Refined X-ray structures of the oxidized, at 1.3 A, and reduced, at 1.17 A,
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Journal Article↗
Biomedical subjects
G Hudry-Clergeon
Publications and source records attributed to G Hudry-Clergeon.
Refined X-ray structures of the oxidized, at 1.3 A, and reduced, at 1.17 A, [2Fe-2S] ferredoxin from the cyanobacterium Anabaena PCC7119 show redox-linked conformational changes.
The chemical sequence of the [2Fe-2S] ferredoxin from the cyanobacterium AnabaenaPCC7119 (Fd7119) and its high-resolution X-ray structures in the oxidized and reduced states have been determined. The Fd7119 sequence is identical to that of the ferredoxin from the PCC7120 strain (Fd7120). X-ray diffraction data were collected at 100 K with an oxidized trigonal Fd7119 crystal, at 1.3 A resolution, and with an orthorhombic crystal, previously reduced with dithionite and flash frozen under anaerobic conditions, at 1.17 A resolution. The two molecular models were determined by molecular replacement with the [2Fe-2S] ferredoxin from the strain PCC7120 (Rypniewski, W. R., Breiter, D. R., Benning, M. M., Wesenberg, G., Oh, B.-H., Markley, J. L., Rayment, I., and Holden, H. M. (1991) Biochemistry 30, 4126-4131.) The final R-factors are 0. 140 (for the reduced crystal) and 0.138 (for the oxidized crystal). The [2Fe-2S] cluster appears as a significantly distorted lozenge in the reduced and oxidized redox states. The major conformational difference between the two redox forms concerns the peptide bond linking Cys46 and Ser47 which points its carbonyl oxygen away from the [2Fe-2S] cluster ("CO out") in the reduced molecule and toward it ("CO in") in the oxidized one. The "CO out" conformation could be the signature of the reduction of the iron atom Fe1, which is close to the molecular surface. Superposition of the three crystallographically independent molecules shows that the putative recognition site with the physiological partner (FNR) involves charged, hydrophobic residues and invariant water molecules.
Amino Acid Sequence↗
The protein sequence of an archaeal catalase-peroxidase.
The gene encoding a catalase-peroxidase of archaeal origin, the halophilic catalase-peroxidase from Haloarcula marismortui, was sequenced. The primary structure proposed was confirmed by Edman degradation and mass spectrometry analyses of proteolytic fragments of the purified protein. The open reading frame in the gene corresponds to 731 amino acids and the calculated mass of the mature protein (deleted of the N-terminal methionine) is 81,253.65 Da, in reasonable agreement with the value of 81,292 +/- 9 Da previously measured by mass spectrometry. Southern and Northern blot analyses showed that the protein is encoded by a single gene as a monocistronic transcript. The protein sequence shows a high level of identity with bacterial catalase-peroxidases, with strongly conserved regions around the heme binding histidines. Similarly to other soluble halophilic proteins, it shows the excess of acidic residues that has been associated with solvation in halophilic adaptation.
Amino Acid Sequence↗
Can immunoglobulin C(H)1 constant region domain modulate antigen binding affinity of antibodies?
Although the switch process is frequently associated with affinity maturation, the constant region is not assumed to play a role in Ag-Ab binding. In the present work, we demonstrate that two clonally related human monoclonal Igs sharing identical V(H) and V(L) sequences, but expressing different isotypes (IgA1kappa(PER) and IgG1kappa(PER)), bind tubulin with significantly different affinities. This difference was mainly accounted for by a disparity in the association rate constants. These results suggest that affinity maturation of this clone could be achieved through class switching in the absence of further somatic mutations. Since the differences observed were found at the Fab level, they also suggest a role for the C(H)1 domain in structuring the Ag-binding site into a more kinetically competent form.
Amino Acid Sequence↗
Identification of a cryptic protein kinase CK2 phosphorylation site in human complement protease Clr, and its use to probe intramolecular interaction.
Treatment of human (activated)C1r by CK2 resulted in the incorporation of [32P]phosphate into the N-terminal alpha region of its non-catalytic A chain. Fragmentation of 32P-labelled (activated)C1r followed by N-terminal sequence and mass spectrometry analyses allowed identification of Ser189 as the phosphorylation site. Accessibility of Ser189 was low in intact C1r, due in part to the presence of one of the oligosaccharides borne by the alpha region, further reduced in the presence of calcium, and abolished when C1r was incorporated into the C1s-C1r-C1r-C1s tetramer or the C1 complex. In contrast, phosphorylation was enhanced in the isolated alpha fragment and insensitive to calcium. Taken together, these data provide support for the occurrence of a (Ca2+)-dependent interaction between the alpha region and the remainder of the C1r molecule.
Amino Acid Sequence↗
Complete amino acid sequence of the Aa6 subunit of the scorpion Androctonus australis hemocyanin determined by Edman degradation and mass spectrometry.
The primary structure of the hemocyanin Aa6 subunit from the scorpion Androctonus australis was resolved by using protein sequencing and mass spectrometry for analysis of the polypeptide chain and of fragments obtained by CNBr, trypsin, and chymotrypsin cleavage. Due to the high sensitivity of the methodologies used, only a small amount of material, less than 1 mg, was consumed. The complete sequence is composed of 626 amino acid residues and the protein is not glycosylated but probably phosphorylated at Ser374. Its molecular mass measured by mass spectrometry (71,890 +/- 7 Da) is about 30 Da higher than the mass calculated from the sequence data (71,860.1 Da). The origin of this difference is not clear but could result from minor molecular heterogeneities. Within the chelicerates, the Aa6 subunit of the arachnid A. australis shares 405 identical residues with chain e of another arachnid, Eurypelma californicum, and 399 with chain alpha of the merostom Tachypleus tridentatus. The degrees of identity between these three subunits, which are known to occupy the same location in the native hemocyanin oligomers, are significantly higher than those existing between the subunits a, d, and e of E. californicum. This favors the hypothesis that gene duplications, leading to separate chains in one species, have occurred before the divergence between arachnids and merostoms.
Amino Acid Sequence↗
Complete amino acid sequence of Proteus mirabilis PR catalase. Occurrence of a methionine sulfone in the close proximity of the active site.
The catalase of Proteus mirabilis PR, a peroxide-resistant (PR) mutant of Proteus mirabilis, binds strongly NADPH, which is a unique property among known bacterial catalases. The enzyme subunit consists of 484 amino acid residues for a mass of 55,647 daltons. The complete amino acid sequence was resolved through the combination of protein sequencing, mass spectrometry, and nucleotide sequencing of a PCR fragment. The sequence obtained was compared with that of other known catalases. Amino acids of the active site are all conserved as well as essential residues involved in NADPH binding. Among the amino acids interacting with the heme, a methionine sulfone was found at position 53, in place of a valine in most other catalases. The origin of oxidation of this methionine is unknown, but the presence of this modification could change iron accessibility by large substrates or inhibitors. This posttranslational modification was also demonstrated in the wild-type P. mirabilis catalase.
Amino Acid Sequence↗
Amino acid sequences in fibrinogen mediating its interaction with its platelet receptor, GPIIbIIIa.
Platelet membrane GPIIbIIIa is a member of the family receptors named integrins that recognize RGD sequences in their ligands. GPIIbIIIa interacts with at least three different adhesive ligands: fibrinogen, fibronectin, and von Willebrand factor. These interactions are inhibited by RGD-containing peptides and by peptides corresponding to a sequence unique to fibrinogen in the COOH-terminal domain of its gamma chain (HLGGAKQAGDV). Two RGD sequences are present in fibrinogen A alpha chain: an RGDS sequence at A alpha 572-575, and an RGDF sequence at A alpha 95-98. Polyclonal antibodies raised against the RGDF sequence and the gamma COOH-terminal domain both reacted specifically with fibrinogen in solid phase enzyme-linked immunosorbent assays and immunoprecipitated the protein in solution. The Fab fragments prepared from these antibodies inhibited fibrinogen-platelet interaction and aggregation. These results demonstrate that these two sequences are both accessible within the fibrinogen molecule and are both implicated in ligand binding and cell-cell interaction. In addition, by further examining the interaction of the gamma chain peptide with platelets, it was found that RGDF and the gamma peptide produced a similar dose-dependent inhibition of the binding of the labeled gamma peptide to ADP-stimulated platelets. These results provide evidence that the RGDF sequence present at the A alpha 95-98 constitutes with the gamma 401-411 sequence two recognition sites interacting with the same site or with mutually exclusive sites on GPIIbIIIa.
Amino Acid Sequence↗
Differential structural requirements for fibrinogen binding to platelets and to endothelial cells.
The cytoadhesins represent a group of RGD receptors that belongs to the integrin superfamily of adhesion molecules. Members of this cytoadhesin family include the platelet GPIIb-IIIa and the vitronectin receptors. These glycoproteins share the same beta-subunit, which is associated with different alpha subunits to form an alpha/beta heterodimer. In the present study, we have analyzed the fine recognition specificy of the cytoadhesins from platelets and endothelial cells for the adhesive protein, fibrinogen. Two sets of synthetic peptides, RGDX peptides and peptides corresponding to the COOH terminus of the fibrinogen gamma chain, were compared for their structure-function relationships in the two cellular systems. The results indicate that: (a) both RGDX and gamma-chain peptides inhibit the binding of fibrinogen to platelets and endothelial cells; (b) a marked influence of the residue at the COOH- and NH2-terminal positions of each peptide set can be demonstrated on the two types; and (c) RGDX and gamma peptides have differential effects on platelets and endothelial cells with respect to fine structural requirements. These results clearly indicate that while the platelet and endothelial cytoadhesins may interact with similar peptidic sequences, they express a different fine structural recognition.
Actin Cytoskeleton↗
Evidence for two functionally different fibrinogen receptors on hemopoietic cells: the glycoprotein IIb-IIIa and the mitogenic fibrinogen receptor.
We have previously established that the mitogenic effect of fibrinogen on hemopoietic cell lines Raji and JM is mediated via a specific receptor (Levesque, J.-P. et al.: Proc. Natl. Acad. Sci. USA 83:6494-6498, 1986). In this study, we have further characterized the fibrinogen domain involved in the binding to the mitogenic receptor. This binding was not inhibited either by a monoclonal antibody against the C-terminal sequence of the fibrinogen gamma chains or by synthetic peptides containing the Arg-Gly-Asp sequence. Such inhibition is specific of the platelet fibrinogen receptor, the glycoprotein IIb-IIIa complex. Fragments containing the fibrinogen D domain were the only plasmin degradation products of fibrinogen which were mitogenic. These fragments acted via direct binding on the mitogenic receptor with a Kd of 2.24 X 10(-6) M. This value was similar to the KI value of unlabeled fragments D (2.47 X 10(-6) M). Our results suggest the presence of two different functional types of fibrinogen receptors: the glycoprotein IIb-IIIa receptor responsible both for platelet aggregation and leukocyte adhesion and killing, and the mitogenic receptor involved in proliferation control of hemopoietic cells.
Antibodies, Monoclonal↗
Fibrinogen and fibrin in strong magnetic fields. Complementary results and discussion.
When fibrin polymerizes in a strong magnetic field, it can be highly oriented. The structural diffraction study of the oriented polymer becomes thus possible. The magnetic birefringence can also be used to study the development of the polymer Fibrinogen in solution is weakly oriented in high magnetic fields. In this work we present complementary results and discussion. The validity of the comparison of the orientation parameters of fibrinogen and fibrin with those of other orientable known biological structures is discussed. The orientation of fibrin formed from fibrin monomer solution is compared to that of fibrin formed by the action of thrombin on fibrinogen. The conditions to obtain highly oriented fibrin gels suitable for three dimensional structure studies are also briefly discussed.
Animals↗
Molecular characterization of an abnormal fibrinogen by two-dimensional electrophoresis.
We examined normal and abnormal fibrinogen (fibrinogen "Grenoble") by two-dimensional gel electrophoresis to obtain data on possible defects at the molecular level. Fibrinogen Grenoble is characterized by an abnormal rate of fibrin monomer aggregation. The electrophoretic analysis revealed the presence of abnormal gamma chains. Two kinds of gamma chains can be detected in fibrinogen Grenoble: (a) normal gamma chains and (b) gamma chains Grenoble (gamma G) with a greater molecular mass but no modification in isoelectric point. The latter chain can be detected in whole plasma by two-dimensional gel electrophoresis. Metrological analysis was performed in an attempt to quantify observed differences between normal fibrinogen and fibrinogen Grenoble. On use of gels stained either with Coomassie Brilliant Blue or with silver, the partly qualified evaluation gives about 60% normal gamma chain and 40% gamma chain Grenoble.
Blood Coagulation Disorders↗
Orientation of fibrin in strong magnetic fields.
Magnetic field orientation can be considered as a new valuable technique for the study of fibrin. Birefringence measurements during polymerization in the magnetic field enables a new approach to study the mode of association of fibrin monomers. Stable fully oriented clots can be obtained for investigation of the three-dimensional and the secondary structures of the fibrin fiber. Such experiments could be useful to assess the structure of other biopolymers diamagnetically anisotropic.
Animals↗
Fibrinogen and fibrin structure and fibrin formation measured by using magnetic orientation.
Accurate birefringence measurements show that fibrinogen orients to a small degree in high magnetic fields. This effect can be explained as due to the molecule having about 30% (by weight) alpha-helix oriented relatively parallel to the long axis. Birefringence measurements on fully oriented fibrin suggest that aligned alpha-helical content is less than that estimated for fibrinogen. But because of limitations in the analysis this difference must be viewed with caution. Highly oriented fibrin results when polymerization takes place slowly in a strong magnetic field. Low-angle neutron diffraction patterns from oriented fibrin made in the presence of EDTA, made in the presence of calcium, or stabilized with factor XIIIa are very similar, showing that the packing of the molecules within the fibers is the same or very similar in these different preparations. The induced magnetic birefringence was used to follow fibrin formation under conditions in which thrombin was rate limiting. The fiber network formed by approximately the gelation point constitutes a kind of matrix or frame that is largely built upon during the remaining approximately 85% of the reaction. After gelation the reaction is pseudo-first order.
Animals↗
Similarity of CSF of media conditioned by different human tissues.
Conditioned media prepared using human placenta, spleen, bone marrow and peripheral blood leucocytes revealed a common pattern of two distinct species of colony-stimulating factors (CSF) separable by gel filtration. The peak of greatest activity, active against both human and mouse marrow, had an apparent molecular weight (MW) of 24,000-28,000 Daltons. A peak of low activity detected only against mouse marrow had an apparent MW of approximately 150,000 Daltons. The type of progenitor cells stimulated by the crude medium, by the low MW CSF species, and by the high MW species were similar for the four conditioned media despite their different origins. No difference was found in either the MW of the human active CSF present or the type of progenitor cells stimulated by media conditioned with cells of leukemic origin.
Animals↗
Human factor VIII procoagulant activity and phospholipid interaction.
The interaction between purified human factor VIII and phospholipid vesicles was investigated. The binding of factor VIII to an equimolecular mixture of phosphatidylserine (PS) and phosphatidylcholine (PC) was studied by sucrose gradient ultracentrifugation (10-40% w/v saccharose in 0.01 M Tris-HC1/0.15 M NaCl buffer (pH 7). In the absence of phospholipids all factor VIII activities (VIII R : WF and VIII R : AG) were found in the zone of highest sucrose density including the factor VIII related protein subunit (200 000 molecular weight). In the presence of an equimolecular mixture of PS/PC VIII R : WF activity, VIII R: AG and factor VIII related protein still migrated to the bottom of the tube, while VIII: C activity remained at the top where phospholipids were found. Thus a dissociation phenomenon between VIII : C and the other factor VIII related activities was apparent in the presence of phospholipids. These results also demonstrate the binding of factor VIII: C to certain active phospholipids.
Antigens↗
The fibrinolytic action of the synthetic agent S-1623 in a purified system of fibrin and plasminogen.
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Animals↗
Oriented fibrin gels formed by polymerization in strong magnetic fields.
Fibrinogen is a soluble plasma protein which, after cleavage by the specific proteolytic enzyme thrombin, polymerizes to form the filamentous fibrin network during blood clotting (see refs 1 and 2 for reviews). Fibrinogen has a molecular weight of 340,000 and is composed of two identical halves, each containing three peptide chains designated A alpha, B beta and gamma. Fibrin monomers are produced by thrombin which releases the small negatively charged fibrinopeptides A and B. The overall shape of the fibrinogen molecule has not been unequivocally established. The trinodular, elongated (approximately 450 A long) structure proposed by Hall and Slayter is the most widely accepted model and it has obtained additional support from recent work. Fibrin monomers are also about 450 A long and in fibres they probably have a half-staggered arrangement along the axis. The fibres are an assembly of protofibrils whose structure and packing are not reliably known. We report here that highly oriented fibrin gels are formed when polymerization takes place slowly in a strong magnetic field. It is shown that the protofibrils pack into a three-dimensional crystalline lattice. We introduce magnetically induced birefringence as a potential tool for studying polymerization and briefly speculate on the applications of strong magnetic fields.
Biopolymers↗