinity. We calculated the Gibbs free energy of solvation of the analogue isoquino[3,4-b]phenanthridine (IC50 = 137 nM), relative to that of dibenz[a,h]anthracene (IC50 = 2.5 nM), in water to be -6 kcal/mol by free energy perturbation, which indicates that the most important explanation for the observed difference in binding affinity is the smaller difference in relative free energy of binding at the binding sites, compared with the Gibbs free energy of solvation of the two compounds.

Animals↗

PubMed1992

Solution conformation of human neuropeptide Y by 1H nuclear magnetic resonance and restrained molecular dynamics.

The solution structure of human neuropeptide Y has been solved by conventional two-dimensional NMR techniques followed by distance-geometry and molecular-dynamics methods. The conformation obtained is composed of two short contiguous alpha-helices comprising residues 15-26 and 28-35, linked by a hinge inducing a 100 degree angle. The first helix (15-26) is connected to a polyproline stretch (residues 1-10) by a tight hairpin (residues 11-14). The helices and the polyproline stretch are packed together by hydrophobic interactions. This structure is related to that of the homologous avian pancreatic polypeptide and bovine pancreatic polypeptide. The C- and N-terminii, known to be involved in the biological activity for respectively the receptor binding and activation, are close together in space. The side chains of residues Arg33, Arg35 and Tyr36 on the one hand, and Tyr1 and Pro2 on the other, form a continuous solvent-exposed surface of 4.9 mm2 which is supposed to interact with the receptor for neuropeptide Y.

Amino Acid Sequence↗

PubMed1992

Crystallization and preliminary X-ray diffraction study of Lathyrus ochrus isolectin II complexed to the human lactotransferrin N2 fragment.

Isolectin II (LOL II) isolated from the seeds of Lathyrus ochrus has been crystallized in the presence of the N2 fragment (18,500 Da) isolated from human lactotransferrin, which contains an N-acetyllactosamine type biantennary glycan linked to Asn137. This is the first example of a legume lectin crystallized with an N-glycosylprotein. Crystals of the LOL II-N2 complex belong to the tetragonal space group (P4(1)2(1)2 or the enantiomorph) with cell dimensions: a = b = 63.5 A, c = 251.9 A. They diffract well up to at least 3.5 A resolution and more weakly up to 2.8 A resolution. Assuming one functional half-entity in the asymmetric unit, an alpha, beta monomer complexed to one N2 fragment (24,500 Da + 18,500 Da) would give a Vm of 2.95 A3/Da and a solvent content of approximately 58%. SDS/polyacrylamide gels of the dissolved crystals show the presence of both the LOL II and N2 fragment.

Crystallization↗

PubMed1992

Structure of the pancreatic lipase-procolipase complex.

Interfacial adsorption of pancreatic lipase is strongly dependent on the physical chemical properties of the lipid surface. These properties are affected by amphiphiles such as phospholipids and bile salts. In the presence of such amphiphiles, lipase binding to the interface requires a protein cofactor, colipase. We obtained crystals of the pancreatic lipase-procolipase complex and solved the structure at 3.04 A resolution. Here we describe the structure of procolipase, which essentially consists of three 'fingers' and is topologically comparable to snake toxins. The tips of the fingers contain most of the hydrophobic amino acids and presumably form the interfacial binding site. Lipase binding occurs at the opposite side to this site and involves polar interactions. Determination of the three-dimensional structure of pancreatic lipase has revealed the presence of two domains: an amino-terminal domain, at residues 1-336 containing the active site and a carboxy-terminal domain at residues 337-449 (ref. 6). Procolipase binds exclusively to the C-terminal domain of lipase. No conformational change in the lipase molecule is induced by the binding of procolipase.

Colipases↗

PubMed1992

Stimulation of a membrane tyrosine phosphatase activity by somatostatin analogues in rat pancreatic acinar cells.

A phosphoryl protein tyrosine phosphatase (PTPase) activity has been characterized in rat pancreatic acinar membranes using 32P-labeled poly(Glu,Tyr) as substrate. Acinar membranes exhibited a high affinity for the substrate, with an apparent Km of 0.46 microM and an apparent Vmax of 0.9 nmol.mg protein-1.min-1. Acinar membrane PTPase activity displayed specific characteristics of other PTPases; it was inhibited by the inhibitors Zn2+, orthovanadate and by the divalent cations Mn2+ and Mg2+, and was stimulated by the reducing-agent dithiothreitol. It was also inhibited by soybean trypsin inhibitor and stimulated by trypsin. Gel permeation of pancreatic acinar membranes gave a single peak of enzyme activity with an apparent molecular mass of 70 000 Da. Further purification by HPLC on DEAE revealed two peaks of PTPase activity at 120 mM and 180 mM NaCl. These two peaks reacted in a Western-blot procedure with anti-(peptide) serum directed towards conserved domain of PTPase as a common 67-kDa form associated with lower-molecular-mass proteolytic fragments (31-56 kDa). Incubation of pancreatic acini with somatostatin analogues, SMS 201-995 or BIM 23014, resulted in a stimulation of membrane PTPase activity. The stimulation was rapid and transient, with a maximal level reached within 15 min of addition. The two analogs stimulated PTPase activity in a dose-dependent manner with half-maximal activation occurring at 7 pM and 37 pM and maximal activation at 0.1 nM and 0.1-1 nM for SMS 201-995 and BIM 23014, respectively. The stimulated-membrane PTPase activity also eluted at an apparent molecular mass of 70 kDa in gel-permeation chromatography. The two analogs inhibited the binding of [125I-Tyr3]SMS 201-995 to pancreatic acinar membranes with similar relative potencies to that observed on stimulation of PTPase activity. We conclude that pancreatic acinar membranes possess a low-molecular-mass PTPase which is stimulated by somatostatin analogs at concentrations involving activation of membrane somatostatin receptors.

Amino Acid Sequence↗

PubMed1992

Isoform purification of gastric lipases. Towards crystallization.

Several isoforms of rabbit and human gastric lipases have been purified. These isoforms have the same apparent molecular weight (Mr approximately 50,000), but very different isoelectric points. Some of these isoforms were purified: pI 7.2 and 6.5 in the case of rabbit gastric lipase; and pI 7.4 and 7.2 in that of human gastric lipase. All the purified isoforms were found to have the same specific lipase activity (around 1200 units per mg of protein, measured on tributyrin as substrate). The isoforms of dog gastric lipase are more closely related, and could not be separated. Partial enzymatic deglycosylation of human gastric lipase reduced the apparent molecular weight from Mr approximately 50,000 to Mr approximately 43,000 and induced a change in the isoelectrofocusing pattern and the emergence of a new isoform (pI 7.3). It is concluded that the charge heterogeneity of gastric lipases is at least partly due to the glycan moiety of the molecule, which amounts to approximately 14% of the total molecular weight. Several crystallization trials on purified native preparations of rabbit and human gastric lipases were unsuccessful, whereas crystals were obtained from native dog gastric lipase and all the purified isoforms of rabbit and human gastric lipases, some of which were crystallographically characterized.

Animals↗

PubMed1992

Fusarium solani cutinase is a lipolytic enzyme with a catalytic serine accessible to solvent.

Lipases belong to a class of esterases whose activity on triglycerides is greatly enhanced at lipid-water interfaces. This phenomenon, called interfacial activation, has a structural explanation: a hydrophobic lid, which at rest covers the catalytic site, is displaced on substrate or inhibitor binding and probably interacts with the lipid matrix. Fusarium solani pisi cutinase belongs to a group of homologous enzymes of relative molecular mass 22-25K (ref. 7) capable of degrading cutin, the insoluble lipid-polyester matrix covering the surface of plants, and hydrolysing triglycerides. Cutinases differ from classical lipases in that they do not exhibit interfacial activation; they are active on soluble as well as on emulsified triglycerides. Cutinases therefore establish a bridge between esterases and lipases. We report here the three-dimensional structure of a recombinant cutinase from F. solani pisi, expressed in Escherichia coli. Cutinase is an alpha-beta protein; the active site is composed of the triad Ser 120, His 188 and Asp 175. Unlike other lipases, the catalytic serine is not buried under surface loops, but is accessible to solvent. This could explain why cutinase does not display interfacial activation.

Amino Acid Sequence↗

PubMed1992

Basic fibroblast growth factor induces proliferation of a rat pancreatic cancer cell line. Inhibition by somatostatin.

AR4-2J, a rat pancreatic acinar-tumor cell line, was used to investigate long-term effects of basic fibroblast growth factor (bFGF) and somatostatin on pancreatic cancer cells. We observed that bFGF stimulated cell proliferation when cells were cultured in serum-free medium. The effect was dose-dependent with half-maximal and maximal effects at 25 pM and I nM bFGF, respectively. The somatostatin analog SMS 201-995 (SMS) decreased the growth-promoting effect of bFGF. The maximal effect was observed at I nM SMS and the half-maximal effect at 20 pM SMS. Characterization of bFGF receptor-binding properties with [125I]bFGF revealed that AR4-2J cells exhibited 2 classes of bFGF binding site with respective KD values of 47 pM and 3 nM and binding capacities of 14 fmol and 0.9 pmol/10(6) cells. High-affinity receptors correlated with bFGF stimulation of AR4-2J cell growth, suggesting that the effects of bFGF are receptor-mediated.

Animals↗