Molecular mechanisms of initiation of fibrinolysis by fibrin

2003 ◽  
Vol 89 (03) ◽  
pp. 409-419 ◽  
Author(s):  
Willem Nieuwenhuizen ◽  
Leonid Medved
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Binding Sites ◽  
Molecular Mechanisms ◽  
Intramolecular Interaction ◽  
Affinity Binding ◽  
Fibrin Deposition ◽  
High Affinity ◽  
Physiological Processes ◽  
D Region

SummaryFibrinogen is rather inert in the circulation, however, after conversion into fibrin it participates in various physiological processes including fibrinolysis. Initiation of fibrinolysis occurs through a number of orchestrated interactions between fibrin, plasminogen and its activator tPA which result in generation of plasmin. Numerous studies localized a set of specific low affinity tPA- and plasminogen-binding sites in each D region of fibrin(ogen). The tPA-binding site includes residues γ312-324 and the plasminogen-binding site includes residues Aα148-160; they bind tPA and plasminogen with a Kd of about 1 μM. Another set of high affinity tPA- and plasminogen-binding sites (Kds = 16-33 nM) was identified in the compact portion of each fibrin(ogen) αC-domain within residues Aα392-610. All these sites are cryptic in fibrinogen and become exposed in fibrin. Recent studies with recombinant and proteolytic fibrin(ogen) fragments clarified the molecular mechanisms by which these sites become exposed. Namely, upon fibrin assembly, the interaction between the D and E regions causes conformational changes in the former that expose the low affinity binding sites. The exposure of the high affinity binding sites in the αC-domains is connected most probably with their switch from an intramolecular interaction in fibrinogen to an intermolecular one in fibrin. These mechanisms serve to minimize degradation of circulating fibrinogen and confine fibrinolysis to places of fibrin deposition.

scholarly journals Characterization of Ca2+ interactions with matrix metallopeptidase-12: implications for matrix metallopeptidase regulation

2006 ◽  
Vol 398 (3) ◽  
pp. 393-398 ◽  
Author(s):  
Thomas Gossas ◽  
U. Helena Danielson
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Calcium Binding ◽  
Three Dimensional ◽  
Zinc Ion ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Matrix Metallopeptidase

Matrix metallopeptidase-12 (MMP-12) binds three calcium ions and a zinc ion, in addition to the catalytic zinc ion. These ions are thought to have a structural role, stabilizing the active conformation of the enzyme. To characterize the importance of Ca2+ binding for MMP-12 activity and the properties of the different Ca2+ sites, the activity as a function of [Ca2+] and the effect of pH was investigated. The enzymatic activity was directly correlated to calcium binding and a Langmuir isotherm for three binding sites described the activity as a function of [Ca2+]. The affinities for two of the binding sites were quantified at several pH values. At pH 7.5, the KD was 0.1 mM for the high-affinity binding site, 5 mM for the intermediate-affinity binding site and >100 mM for the low-affinity binding site. For all three sites, the affinity for calcium decreased with reduced pH, in accordance with the loss of interactions upon protonation of the calcium-co-ordinating aspartate and glutamate carboxylates at acidic pH. The pKa values of the calcium binding sites with the highest and intermediate affinities were determined to be 4.3 and 6.5 respectively. Optimal pH for catalysis was above 7.5. The low-, intermediate- and high-affinity binding sites were assigned on the basis of analysis of three-dimensional-structures of MMP-12. The strong correlation between MMP-12 activity and calcium binding for the physiologically relevant [Ca2+] and pH ranges studied suggest that Ca2+ may be involved in controlling the activity of MMP-12.

scholarly journals Self-association configures the NAD+-binding site of plant NLR TIR domains

2021 ◽  
Author(s):  
Hayden Burdett ◽  
Xiahao Hu ◽  
Maxwell X Rank ◽  
Natsumi Maruta ◽  
Bostjan Kobe
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Active Site ◽  
Binding Sites ◽  
Molecular Mechanisms ◽  
Structural Features ◽  
Active Conformation ◽  
Effector Triggered Immunity ◽  
Self Association ◽  
Tir Domains

TIR domains are signalling domains present in plant nucleotide-binding leucine-rich repeat receptors (NLRs), with key roles in plant innate immunity. They are required for the induction of a hypersensitive response (HR) in effector-triggered immunity, but the mechanism by which this occurs is not yet fully understood. It has been recently shown that the TIR domains from several plant NLRs possess NADase activity. The oligomeric structure of TIR-containing NLRs ROQ1 and RPP1 reveals how the TIR domains arrange into an active conformation, but low resolution around the NAD+ binding sites leaves questions unanswered about the molecular mechanisms linking self-association and NADase activity. In this study, a number of crystal structures of the TIR domain from the grapevine NLR RUN1 reveal how self-association and enzymatic activity may be linked. Structural features previously proposed to play roles involve the ″AE interface″ (mediated by helices A and E), the ″BB-loop″ (connecting β-strand B and helix B in the structure), and the ″BE interface″ (mediated by the BB-loop from one TIR and the ″DE surface″ of another). We demonstrate that self-association through the AE interface induces conformational changes in the NAD+-binding site, shifting the BB-loop away from the catalytic site and allowing NAD+ to access the active site. We propose that an intact ″DE surface″ is necessary for production of the signalling product (variant cyclic ADPR), as it constitutes part of the active site. Addition of NAD+ or NADP+ is not sufficient to induce self-association, suggesting that NAD+ binding occurs after TIR self-association. Our study identifies a mechanistic link between TIR self-association and NADase activity.

Fragment D Immunoreacivity: The Influence of Iodination and Calcium Environment

1979 ◽  
Author(s):  
B. Kudryk ◽  
M. Blombäck
Keyword(s):  
Conformational Changes ◽  
Binding Sites ◽  
Specific Activity ◽  
High Specific Activity ◽  
Antigenic Determinants ◽  
Affinity Binding ◽  
Compact Structure ◽  
High Affinity Binding ◽  
High Affinity ◽  
Chloramine T

Human fragment D (Fg-Ds) has heen iodinated using both the Chloramine-T and lactoperoildaae methods. The specific activity was similar regardless of the method used. However, binding to a specific antibody was different for each preparation. The antigen labeled by the Chloramine-T method bound to a maximum of 40% the other labeled product bound up to 85%. A correlation between the decree of immunoreactivity and avidity for a fihrinmcnomer conjugate vas found also. Fibrinmonomer bound about twice the ajnount of lactoperoxidase iodinated Fg-Da ae it did the Chloramine-T product. The use of these conjugates in the purification of immunoreactive Fg-Ds of high specific activity will be discussed. High affinity binding sites for calcium have recently been demonstrated in fibrinogen. Tha presence of bound calcium is also believed to protect Fg-Ds f m further digestion by plasmin. This is probably due to the formation of a more compact structure. However, conformational changes for calcium bound fibrinogen or Fg-Ds have not been observed. We tested the immunoreactivity of the lactoperoxidase iodinated Fg-Ds in presence and absence of calcium. Differences were found and this data suggests that soma modification of antigenic determinants takes place as a consequence of calcium in the environment.

scholarly journals The TorR High-Affinity Binding Site Plays a Key Role in Both torR Autoregulation and torCADOperon Expression in Escherichia coli

2000 ◽  
Vol 182 (4) ◽  
pp. 961-966 ◽  
Author(s):  
Mireille Ansaldi ◽  
Gwénola Simon ◽  
Michèle Lepelletier ◽  
Vincent Méjean
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Binding Sites ◽  
Response Regulator ◽  
Regulatory System ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site

ABSTRACT In the presence of trimethylamine N-oxide (TMAO), the TorS-TorR two-component regulatory system induces thetorCAD operon, which encodes the TMAO respiratory system ofEscherichia coli. The sensor protein TorS detects TMAO and transphosphorylates the response regulator TorR which, in turn, activates transcription of torCAD. The torRgene and the torCAD operon are divergently transcribed, and the short torR-torC intergenic region contains four direct repeats (the tor boxes) which proved to be TorR binding sites. The tor box 1-box 2 region covers thetorR transcription start site and constitutes a TorR high-affinity binding site, whereas box 3 and box 4 correspond to low-affinity binding sites. By using torR-lacZ operon fusions in different genetic backgrounds, we showed that thetorR gene is negatively autoregulated. Surprisingly, TorR autoregulation is TMAO independent and still occurs in atorS mutant. In addition, this negative regulation involves only the TorR high-affinity binding site. Together, these data suggest that phosphorylated as well as unphosphorylated TorR binds the box 1-box 2 region in vivo, thus preventing RNA polymerase from binding to the torR promoter whatever the growth conditions. By changing the spacing between box 2 and box 3, we demonstrated that the DNA motifs of the high- and low-affinity binding sites must be close to each other and located on the same side of the DNA helix to allow induction of the torCAD operon. Thus, prior TorR binding to the box 1-box 2 region seems to allow cooperative binding of phosphorylated TorR to box 3 and box 4.

scholarly journals Alternative ligands as probes for the carotenoid-binding site of lobster carapace crustacyanin

1990 ◽  
Vol 265 (3) ◽  
pp. 919-921 ◽  
Author(s):  
J B Clarke ◽  
E E Eliopoulos ◽  
J B C Findlay ◽  
P F Zagalsky
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Binding Sites ◽  
Amino Acid Sequences ◽  
Fluorescence Probes ◽  
Affinity Binding ◽  
Hydrophobic Pocket ◽  
High Affinity Binding ◽  
High Affinity ◽  
Parinaric Acid

The apoproteins of the lobster carotenoprotein, crustacyanin, show single high-affinity binding sites for the hydrophobic fluorescence probes 8-anilo-1-naphthalenesulphonic acid and cis-parinaric acid, and exhibit fluorescence transfer from tryptophan to the ligands. These results, together with information from the amino acid sequences, infer that the native carotenoid, astaxanthin, is bound to each apoprotein within an internal hydrophobic pocket, or calyx.

scholarly journals The low C5 convertase activity of the C4A6 allotype of human complement component C4

1989 ◽  
Vol 261 (3) ◽  
pp. 743-748 ◽  
Author(s):  
T Kinoshita ◽  
A W Dodds ◽  
S K A Law ◽  
K Inoue
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Alternative Pathway ◽  
Covalent Attachment ◽  
Haemolytic Activity ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
High Affinity Binding Site ◽  
Covalently Linked

We have compared the C5-convertase-forming ability of different C4 allotypes, including the C4A6 allotype, which has low haemolytic activity and which has previously been shown to be defective in C5-convertase formation. Recent studies suggest that C4 plays two roles in the formation of the C5 convertase from the C3 convertase. Firstly, C4b acts as the binding site for C3 which, upon cleavage by C2, forms a covalent linkage with the C4b. Secondly, C4b with covalently attached C3b serves to form a high-affinity binding site for C5. Purified allotypes C4A3, C4B1 and C4A6 were used to compare these two activities of C4. Covalently linked C4b-C3b complexes were formed on sheep erythrocytes with similar efficiency by using C4A3 and C4B1, indicating that the two isotypes behave similarly as acceptors for covalent attachment of C3b. C4A6 showed normal efficiency in this function. However, cells bearing C4b-C3b complexes made from C4A6 contained only a small number of high-affinity binding sites for C5. Therefore a lack of binding of C5 to the C4b C3b complexes is the reason for the inefficient formation of C5 convertase by C4A6. The small number of high-affinity binding sites created, when C4A6 was used, were tested for inhibition by anti-C3 and anti-C4. Anti-C4 did not inhibit C5 binding, whereas anti-C3 did. This suggests that the sites created when C4A6 is used to make C3 convertase may be C3b-C3b dimers, and hence the low haemolytic activity of C4A6 results from the creation of low numbers of alternative-pathway C5-convertase sites.

scholarly journals Cyanovirin-N Binds Viral Envelope Proteins at the Low-Affinity Carbohydrate Binding Site without Direct Virus Neutralization Ability

Molecules ◽  
2021 ◽  
Vol 26 (12) ◽  
pp. 3621
Author(s):  
Irene Maier ◽  
Robert H. Schiestl ◽  
Georg Kontaxis
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Affinity Maturation ◽  
Viral Envelope ◽  
Biologically Active ◽  
Carbohydrate Binding ◽  
Affinity Binding ◽  
Protein Patterns ◽  
High Affinity Binding ◽  
High Affinity

Glycan-targeting antibodies and pseudo-antibodies have been extensively studied for their stoichiometry, avidity, and their interactions with the rapidly modifying glycan shield of influenza A. Broadly neutralizing antiviral agents bind in the same order when they neutralize enveloped viruses regardless of the location of epitopes to the host receptor binding site. Herein, we investigated the binding of cyanovirin-N (CV–N) to surface-expressed glycoproteins such as those of human immunodeficiency virus (HIV) gp120, hemagglutinin (HA), and Ebola (GP)1,2 and compared their binding affinities with the binding response to the trimer-folded gp140 using surface plasmon resonance (SPR). Binding-site knockout variants of an engineered dimeric CV–N molecule (CVN2) revealed a binding affinity that correlated with the number of (high-) affinity binding sites. Binding curves were specific for the interaction with N-linked glycans upon binding with two low-affinity carbohydrate binding sites. This biologically active assembly of a domain-swapped CVN2, or monomeric CV–N, bound to HA with a maximum KD of 2.7 nM. All three envelope spike proteins were recognized at a nanomolar KD, whereas binding to HIV neutralizing 2G12 by targeting HA and Ebola GP1,2 was measured in the µM range and specific for the bivalent binding scheme in SPR. In conclusion, invariant structural protein patterns provide a substrate for affinity maturation in the membrane-anchored HA regions, as well as the glycan shield on the membrane-distal HA top part. They can also induce high-affinity binding in antiviral CV–N to HA at two sites, and CVN2 binding is achieved at low-affinity binding sites.

scholarly journals Lactoferrin-lipopolysaccharide interaction: involvement of the 28-34 loop region of human lactoferrin in the high-affinity binding to Escherichia coli 055B5 lipopolysaccharide

1995 ◽  
Vol 312 (3) ◽  
pp. 839-845 ◽  
Author(s):  
E Elass-Rochard ◽  
A Roseanu ◽  
D Legrand ◽  
M Trif ◽  
V Salmon ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Binding Site ◽  
Binding Sites ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
Tryptic Fragment ◽  
High Affinity ◽  
Loop Region ◽  
High Affinity Binding Site ◽  
Lps Binding

The ability of lactoferrin (Lf), an iron-binding glycoprotein that is also called lactotransferrin, to bind lipopolysaccharide (LPS) may be relevant to some of its biological properties. A knowledge of the LPS-binding site on Lf may help to explain the mechanism of its involvement in host defence. Our report reveals the presence of two Escherichia coli 055B5 LPS-binding sites on human Lf (hLf): a high-affinity binding site (Kd 3.6 +/- 1 nM) and a low-affinity binding site (Kd 390 +/- 20 nM). Bovine Lf (bLf), which shares about 70% amino acid sequence identity with hLf, exhibits the same behaviour towards LPS. Like hLf, bLf also contains a low- and a high-affinity LPS-binding site. The Kd value (4.5 +/- 2 nM) corresponding to the high-affinity binding site is similar to that obtained for hLf. Different LPS-binding sites for human serum transferrin have been suggested, as this protein, which is known to bind bacterial endotoxin, produced only 12% inhibition of hLf-LPS interaction. Binding and competitive binding experiments performed with the N-tryptic fragment (residues 4-283), the C-tryptic fragment (residues 284-692) and the N2-glycopeptide (residues 91-255) isolated from hLf have demonstrated that the high-affinity binding site is located in the N-terminal domain I of hLf, and the low-affinity binding site is present in the C-terminal lobe. The inhibition of hLf-LPS interaction by a synthetic octadecapeptide corresponding to residues 20-37 of hLf and lactoferricin B (residues 17-41), a proteolytic fragment from bLf, revealed the importance of the 28-34 loop region of hLf and the homologous region of bLf for LPS binding. Direct evidence that this amino acid sequence is involved in the high-affinity binding to LPS was demonstrated by assays carried out with EGS-loop hLf, a recombinant hLf mutated at residues 28-34.

scholarly journals Role of Mg2+ in the structure and activity of maize (Zea mays L.) isocitrate lyase: indications for hysteretic behaviour

1997 ◽  
Vol 327 (1) ◽  
pp. 171-176 ◽  
Author(s):  
Sonia BEECKMANS ◽  
A. Salam KHAN ◽  
Edilbert VAN DRIESSCHE
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Isocitrate Lyase ◽  
Affinity Binding ◽  
High Affinity Binding ◽  
High Affinity ◽  
Activity Measurements ◽  
Enzyme Molecules ◽  
Structure And Activity

The role of Mg2+ in the structure and activity of maize isocitrate lyase has been studied by CD, limited proteolysis, protection by ligands against inactivation, and activity measurements at various metal concentrations. From CD and trypsinolysis experiments, the existence of high-affinity binding sites for Mg2+ was demonstrated, and a KdME of 200 μM was determined. Both free enzyme (E) and enzyme molecules with high-affinity sites occupied (ME) are catalytically competent, the former showing 40% of the activity of the latter. Mg2+ thus acts as a non-essential activator. A second Mg2+-binding site with a KdMEM of 6 mM was revealed from protection experiments by increasing Mg2+ concentrations against inactivation. From activity measurements at different Mg2+ concentrations, the affinity of the enzyme for the Mg2+–isocitrate complex (MI) was determined to be KdE(MI) = 9 μM. Maize isocitrate lyase was shown to display hysteretic behaviour. Filling the low-affinity binding sites with Mg2+ induces a conformational change in the high-affinity binding sites resulting in an even higher affinity for Mg2+ (KdME* = 40 μM). On lowering the Mg2+ concentration again, the enzyme only responds slowly: the time needed for all enzyme molecules to return to the conformation at which KdME is 200 μM was found to be 60 min. Finally it was shown that the high-affinity binding site for Mg2+ is not formed at low (4 °C) temperature.

scholarly journals Covalent association of C3b with C4b within C5 convertase of the classical complement pathway.

1987 ◽  
Vol 165 (6) ◽  
pp. 1494-1507 ◽  
Author(s):  
Y Takata ◽  
T Kinoshita ◽  
H Kozono ◽  
J Takeda ◽  
E Tanaka ◽  
...  
Keyword(s):  
Binding Site ◽  
Binding Sites ◽  
Association Constant ◽  
Affinity Binding ◽  
Ester Bond ◽  
Complement Pathway ◽  
High Affinity Binding ◽  
Classical Complement Pathway ◽  
High Affinity ◽  
Classical Complement

The C5 convertase of the classical complement pathway is a complex enzyme consisting of three complement fragments, C4b, C2a, and C3b. Previous studies have elucidated functional roles of each subunit (4, 6, 7), but little is known about how the subunits associate with each other. In this investigation, we studied the nature of the classical C5 convertase that was assembled on sheep erythrocytes. We found that one of the nascent C3b molecules that had been generated by the C3 convertase directly bound covalently to C4b. C3b bound to the alpha' chain of C4b through an ester bond, which could be cleaved by treatment with hydroxylamine. The ester bond was rather unstable, with a half-life of 7.9 h at pH 7.4 and 37 degrees C. Formation of the C4b-C3b dimer is quite efficient; e.g., 54% of the cell-bound C3b was associated with C4b when 25,000 molecules of C4b and 12,000 molecules of C3b were present per cell. Kinetic analysis also showed the efficient formation of the C4b-C3b dimer; the rate of dimer formation was similar to or even faster than that of cell-bound monomeric C3b molecules. These results indicate that C4b is a highly reactive acceptor molecule for nascent C3b. High-affinity C5-binding sites with an association constant of 2.1 X 10(8) L/M were demonstrated on C4b-C3b dimer-bearing sheep erythrocytes, EAC43 cells. The number of high-affinity C5-binding sites coincided with the number of C4b-C3b dimers, but not with the total number of cell-bound C3b molecules. Anti-C4 antibodies caused 80% inhibition of the binding of C5 to EAC43 cells. These results suggest that only C4b-associated C3b serves as a high-affinity C5 binding site. EAC14 cells had a small amount of high-affinity C5 binding sites with an association constant of 8.1 X 10(7) L/M, 100 molecules of bound C4b being necessary for 1 binding site. In accordance with the hypothesis that C4b-associated C4b might also serve as a high-affinity C5-binding site, a small amount of C4b-C4b dimer was detected on EAC14 cells by SDS-PAGE analysis. Taken together, these observations indicate that the high-affinity binding of C5 is probably divalent, in that C5 recognizes both protomers in the dimers. The high-affinity binding may allow selective binding of C5 to the convertase in spite of surrounding monomeric C3b molecules.

Sign in / Sign up

Export Citation Format

Share Document