scholarly journals Significance of RGD Loop and C-Terminal Domain of Echistatin for Recognition of αIIbβ3 and αvβ3 Integrins and Expression of Ligand-Induced Binding Site

Blood ◽  
1997 ◽  
Vol 90 (4) ◽  
pp. 1565-1575 ◽  
Author(s):  
Cezary Marcinkiewicz ◽  
Senadhi Vijay-Kumar ◽  
Mary Ann McLane ◽  
Stefan Niewiarowski
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Inhibitory Effect ◽  
Selective Recognition ◽  
Αvβ3 Integrin ◽  
Inhibit Platelet Aggregation ◽  
Disulfide Bridges ◽  
Terminal Domain ◽  
Αvβ3 Integrins ◽  
Terminal Amino

Echistatin is a viper venom disintegrin containing RGD loop maintained by disulfide bridges. It binds with a high affinity to αvβ3 and αIIbβ3 and it induces extensive conformational changes in these integrins resulting in expression of ligand-induced binding site (LIBS) epitopes. We investigated the activities of echistatin and its three analogues (R24A, D27W, echistatin 1-41). R24A echistatin did not react with αIIbβ3 and αvβ3 integrins and did not cause LIBS effect. D27W echistatin showed increased binding to αIIbβ3 and decreased binding to αvβ3. This substitution impaired the ability of echistatin to induce LIBS in αvβ3 integrin. Deletion of nine C-terminal amino acids of echistatin decreased its ability to bind αIIbβ3 and inhibit platelet aggregation. Truncated echistatin failed to induce LIBS epitopes on cells transfected with αIIbβ3 and αvβ3 genes. The ability of echistatin 1-41 to compete with binding of vitronectin to immobilized αvβ3 and monoclonal antibody 7E3 to platelets and to VNRC3 cells was decreased, although this analogue, after immobilization, retained its ability to bind purified αvβ3. We propose a hypothesis in which echistatin's RGD loop determines selective recognition of αIIbβ3 and αvβ3 integrin, whereas the C-terminal domain supports its binding to resting integrin and significantly contributes to the expression of LIBS epitope and to conformational changes of the receptor, leading to a further increase of the binding affinity of echistatin and of the inhibitory effect.

scholarly journals Significance of RGD Loop and C-Terminal Domain of Echistatin for Recognition of αIIbβ3 and αvβ3 Integrins and Expression of Ligand-Induced Binding Site

Blood ◽  
1997 ◽  
Vol 90 (4) ◽  
pp. 1565-1575 ◽  
Author(s):  
Cezary Marcinkiewicz ◽  
Senadhi Vijay-Kumar ◽  
Mary Ann McLane ◽  
Stefan Niewiarowski
Keyword(s):  
Binding Site ◽  
Conformational Changes ◽  
Inhibitory Effect ◽  
Selective Recognition ◽  
Αvβ3 Integrin ◽  
Inhibit Platelet Aggregation ◽  
Disulfide Bridges ◽  
Terminal Domain ◽  
Αvβ3 Integrins ◽  
Terminal Amino

Abstract Echistatin is a viper venom disintegrin containing RGD loop maintained by disulfide bridges. It binds with a high affinity to αvβ3 and αIIbβ3 and it induces extensive conformational changes in these integrins resulting in expression of ligand-induced binding site (LIBS) epitopes. We investigated the activities of echistatin and its three analogues (R24A, D27W, echistatin 1-41). R24A echistatin did not react with αIIbβ3 and αvβ3 integrins and did not cause LIBS effect. D27W echistatin showed increased binding to αIIbβ3 and decreased binding to αvβ3. This substitution impaired the ability of echistatin to induce LIBS in αvβ3 integrin. Deletion of nine C-terminal amino acids of echistatin decreased its ability to bind αIIbβ3 and inhibit platelet aggregation. Truncated echistatin failed to induce LIBS epitopes on cells transfected with αIIbβ3 and αvβ3 genes. The ability of echistatin 1-41 to compete with binding of vitronectin to immobilized αvβ3 and monoclonal antibody 7E3 to platelets and to VNRC3 cells was decreased, although this analogue, after immobilization, retained its ability to bind purified αvβ3. We propose a hypothesis in which echistatin's RGD loop determines selective recognition of αIIbβ3 and αvβ3 integrin, whereas the C-terminal domain supports its binding to resting integrin and significantly contributes to the expression of LIBS epitope and to conformational changes of the receptor, leading to a further increase of the binding affinity of echistatin and of the inhibitory effect.

MODULATION OF ANTITHROMBIN III ACTIVITY AND ANTITHROMBIN III-THROMBIN COMPLEXES BINDING TO CULTURED CELLS BY MONOCLONAL ANTIBODIES AGAINST ANTITHROMBIN III

1987 ◽  
Author(s):  
S Knoller ◽  
N Savion
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Conformational Changes ◽  
Antithrombin Iii ◽  
Cultured Cells ◽  
Inhibitory Effect ◽  
National Council ◽  
Corneal Endothelial Cells ◽  
Inhibitory Effects ◽  
Antithrombin Iii Activity

Two monoclonal antibodies (mAb's) against antithrombin III (ATIII) were characterized with respect to their ability to interfere with ATIII activity. AT III activity was measured by its ability to inhibit the amidolitic activity of thrombin on the substrate BCP-100. Incubation of 150 ng of ATIII with 28pg mAb A36R2 prior to addition of 50 ng thrombin totally abolishes the inhibitory effect of ATIII on thrombin. Incubation of 200ng of ATIII with 10 μg of mAb B26R4 prior to addition of 75 ng thrombin raises the inhibitory effects of ATIII from 37% to 100%. We examined the effect of these mAb's on binding of antithrombin III-thrombin (ATIII-Th) complexes to bovine corneal endothelial cells. 120 pg/ml mAb's are reacted with 2 μg/ml ATIII-Th complexes prior to their addition to the cells. mAb A36R2 completely blocks ATIII-Th complexes binding. In contrast, mAb B26R4 enhances binding up to 250% of the control binding.We conclude that mAb A36R2 prevents binding of thrombin to ATIII by recognizing an epitope on ATIII close to thrombin binding site or that its binding to ATIII induces a conformational change in the thrombin binding site thus it no longer recognizes thrombin. mAb B26R4 has a heparin-like effect on ATIII: Its binding to ATIII induces conformational changes which improve thrombin binding to ATIII. There is a correlation between inhibition and enhancement of thrombin binding to ATIII and of ATIII-Th complexes binding to cells by the two mAb's. These mAb's may provide a new tool to control the activity of ATIII and to identify the cellular binding site on the ATIII-Th complex.This research was supported by a grant from the National Council for Research and Development, Israel and G.S.F. München, Germany.

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

2019 ◽  
Vol 476 (21) ◽  
pp. 3227-3240 ◽  
Author(s):  
Shanshan Wang ◽  
Yanxiang Zhao ◽  
Long Yi ◽  
Minghe Shen ◽  
Chao Wang ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Magnaporthe Oryzae ◽  
Structural Information ◽  
Complex Structure ◽  
Critical Role ◽  
Catalytic Process ◽  
Structural Basis ◽  
Salt Bridges ◽  
Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

Inhibitory Effect of Staphylokinase on Platelet Aggregation

1993 ◽  
Vol 70 (05) ◽  
pp. 834-837 ◽  
Author(s):  
Akira Suehiro ◽  
Yoshio Oura ◽  
Motoo Ueda ◽  
Eizo Kakishita
Keyword(s):  
Platelet Aggregation ◽  
Human Platelet ◽  
Degradation Products ◽  
Platelet Rich Plasma ◽  
Inhibitory Effect ◽  
Effective Concentration ◽  
Inhibit Platelet Aggregation ◽  
Platelet Suspension ◽  
Washed Platelet ◽  
Human Platelet Aggregation

SummaryWe investigated the effect of staphylokinase (SAK), which has specific thrombolytic properties, on human platelet aggregation. Platelet aggregation induced with collagen was observed following preincubation of platelets in platelet-rich plasma (PRP) or washed platelet suspension (WP) with SAK at 37° C for 30 min. SAK inhibited platelet aggregation in PRP only at the highest examined concentration (1 x 10-4 g/ml). Although SAK did not inhibit platelet aggregation in WP which contained fibrinogen, it did when the platelets had been preincubated with SAK and plasminogen. The most effective concentration in WP was 1 x 10-6 g/ml. The effect could be inhibited by adding aprotinin or α2-antiplasmin. The highest generation of plasmin in the same preincubation fluid was detected at 1 x 10-6 g/ml SAK. We concluded that SAK can inhibit platelet aggregation in WP by generating plasmin and/or fibrinogen degradation products, but is only partially effective in PRP because of the existence of α2-antiplasmin.

Synthesis and Characterization of ROSA Dye - A Rhodamine B-type Fluorophore, Suitable for Bioconjugation and Fluorescence Studies in Live Cells

2019 ◽  
Vol 26 (10) ◽  
pp. 758-767
Author(s):  
Vicente Rubio ◽  
Vijaya Iragavarapu ◽  
Maciej J. Stawikowski
Keyword(s):  
Quantum Yield ◽  
Cancer Cells ◽  
Fluorescent Probe ◽  
Extinction Coefficient ◽  
Rhodamine B ◽  
Solid Phase ◽  
Total Yield ◽  
Molar Extinction Coefficient ◽  
Αvβ3 Integrin ◽  
Αvβ3 Integrins

Background: Herein we report the multigram-scale synthesis, characterization and application of a rhodamine B-based fluorophore (ROSA) suitable for fluorescent studies in biological applications. This fluorophore is devoid of rhodamine spirolactone formation and furthermore characterized by a high molar extinction coefficient (ϵ=87250 ± 1630 M-1cm-1) and quantum yield (φ) of 0.589 ± 0.070 in water. Reported here is also the application of ROSA towards synthesis of a ROSA-PEG-GRGDS-NH2 fluorescent probe suitable for live cell imaging of αvβ3 integrins for in vitro assays. Objective: The main objective of this study is to efficiently prepare rhodamine B derivative, devoid of spirolactone formation that would be suitable for bioconjugation and subsequent bioimaging. Methods: Rhodamine B was transformed into rhodamine B succinimide ester (RhoB-OSu) using N-hydroxysuccinimide. RhoB-OSu was further coupled to sarcosine to obtain rhodamine Bsarcosine dye (ROSA) in good yield. The ROSA dye was then coupled to a αvβ3 integrin binding sequence using standard solid-phase conditions. Resulting ROSA-PEG-GRGDS-NH2 probe was used to image integrins on cancer cells. Results: The rhodamine B-sarcosine dye (ROSA) was obtained in multigram scale in good total yield of 47%. Unlike rhodamine B, the ROSA dye does not undergo pH-dependent spirolactone/spirolactam formation as compared with rhodamine B-glycine. It is also characterized by excellent quantum yield (φ) of 0.589 ± 0.070 in water and high molar extinction coefficient of 87250 ± 1630 M-1cm-1. ROSA coupling to the RGD-like peptide was proved to be efficient and straightforward. Imaging using standard filters on multimode plate reader and confocal microscope was performed. The αvβ3 integrins present on the surface of live WM-266-4 (melanoma) and MCF- 7 (breast cancer) cells were successfully imaged. Conclusion: We successfully derivatized rhodamine B to create an inexpensive, stable and convenient to use fluorescent probe. The obtained derivative has excellent photochemical properties and it is suitable for bioconjugation and many imaging applications.

scholarly journals Regulation of Swarming Motility and flhDCSm Expression by RssAB Signaling in Serratia marcescens

2008 ◽  
Vol 190 (7) ◽  
pp. 2496-2504 ◽  
Author(s):  
Po-Chi Soo ◽  
Yu-Tze Horng ◽  
Jun-Rong Wei ◽  
Jwu-Ching Shu ◽  
Chia-Chen Lu ◽  
...  
Keyword(s):  
Serratia Marcescens ◽  
Binding Site ◽  
Promoter Region ◽  
Transcriptional Start Site ◽  
Direct Interaction ◽  
Underlying Mechanism ◽  
Inhibitory Effect ◽  
Start Codon ◽  
Mobility Shift

ABSTRACT Serratia marcescens cells swarm at 30°C but not at 37°C, and the underlying mechanism is not characterized. Our previous studies had shown that a temperature upshift from 30 to 37°C reduced the expression levels of flhDCSm and hagSm in S. marcescens CH-1. Mutation in rssA or rssB, cognate genes that comprise a two-component system, also resulted in precocious swarming phenotypes at 37°C. To further characterize the underlying mechanism, in the present study, we report that expression of flhDCSm and synthesis of flagella are significantly increased in the rssA mutant strain at 37°C. Primer extension analysis for determination of the transcriptional start site(s) of flhDCSm revealed two transcriptional start sites, P1 and P2, in S. marcescens CH-1. Characterization of the phosphorylated RssB (RssB∼P) binding site by an electrophoretic mobility shift assay showed direct interaction of RssB∼P, but not unphosphorylated RssB [RssB(D51E)], with the P2 promoter region. A DNase I footprinting assay using a capillary electrophoresis approach further determined that the RssB∼P binding site is located between base pair positions −341 and −364 from the translation start codon ATG in the flhDCSm promoter region. The binding site overlaps with the P2 “−35” promoter region. A modified chromatin immunoprecipitation assay was subsequently performed to confirm that RssB∼P binds to the flhDCSm promoter region in vivo. In conclusion, our results indicated that activated RssA-RssB signaling directly inhibits flhDCSm promoter activity at 37°C. This inhibitory effect was comparatively alleviated at 30°C. This finding might explain, at least in part, the phenomenon of inhibition of S. marcescens swarming at 37°C.

scholarly journals The Odd Faces of Oligomers: The Case of TRAF2-C, A Trimeric C-Terminal Domain of TNF Receptor-Associated Factor

2021 ◽  
Vol 22 (11) ◽  
pp. 5871
Author(s):  
Almerinda Di Venere ◽  
Eleonora Nicolai ◽  
Velia Minicozzi ◽  
Anna Maria Caccuri ◽  
Luisa Di Paola ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Strong Dependence ◽  
Conformational Dynamics ◽  
Receptor Interaction ◽  
Tnf Receptor ◽  
Oligomeric State ◽  
Dynamic Simulations ◽  
Associated Factor ◽  
Terminal Domain

TNF Receptor Associated Factor 2 (TRAF2) is a trimeric protein that belongs to the TNF receptor associated factor family (TRAFs). The TRAF2 oligomeric state is crucial for receptor binding and for its interaction with other proteins involved in the TNFR signaling. The monomer-trimer equilibrium of a C- terminal domain truncated form of TRAF2 (TRAF2-C), plays also a relevant role in binding the membrane, causing inward vesiculation. In this study, we have investigated the conformational dynamics of TRAF2-C through circular dichroism, fluorescence, and dynamic light scattering, performing temperature-dependent measurements. The data indicate that the protein retains its oligomeric state and most of its secondary structure, while displaying a significative increase in the heterogeneity of the tyrosines signal, increasing the temperature from ≈15 to ≈35 °C. The peculiar crowding of tyrosine residues (12 out of 18) at the three subunit interfaces and the strong dependence on the trimer concentration indicate that such conformational changes mainly involve the contact areas between each pair of monomers, affecting the oligomeric state. Molecular dynamic simulations in this temperature range suggest that the interfaces heterogeneity is an intrinsic property of the trimer that arises from the continuous, asymmetric approaching and distancing of its subunits. Such dynamics affect the results of molecular docking on the external protein surface using receptor peptides, indicating that the TRAF2-receptor interaction in the solution might not involve three subunits at the same time, as suggested by the static analysis obtainable from the crystal structure. These findings shed new light on the role that the TRAF2 oligomeric state might have in regulating the protein binding activity in vivo.

scholarly journals Monoclonal antibodies identify residues 199–216 of the integrin α2 vWFA domain as a functionally important region within α2β1

2000 ◽  
Vol 350 (2) ◽  
pp. 485-493 ◽  
Author(s):  
Danny S. TUCKWELL ◽  
Lyndsay SMITH ◽  
Michelle KORDA ◽  
Janet A. ASKARI ◽  
Sentot SANTOSO ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Binding Site ◽  
Conformational Changes ◽  
Cation Binding ◽  
Inhibitory Antibody ◽  
Collagen Binding ◽  
Collagen Peptide ◽  
Binding Residue ◽  
Important Region ◽  
Domain Mapping

Integrin α2β1 is the major receptor for collagens in the human body, and the collagen-binding site on the α2 subunit von Willebrand factor A-type domain (vWFA domain) is now well defined. However, the biologically important conformational changes that are associated with collagen binding, and the means by which the vWFA domain is integrated into the whole integrin are not completely understood. We have raised monoclonal antibodies against recombinant α2 vWFA domain for use as probes of function. Three antibodies, JA202, JA215 and JA218, inhibited binding to collagen, collagen I C-propeptide and E-cadherin, demonstrating that their function is important for structurally diverse α2β1 ligands. Cross-blocking studies grouped the epitopes into two clusters: (I) JA202, the inhibitory antibody, Gi9, and a non-inhibitory antibody, JA208; (II) JA215 and JA218. Both clusters were sensitive to events at the collagen binding site, as binding of Gi9, JA202, JA215 and JA218 were inhibited by collagen peptide, JA208 binding was enhanced by collagen peptide, and binding of JA202 was decreased after mutagenesis of the cation-binding residue Thr221 to alanine. Binding of cluster I antibodies was inhibited by the anti-functional anti-β1 antibody Mab13, and binding of Gi9 and JA218 to α2β1 was inhibited by substituting Mn2+ for Mg2+, demonstrating that these antibodies were sensitive to changes initiated outside the vWFA domain. Mapping of epitopes showed that JA202 and Gi9 bound between residues 212–216, while JA208 bound between residues 199–216. We have therefore identified two epitope clusters with novel properties; i.e. they are intimately associated with the collagen-binding site, responsive to conformational changes at the collagen-binding site and sensitive to events initiated outside the vWFA domain.

scholarly journals Evidence of a Putative Glycosaminoglycan Binding Site on the Glycosylated SARS-CoV-2 Spike Protein N-terminal domain

2021 ◽  
Author(s):  
Zachariah P. Schuurs ◽  
Edward Hammond ◽  
Stefano Elli ◽  
Timothy R. Rudd ◽  
Courtney J. Mycroft-West ◽  
...  
Keyword(s):  
Binding Site ◽  
Spike Protein ◽  
Terminal Domain
Sign in / Sign up

Export Citation Format

Share Document