Biomedical subjects

C Cambillau

Publications and source records attributed to C Cambillau.

119 records · Page 7Linked to original sources

PubMed1988

Subunit III of ruminant procarboxypeptidase A-S6 complexes and pancreatic proteases E. A new family of pancreatic serine endopeptidases?

Subunit III (BSIII) of the bovine ternary complex of procarboxypeptidase A-S6 (PCPA-S6), a defective serine endopeptidase-like protein, actively synthesized by the pancreas of some ruminant species, is highly homologous to human protease E (HPE). Both proteins possess the same atypical disulfide bridge in position 98-99b. They are structurally related to porcine elastase 1 and human elastase 2 (about 56% identity). However, in contrast to those two enzymes which have an overall positive net charge, BSIII and HPE are negatively charged. Three-dimensional models of BSIII and HPE have been constructed from the crystallographic structure of porcine pancreatic elastase 1. The inhibitor-binding site for TFAI in these three proteins seems to be very similar; the atypical disulfide bridge does not seem to be involved in this binding site. The specific structural features of BSIII and HPE strongly support the assumption that BSIII is a truncated protease E and that both proteins belong to a separate serine endopeptidase family.

Amino Acid Sequence↗

PubMed1988

Amino acid sequence determination and three-dimensional modelling of thioredoxin from the photosynthetic bacterium Rhodobacter sphaeroides Y.

The complete primary structure of thioredoxin from Rhodobacter sphaeroides Y has been determined by analysis of peptides after cleavage with cyanogen bromide, chymotrypsin and trypsin. Peptides were separated by HPLC and analyzed by liquid-phase and gas-phase sequencer degradations. The protein consists of 105 residues (Mr = 11,180); its amino acid sequence shows a clear homology to the five known thioredoxins from plant or bacterial sources, with 40-56% residue identity when the proteins are aligned at the active-site disulfide. Not only the active-site regions are conserved, but also residues which belong to the hydrophobic surface suggested to be important for binding of procaryote thioredoxins in redox interactions with other proteins (residues 75-76; 91-93 in Escherichia coli). A three-dimensional model of Rb. sphaeroides thioredoxin has been derived from the E. coli crystallographic structure with computer graphics. This model indicates that the overall structures as well as the active sites are closely similar; however, the residue substitutions allow both proteins to adopt different local folding as shown in the hydrophobic core.

Amino Acid Sequence↗

PubMed1988

Model of a complex between the tetrahemic cytochrome c3 and the ferredoxin I from Desulfovibrio desulfuricans (Norway strain).

A three-dimensional model of an electron-transfer complex between the tetrahemic cytochrome c3 and the ferredoxin I from the sulfate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) has been generated through computer graphics methods. The model is based on the known X-ray structure of the cytochrome and on a model of the ferredoxin that has been derived through computer graphics modeling and energy minimization methods, from the X-ray structure of the homologous ferredoxin from Peptococcus aerogenes. Four possible models of interaction between the two molecules were examined by bringing in close proximity each of the four hemes and the redox center (4Fe-4S) of the ferredoxin and by optimizing the ion pairs interactions. One of these models shows by far the "best" structure in terms of charges, interactions, and complementarity of the topology of the contact surfaces. In this complex, the distance between the iron atoms of the ferredoxin redox center and the hemic iron atom is 11.8 A, which compares well with those found between redox centers in other complexes. The contact surface area between the two molecules is 170 A2.

Amino Acid Sequence↗

PubMed1988

The binding of methylsulfonyl-polychloro-biphenyls to uteroglobin.

The binding of methylsulfonyl-polychloro-biphenyls (methylsulfonyl-PCBs) to purified uteroglobin was studied by a dextran-coated charcoal assay using 4,4'-bis([ 3H]methylsulfonyl)-2,2',5,5'-tetrachlorobiphenyl [(3H-MeSO2)2TCB] as radiolabeled ligand. The specific binding of this ligand to uteroglobin was enhanced by the presence of dithiothreitol, and the optimal concentration of dithiothreitol for binding was 20 mM. The specific [(3H-MeSO2)2TCB] binding was inhibited by 4-methylsulfonyl-2,2',4',5,5'-pentachlorobiphenyl in a concentration-dependent manner. The molecular structures of methylsulfonyl-PCBs, and progesterone, were fitted into the X-ray crystallographic structure of uteroglobin using the molecular graphics program TOM. In these simulations the water-accessible surfaces of the ligands appeared quite similar, and fitted nicely in the internal water-accessible surface of uteroglobulin. Moreover, it appeared from the computer-supported ligand-binding studies that the sulfone oxygens of the studied methylsulfonyl-PCBs, as well as the carbonyl (C20) of progesterone, may form hydrogen bonds with the hydroxyl group of TYR 21 of uteroglobulin. These findings may explain why both steroids and methylsulfonyl-PCBs interact with the same protein, although these two types of ligands are structurally dissimilar.

Binding, Competitive↗

PubMed1987

Structural assignment of the heme potentials of cytochrome c3, using a specifically modified arginine.

In view of the assignment of the four redox potentials values to the four heme groups in the crystallographic structure of Desulfovibrio desulfuricans Norway cytochrome c3, a biochemical approach is reported. A singly modified cytochrome c3 on arginine 73 has been prepared. The study of the redox properties of the modified cytochrome by electrochemistry together with the graphic modelisation of the molecule allow to assign the highest redox potential (-165 mV) to the heme 4 in the three dimensional structure.

Arginine↗

PubMed1987

Crystallization, preliminary X-ray study and crystal activity of the hydrogenase from Desulfovibrio gigas.

Hydrogenase (EC 1.12) from Desulfovibrio gigas is a dimeric enzyme (26 and 62 (X 10(3) Mr) that catalyzes the reversible oxidation of molecular hydrogen. Single crystals of hydrogenase have been produced using the hanging drop method, with either PEG (polyethylene glycol) 6000 or ammonium sulfate as precipitants at pH 6.5. X-ray examination of the crystals indicates that those obtained with ammonium sulfate are suitable for structure determination to at least 3.0 A resolution when synchrotron radiation Sources are used (1 A = 0.1 nm). The crystals are monoclinic, with space group C2, and cell dimensions a = 257.0 A, b = 184.7 A, c = 148.3 A and beta = 101.3 degrees, and contain between four and ten molecules per asymmetric unit. The enzyme can be reactivated within the crystals under reducing conditions without crystal damage.

Crystallography↗

PubMed1986

Crystallization and preliminary X-ray study of subunit III of the bovine pancreatic procarboxypeptidase A-S6 ternary complex.

Subunit III of the bovine pancreatic procarboxypeptidase A-S6 ternary complex was dissociated from the complex, purified and crystallized using the hanging- or sitting-drop method of vapour diffusion, with ammonium sulphate as the precipitant. The assays were carried out at pH 4.2 (20 mM-acetate buffer). An X-ray examination of the crystals shows that they are monoclinic, with a space group P21 and cell dimensions a = 47.9 A, b = 61.3 A, c = 39.0 A and beta = 95.0 degrees. The asymmetric unit contains one molecule of 25,800 Mr. The crystals are suitable for structure determination to at least 2.8 A resolution.

Animals↗

PubMed1986

The inactive subunit of ruminant procarboxypeptidase A-S6 complexes. Structural basis of inactivity and physiological role.

Subunit III has so far been found only in the pancreas of ruminants in a non-covalent association (procarboxypeptidase A-S6) with two different proteins: the procarboxypeptidase A itself (subunit I) and a C-type chymotrypsinogen (subunit II). In contrast with these latter two proteins, which are zymogens of pancreatic proteases, subunit III seems to be devoid of any activity towards specific substrates of pancreatic proteases. However, it possesses a weakly functional active site which allows it to hydrolyze a non-specific ester, p-nitrophenyl acetate, and to react with several active-site titrants. The binding of proflavin to subunit III shows that this protein owns a non-polar binding site with a very high Kd compared to that of chymotrypsin. The comparison of the amino acid sequences of subunit III and some serine proteases showed that subunit III is closely related to an elastase. Models of the tertiary structure of subunit III suggest a conformational modification that affects the substrate binding and could explain the lack of specific enzymatic activity. The presence of subunit III in the ternary complex is not related to an enzymatic function. This protein does not participate in the activation process of subunit I but prevents the denaturation of this subunit at low pH. This may represent its biological role in the acidic environment of the duodenum in ruminants.

Amino Acid Sequence↗

PubMed1985

Interactions of indoles with specific binding sites for 2,3,7,8-tetrachlorodibenzo-p-dioxin in rat liver.

In order to identify some of the structural requirements for binding of indoles to the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), we have investigated the capacity of various indoles to inhibit specific [1,6-3H]TCDD binding in rat liver cytosol, as analyzed by electrofocusing in polyacrylamide gel. Of these indoles, indolo[3,2-b]carbazole was the most active. The IC50 value for receptor binding of indolo[3,2-b]carbazole as well as for 2,3,7,8-tetrachlorodibenzofuran was 3.6 nM, whereas that of 5,6-benzoflavone was 26 nM. Both indolo[3,2-b]carbazole and 2,3,7,8-tetrachlorodibenzofuran competitively inhibited the binding of [3H]TCDD to the receptor. The well-known microsomal enzyme inducer 3,3'-diindolymethane did not interact significantly with the TCDD receptor. Previous concepts of structure-activity relationships for binding of chlorinated dioxins to the TCDD receptor fail to account for the receptor binding of unhalogenated aryl hydrocarbon hydroxylase inducers such as 5,6-benzoflavone. We have instead considered the true three-dimensional space occupied by some receptor ligands by means of a computer using crystallographic data as inputs. When the atomic van der Waals radii were included, all potent receptor ligands studied could be fitted into a rectangle of 6.8 X 13.7 A.

Animals↗

PubMed1984

Correlation of exons with structural domains in alcohol dehydrogenase.

The intron/exon arrangement in the gene sequence of maize alcohol dehydrogenase has been compared to the three dimensional structure of liver alcohol dehydrogenase. The co-enzyme binding domain is separated from the catalytic domain by introns four and nine. Intron seven separates the co-enzyme binding domain into two structurally similar mononucleotide binding units. The first of these units is divided by introns five and six into three structurally similar alpha beta modules. Implications of these results for protein evolution is discussed. All splice junctions map close to or at the surface of the domains, and several of these cannot be identified by distance maps.

Alcohol Dehydrogenase↗

PubMed1984

Conformational and functional similarities between glutaredoxin and thioredoxins.

The tertiary structures of thioredoxin from Escherichia coli and bacteriophage T4 have been compared and aligned giving a common fold of 68 C alpha atoms with a root mean square difference of 2.6 A. The amino acid sequence of glutaredoxin has been aligned to those of the thioredoxins assuming that glutaredoxin has the same common fold. A model of the glutaredoxin molecule was built on a vector display using this alignment and the T4 thioredoxin tertiary structure. By comparison of the model with those of the thioredoxins, we have identified a molecular surface area on one side of the redox-active S-S bridge which we suggest is the binding area of these molecules for redox interactions with other proteins. This area comprises residues 33-34, 75-76 and 91-93 in E. coli thioredoxin; 15-16, 65-66 and 76-78 in T4 thioredoxin and 12-13, 59-60 and 69-71 in glutaredoxin. In all three molecules, this part of the surface is flat and hydrophobic. Charged groups are completely absent. In contrast, there is a cluster of charged groups on the other side of the S-S bridge which we suggest participates in the mechanisms of the redox reactions. In particular, a lysine residue close to an aromatic ring is conserved in all molecules.

Amino Acid Sequence↗