Activated, But Not Unactivated, αIIbβ3 Can Mediate Adhesion to Fibrinogen Lacking the γ Chain Dodecapeptide

Abstract The interaction of the fibrinogen γ chain dodecapeptide (HHLGGAKQAGDV; γ12) with the αIIbβ3 binding pocket is required for fibrinogen binding to αIIbβ3 and the binding of other αIIbβ3 ligands that contain RGD sequences such as von Willebrand factor and fibronectin. Since the strength of fibrinogen binding to αIIbβ3 increases over time, it is possible that there are additional sites of interaction between αIIbβ3 and fibrinogen, and several studies have proposed potential candidate ancillary sites on fibrinogen, including γ316-322 and γ370-381 (Podolnikova et al. JBC 289;2371,2014 and Remijn et al. Br J Haematol 117;650,2002). If such fibrinogen sites interact with αIIbβ3 at regions separate from the γ12 (RGD) binding site, these αIIbβ3 sites may be attractive targets for developing pure αIIbβ3 antagonists that are specific for fibrinogen. To identify potential additional interaction sites on fibrinogen and αIIbβ3, we studied adhesion of HEK293 cells expressing normal αIIbβ3 (αIIbβ3-HEK) to either fibrinogen or the D98 plasmin cleavage fragment of fibrinogen (which lacks γ12) in the presence of 2 mM Ca2+/1 mM Mg2+. The D98 fragment did not contain the γ12 peptide as judged by both immunoblotting with mAb 7E9 (anti-γ12) and mass spectroscopy. We also studied HEK293 cells expressing αIIbβ3 containing: 1. The double αIIb F992A + F993A mutations (αIIb-FF), which causes constitutive activation and ligand binding 2. Other activating mutations of αIIbβ3, including αIIb truncation at 991, β3 N339S, and β3 truncation at 717, 3. The β3 D119A mutation, which disrupts the β3 metal ion-dependent adhesion site (MIDAS) and eliminates adhesion of cells expressing normal αIIbβ3 to fibrinogen, or 4. Combined αIIb FF and β3 D119 mutations (FF+D119). αIIbβ3-HEK and αIIb-FF both bound to immobilized fibrinogen (10 µg/ml coating concentration). αIIbβ3-HEK did not adhere to immobilized D98 (10 µg/ml coating concentration), whereas αIIb-FF did adhere [αIIbβ3-HEK: 201 ± 295 vs. αIIb-FF: 8,221 ± 1,585 arbitrary fluorescence intensity units (AFU); n=7; p=0.003]. HEK cells expressing the other activating mutations also adhered to both fibrinogen and D98. HEK cells expressing the D119 mutation did not adhere to fibrinogen or D98 and adding the D119 mutation to the αIIb FF mutant led to loss of adhesion to both fibrinogen and D98. Adhesion of αIIb-FF to D98 was inhibited by mAb 10E5, which inhibits fibrinogen binding to αIIbβ3 and binds to the αIIb cap domain (89% ± 18%; n=7; p=0.003) and by mAb 7E3, which inhibits fibrinogen binding and binds to the β3 subunits of both αIIbβ3 and αVβ3 (95% ± 10%; n=7; p=0.006), but not by mAb 7E9 (28% ± 29%; n=7; p=0.299). Since cells expressing activated, but not unactivated αIIbβ3 were able to adhere to D98, our data are consistent with a model in which the initial interaction between αIIbβ3 and fibrinogen is mediated by γ12 binding to the αIIbβ3 ligand binding site followed by a conformational change that exposes additional site(s) on αIIbβ3 for another region or regions of fibrinogen. Refrences: Podolnikova NP, Yakovlev S, Yakubenko VP, Wang X, Gorkun OV, Ugarova TP. The interaction of integrin alphaIIbbeta3 with fibrin occurs through multiple binding sites in the alphaIIb beta-propeller domain. J Biol Chem. 2014;289:2371-2383. Remijn JA, Ijsseldijk MJ, van Hemel BM, Galanakis DK, Hogan KA, Lounes KC, Lord ST, Sixma JJ, de Groot PG. Reduced platelet adhesion in flowing blood to fibrinogen by alterations in segment gamma316-322, part of fibrin-specific region. Br J Haematol 2002;117:650-657. Disclosures No relevant conflicts of interest to declare.

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Functional and Computational Analysis of the Role of the Adjacent to Metal Ion-Dependent Adhesion Site (ADMIDAS) of Integrin αIIbβ3.

Blood ◽

10.1182/blood.v108.11.143.143 ◽

2006 ◽

Vol 108 (11) ◽

pp. 143-143

Author(s):

Marketa Jirouskova ◽

Marta Murcia ◽

JiHong Li ◽

Marta Filizola ◽

Barry S. Coller

Keyword(s):

Ligand Binding ◽

Binding Site ◽

Metal Ion ◽

Md Simulations ◽

Hek293 Cells ◽

Normal Cells ◽

Shear Rates ◽

Static Conditions ◽

Mutant Cells

Abstract The role of the β3 MIDAS in αIIbβ3 ligand binding is well established, but the role of the nearby ADMIDAS is less well defined. Thus, we studied HEK293 cells expressing normal αIIbβ3 (normal cells) or the ADMIDAS mutants β3 D126A and D127A (mutant cells). Both mutant cells adhered as well or better than normal cells to immobilized fibrinogen under static conditions in the presence of either Ca2+/Mg2+ or Mn2+. Under low shear flow conditions (0.15 dyne/cm2), adhesion of normal cells and D126A mutant cells to fibrinogen was similar in the presence of either Ca2+/Mg2+ or Mn2+. Adherent D126A mutant cells, however, demonstrated greater resistance to detachment at increasing shear rates in the presence of Ca2+/Mg2+ (e.g., at 20.4 dynes/cm2, only 40 ± 10% of normal cells remained vs 85 ± 8% of D126A mutant cells; mean ± SD; p<0.001). Substituting Mn2+ for Ca2+/Mg2+ increased the resistance to detachment of the normal cells (60 ± 20% remaining at 20.4 dynes/cm2; p=0.01), but the value was still less than the mutant cells in the presence of either Ca2+/Mg2+ (see above; p<0.01) or Mn2+ (84 ± 4%; p<0.01). The increased strength of adhesion we observed in the αIIbβ3 ADMIDAS mutant cells is similar to that found in α4β7 ADMIDAS mutant cells (Chen et al, JBC 2004) and is consistent with the findings in isolated β3 βA (I-like) domains (Pesho et al. JBC 2006). The binding of 7E3, whose epitope is near the ADMIDAS, to the D126 mutant cells was similar to its binding to the normal cells, but 7E3 binding to the D127A mutant cells was reduced by 89 ± 7% (n = 4; p<0.001). 7E3 decreased adhesion of normal cells to fibrinogen by 88 ± 4%, but it only decreased adhesion of D126A mutant cells by 3 ± 9%, and it did not inhibit adhesion of D127A cells at all. To provide a structural context for the role of the ADMIDAS in ligand binding to αIIbβ3, we compared results from nanosecond time-scale molecular dynamics (MD) simulations of the cyclic peptide ligand eptifibatide in complex with either the fully hydrated normal αIIbβ3 or the D126A mutant in the presence of Ca2+/Mg2+. Calculations were carried out using the OPLS all-atom force-field of the GROMACS simulation package. With respect to normal αIIbβ3, the mutant receptor demonstrated reduced fluctuations in the β3 207–210 and 335 regions and increased fluctuations in the β3 282–284 region. In addition, the ADMIDAS metal ion moved ~3 Å away from the MIDAS and became more solvent exposed. Rearrangements of the coordination of the ADMIDAS involving S123, D251, and D127 were also observed in the D126A mutant compared to normal αIIbβ3. Steered MD simulations were used to investigate the unbinding of eptifibatide from its binding site. The unbinding force for the D126A mutant was similar to that for the normal αIIbβ3. Quantitative estimations of the binding energies of eptifibatide to normal and D126A mutant αIIbβ3 from Molecular Mechanics/Poisson Boltzman Surface Area analysis of the MD trajectories also yielded similar results. Thus, the much greater resistance of D126A mutant cells to detachment from fibrinogen at increasing shear rates does not appear to be explained by differences in fibrinogen-αIIbβ3 interactions at the sites involved in the binding of eptifibatide. Potential alternative mechanisms involve differences in fibrinogen’s access to the binding site, interactions with other sites, or changes in fibrinogen avidity due to receptor clustering.

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Equilibrium and Non-Equilibrium Molecular Dynamics Simulations Provide Potential Mechanisms for the Loss of Ligand Binding to αIIbβ3 Mutants Affecting the Ligand-Induced Metal Binding Site (LIMBS).

Blood ◽

10.1182/blood.v108.11.1805.1805 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1805-1805

Author(s):

Marta Murcia ◽

Marketa Jirouskova ◽

Jihong Li ◽

Barry S. Coller ◽

Filizola Marta

Keyword(s):

Molecular Dynamics ◽

Ligand Binding ◽

Binding Site ◽

Metal Binding ◽

Metal Ion ◽

Cyclic Peptide ◽

Hek293 Cells ◽

Peptide Ligand ◽

Non Equilibrium

Abstract Although the role of the β3 MIDAS metal ion in ligand binding to αIIbβ3 is well established, serving as the site of interaction of the ligand Asp residue, the role of the nearby LIMBS metal ion is less well defined. Previous studies suggested a role for the LIMBS in ligand binding. We confirmed this by showing that HEK293 cells expressing normal αIIbβ3 adhered to both immobilized fibrinogen and the RGD-containing venom echistatin in the presence of either Mg++/Ca++ or Mn++, whereas two different αIIbβ3 LIMBS mutants (β3 N215A and D217A) failed to adhere to either protein. In addition, we found that both mutations also increased the binding of mAb AP5, which recognizes a ligand-induced binding site (LIBS) in the β3 PSI domain (normal 7±4% vs N215A 46±12% and D217A 41±20% of mAb anti-αIIb (HIP8) binding; mean±SD, n=6, p<0.05 for both), indicating that the mutations caused allosteric changes in the conformation of the receptor. To define the mechanism(s) by which the LIMBS mutants affect ligand binding, we carried out equilibrium and non-equilibrium (steered) molecular dynamics (MD) simulations of the cyclic peptide ligand eptifibatide in complex with either the fully hydrated normal αIIbβ3 integrin headpiece (PDB 1TY6) or the equivalent β3 D217A mutant, with and without the LIMBS metal ion. Simulations were carried out using the GROMACS package with the OPLS all-atom force-field. During the simulation, the hybrid domain of the D217A mutant demonstrated greater structural fluctuations than the normal αIIbβ3. Although Craig et al. have reported the appearance of a new contact between the RGD peptide ligand Asp carboxyl and the LIMBS metal ion in αVβ3 after 10 ps of a 1 ns simulation, we did not observe the appearance of such an interaction between the eptifibatide carboxyl and the normal αIIbβ3 LIMBS metal ion even after 20 ns. We did, however, observe such an interaction with the LIMBS metal ion in the D217A mutant. This interaction was facilitated by the movement of the LIMBS ~ 2 Å closer to the MIDAS, and was accompanied by rearrangements of the LIMBS coordinating residues D158 and N215. When the D217A mutant simulation was performed in the absence of the LIMBS metal ion, changes in the orientation of E220 were also observed. The D217A mutant demonstrated increased fluctuations in the C177–C184 specificity-determining loop (SDL), which has been implicated in ligand binding, and decreased fluctuations in K209. Steered MD were used to investigate the pulling forces required to unbind eptifibatide from its binding site. Notably, although the unbinding force decreased modestly when the LIMBS metal ion was removed, it required removal of both the LIMBS and MIDAS metal ions to effect a marked reduction in unbinding force. The binding free energies of the association of the αIIb and β3 subunits were also calculated, and the D217A mutant in the presence of the LIMBS metal ion demonstrated much tighter binding than normal integrin αIIbβ3 (ΔGb −162±6 vs −119±6 Kcal/mol; mean±SD; n=500). We conclude that the LIMBS plays a crucial role in ligand binding to αIIbβ3, perhaps by virtue of its effects on the coordination of the MIDAS, the accentuated mobility of specific domains (e.g., the SDL and the hybrid domains), and/or the number and strength of contacts between αIIb and β3.

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Inverse Effects of EDTA on αIIbβ3-Mediated Adhesion to Immobilized Fibrinogen Versus the D98 Plasmin Fragment of Fibrinogen

Blood ◽

10.1182/blood.v126.23.1036.1036 ◽

2015 ◽

Vol 126 (23) ◽

pp. 1036-1036

Author(s):

Hina Zafar ◽

Jihong Li ◽

George A David ◽

Barry S. Coller

Keyword(s):

Binding Sites ◽

Metal Ion ◽

Conflicts Of Interest ◽

Divalent Cations ◽

Hek293 Cells ◽

Clot Retraction ◽

Ligand Interaction ◽

Fibrinogen Binding ◽

A Site ◽

Adhesion Of Platelets

Abstract αIIbβ3 is known to mediate adhesion of platelets to immobilized fibrinogen through its interaction with the C-terminal γ chain dodecapeptide (γ12) and EDTA inhibits the adhesion by binding divalent cations required for ligand interaction with the β3 metal ion-dependent adhesion site (MIDAS) cation. Studies by several groups, however, suggest that αIIβb3 can also interact with other sites on fibrin(ogen). To identify potential additional sites of interaction between fibrinogen and αIIbβ3, we studied the adhesion of HEK293 cells expressing αIIbβ3 (αIIbβ3-HEK) to the D98 plasmin fragment of fibrinogen, which lacks the γ12 peptide. The D98 fragment did not contain the γ12 peptide as judged by both immunoblotting with mAb 7E9 (anti-γ12) and mass spectroscopy. αIIbβ3-HEK did not bind to immobilized D98 (10 µg/ml coating concentration) in the presence of 2 mM Ca2+/1 mM Mg2+, but they did bind to immobilized intact fibrinogen (10 µg/ml coating concentration) and the adhesion was inhibited by mAbs 10E5 (anti-αIIbβ3), 7E3 (anti-αIIbβ3 + αVβ3), and 7E9. Adhesion of αIIbβ3-HEK to fibrinogen was nearly eliminated by 10 mM EDTA [13,007 ± 3,676 vs 304 ± 331 arbitrary fluorescence intensity units (AFU); n=9; p<0.001]. Unexpectedly, and in dramatic contrast, 10 mM EDTA increased adhesion of αIIbβ3-HEK to D98 nearly 25-fold, from 458 ± 601 to 10,718 ± 3,106 AFU (n=9; p=0.001). The adhesion to D98 in the presence of EDTA was not inhibited by mAb 7E9 or mAb LM609 (anti-αVβ3), and was inhibited by mAb 7E3 by less than 10%. EDTA-dependent adhesion was, however, inhibited by mAb 10E5, which binds to the αIIb cap domain and inhibits fibrinogen binding to αIIbβ3, by 85% ± 4% (n=7; p=0.003). Dose-response experiments demonstrated that 3 mM EDTA was sufficient to block adhesion to fibrinogen and 3-4 mM EDTA was required to enhance adhesion to D98. Adding the β3 D119 mutation to αIIbβ3-HEK (αIIbβ3-D119-HEK), which disrupts the MIDAS, eliminated adhesion to fibrinogen (αIIbβ3-HEK: 17,342 ± 6,148 vs. αIIbβ3-D119-HEK: 0 ± 241 AFU; n=3; p=0.006), but had little or no effect on the binding to D98 in the presence of EDTA (αIIbβ3-HEK: 11,363 ± 3,700 vs. αIIbβ3-D119-HEK: 9,026 ± 3,252 AFU; n=3; p=0.054). However, unlike EDTA-dependent adhesion of αIIbβ3-HEK to D98, the adhesion of αIIbβ3-D119-HEK was inhibited by mAb 10E5 by only 20% ± 19% (n=3; p=0.247). We conclude that EDTA exposes one or more sites on αIIbβ3 that bind(s) to a site(s) on immobilized D98 that is either not accessible or not expressed on intact fibrinogen. These data are consistent with the known effect of EDTA in altering the conformation of αIIbβ3 as judged by its exposing "ligand-induced" binding sites recognized by mAbs such as AP5 and PMI-1, even in the absence of ligand, and the ability of αIIbβ3 to mediate interactions with fibrin to support clot retraction even in the presence of EDTA. EDTA-treated αIIbβ3 may, therefore, provide insights into potential ancillary sites of interaction between αIIbβ3 and fibrin(ogen). Disclosures No relevant conflicts of interest to declare.

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Identification of a ligand-binding site in an immunoglobulin fold domain of the Saccharomyces cerevisiae adhesion protein alpha-agglutinin.

Molecular Biology of the Cell ◽

10.1091/mbc.7.1.143 ◽

1996 ◽

Vol 7 (1) ◽

pp. 143-153 ◽

Cited By ~ 17

Author(s):

H de Nobel ◽

P N Lipke ◽

J Kurjan

Keyword(s):

Saccharomyces Cerevisiae ◽

Ligand Binding ◽

Binding Site ◽

Mutational Analysis ◽

Homology Model ◽

Ligand Binding Site ◽

Adhesion Protein ◽

Interaction Sites ◽

Critical Residue ◽

Immunoglobulin Fold

The Saccharomyces cerevisiae adhesion protein alpha-agglutinin (Ag alpha 1p) is expressed by alpha cells and binds to the complementary a-agglutinin expressed by a cells. The N-terminal half of alpha-agglutinin is sufficient for ligand binding and has been proposed to contain an immunoglobulin (Ig) fold domain. Based on a structural homology model for this domain and a previously identified critical residue (His292), we made Ag alpha 1p mutations in three discontinuous patches of the domain that are predicted to be in close proximity to His292 in the model. Residues in each of the three patches were identified that are important for activity and therefore define a putative ligand binding site, whereas mutations in distant loops had no effect on activity. This putative binding site is on a different surface of the Ig fold than the defined binding sites of immunoglobulins and other members of the Ig superfamily. Comparison of protein interaction sites by structural and mutational analysis has indicated that the area of surface contact is larger than the functional binding site identified by mutagenesis. The putative alpha-agglutinin binding site is therefore likely to identify residues that contribute to the functional binding site within a larger area that contacts a-agglutinin.

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Platelet-associated anti–GPIIb-IIIa autoantibodies in chronic immune thrombocytopenic purpura recognizing epitopes close to the ligand-binding site of glycoprotein (GP) IIb

Blood ◽

10.1182/blood.v98.6.1819 ◽

2001 ◽

Vol 98 (6) ◽

pp. 1819-1827 ◽

Cited By ~ 28

Author(s):

Satoru Kosugi ◽

Yoshiaki Tomiyama ◽

Shigenori Honda ◽

Hisashi Kato ◽

Teruo Kiyoi ◽

...

Keyword(s):

Ligand Binding ◽

Binding Site ◽

Immune Thrombocytopenic Purpura ◽

Tertiary Structure ◽

Enzyme Linked Immunosorbent Assay ◽

Ligand Binding Site ◽

Thrombocytopenic Purpura ◽

Chronic Immune Thrombocytopenic Purpura ◽

Fibrinogen Binding ◽

Impaired Binding

Abstract Localization of epitopes for platelet-associated (PA) anti–GPIIb-IIIa (αIIbβ3) autoantibodies in chronic immune thrombocytopenic purpura remains elusive. Previous studies suggest that PA antibodies recognize the tertiary structure of intact glycoprotein (GP) IIb-IIIa. To localize their epitopes using antigen-capture enzyme-linked immunosorbent assay (ELISA), the reactivity of 34 PA anti–GPIIb-IIIa antibodies was examined with recombinant GPIIb-IIIa having a defect in ligand-binding sites in either GPIIb or GPIIIa, and no major conformational change was induced: KO variant GPIIb-IIIa was attributed to a 2–amino acid insertion between residues 160 and 161 in the W3 4-1 loop in GPIIb, and CAM variant GPIIb-IIIa was attributed to D119Y in GPIIIa. In one third (11 of 34) of the patients, PA antibodies showed a marked decrease (less than 50%) in reactivity with KO compared with wild-type GPIIb-IIIa. Their reactivity was also impaired against GPIIbD163A-IIIa. In sharp contrast, they reacted normally with CAM GPIIb-IIIa. OP-G2, a ligand-mimetic monoclonal antibody, markedly inhibited their binding to GPIIb-IIIa in patients with impaired binding to KO GPIIb-IIIa, but small GPIIb-IIIa antagonists did not. In addition, a newly developed sensitive ELISA indicated that autoantibodies showing impaired binding to KO are more potent inhibitors for fibrinogen binding. The present data suggest that certain PA anti–GPIIb-IIIa autoantibodies recognize epitopes close to the ligand-binding site in GPIIb, but not in GPIIIa.

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Unraveling the structural elements of pH sensitivity and substrate binding in the human zinc transporter SLC39A2 (ZIP2)

Journal of Biological Chemistry ◽

10.1074/jbc.ra118.006113 ◽

2019 ◽

Vol 294 (20) ◽

pp. 8046-8063 ◽

Cited By ~ 5

Author(s):

Gergely Gyimesi ◽

Giuseppe Albano ◽

Daniel G. Fuster ◽

Matthias A. Hediger ◽

Jonai Pujol-Giménez

Keyword(s):

Binding Site ◽

Metal Binding ◽

In Silico ◽

Metal Ion ◽

Substrate Binding ◽

Functional Characterization ◽

Ph Sensitivity ◽

Hek293 Cells ◽

Substrate Binding Site

The transport and ion-coupling mechanisms of ZIP transporters remain largely uncharacterized. Previous work in our laboratory has revealed that the solute carrier family 39 member A2 (SLC39A2/ZIP2) increases its substrate transport rate in the presence of extracellular H+. Here, we used a combination of in silico and in vitro techniques involving structural modeling, mutagenesis, and functional characterization in HEK293 cells to identify amino acid residues potentially relevant for both the ZIP2–H+ interaction and substrate binding. Our ZIP2 models revealed a cluster of charged residues close to the substrate–translocation pore. Interestingly, the H63A substitution completely abrogated pH sensitivity, and substitutions of Glu-67 and Phe-269 altered the pH and voltage modulation of transport. In contrast, substitution of Glu-106, which might be part of a dimerization interface, altered pH but not voltage modulation. Substitution of Phe-269, located close to the substrate-binding site, also affected substrate selectivity. These findings were supported by an additional model of ZIP2 that was based on the structure of a prokaryotic homolog, Bordetella bronchiseptica ZrT/Irt-like protein (bbZIP), and in silico pKa calculations. We also found that residues Glu-179, His-175, His-202, and Glu-276 are directly involved in the coordination of the substrate metal ion. We noted that, unlike bbZIP, human ZIP2 is predicted to harbor a single divalent metal-binding site, with the charged side chain of Lys-203 replacing the second bound ion. Our results provide the first structural evidence for the previously observed pH and voltage modulation of ZIP2-mediated metal transport, identify the substrate-binding site, and suggest a structure-based transport mechanism for the ZIP2 transporter.

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Structure and ligand binding of the glutamine-II riboswitch

Nucleic Acids Research ◽

10.1093/nar/gkz539 ◽

2019 ◽

Vol 47 (14) ◽

pp. 7666-7675 ◽

Cited By ~ 9

Author(s):

Lin Huang ◽

Jia Wang ◽

Andrew M Watkins ◽

Rhiju Das ◽

David M J Lilley

Keyword(s):

Ligand Binding ◽

Binding Site ◽

Metal Ion ◽

Titration Calorimetry ◽

Water Molecules ◽

Molecular Replacement ◽

Major Groove ◽

X Ray Crystallography ◽

Carboxylate Oxygen ◽

Hydrogen Bonding Interactions

Abstract We have determined the structure of the glutamine-II riboswitch ligand binding domain using X-ray crystallography. The structure was solved using a novel combination of homology modeling and molecular replacement. The structure comprises three coaxial helical domains, the central one of which is a pseudoknot with partial triplex character. The major groove of this helix provides the binding site for L-glutamine, which is extensively hydrogen bonded to the RNA. Atomic mutation of the RNA at the ligand binding site leads to loss of binding shown by isothermal titration calorimetry, explaining the specificity of the riboswitch. A metal ion also plays an important role in ligand binding. This is directly bonded to a glutamine carboxylate oxygen atom, and its remaining inner-sphere water molecules make hydrogen bonding interactions with the RNA.

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Structures of T Cell Immunoglobulin Mucin Protein 4 Show a Metal-Ion-Dependent Ligand Binding Site where Phosphatidylserine Binds

Immunity ◽

10.1016/j.immuni.2007.11.008 ◽

2007 ◽

Vol 27 (6) ◽

pp. 941-951 ◽

Cited By ~ 147

Author(s):

César Santiago ◽

Angela Ballesteros ◽

Laura Martínez-Muñoz ◽

Mario Mellado ◽

Gerardo G. Kaplan ◽

...

Keyword(s):

T Cell ◽

Ligand Binding ◽

Binding Site ◽

Metal Ion ◽

Ligand Binding Site

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Recognition of ion ligand binding sites based on amino acid features with the fusion of energy, physicochemical and structural features

Current Pharmaceutical Design ◽

10.2174/1381612826666201029100636 ◽

2020 ◽

Vol 26 ◽

Author(s):

Shan Wang ◽

Xiuzhen Hu ◽

Zhenxing Feng ◽

Liu Liu ◽

Kai Sun ◽

...

Keyword(s):

Amino Acid ◽

Ligand Binding ◽

Binding Site ◽

Binding Sites ◽

Metal Ion ◽

Structural Features ◽

Support Vector ◽

Sequence Information ◽

Ligand Binding Site ◽

Ligand Binding Sites

Background: Rational drug molecular design based on virtual screening requires the ligand binding site to be known. Recently, the recognition of ion ligand binding site has become an important research direction in pharmacology. Methods: In this work, we selected the binding residues of 4 acid radical ion ligands(NO2 - , CO3 2- , SO4 2- and PO4 3- ) and 10 metal ion ligands (Zn2+,Cu2+, Fe2+, Fe3+, Ca2+, Mg2+, Mn2+, Na+ , K+ and Co2+) as research objects. Based on the protein sequence information, we extracted amino acid features, energy, physicochemical and structure features. Then we incorporating the above features and input them into the MultilayerPerceptron (MLP) and support vector machine (SVM) algorithms. Results: In the independent test, the best accuracy was higher than 92.5%, which was better than the previous result on Conclusion: Finally, we set up a free web server for the prediction of protein-ion ligand binding sites (http://39.104.77.103:8081/lsb/HomePage/HomePage.html). This study is helpful for molecular drug design.

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Application of High Throughput Screening To Identify Novel Small Molecule Inhibitors of αIIbβ3-Mediated Platelet Adhesion to Fibrinogen.

Blood ◽

10.1182/blood.v108.11.144.144 ◽

2006 ◽

Vol 108 (11) ◽

pp. 144-144

Author(s):

Robert Blue ◽

Markéta Jiroušková ◽

Charles Karan ◽

Barry S. Coller

Keyword(s):

Platelet Aggregation ◽

Ligand Binding ◽

Binding Site ◽

Small Molecule ◽

High Throughput ◽

Conformational Changes ◽

High Throughput Screening ◽

Metal Ion ◽

Platelet Adhesion ◽

Small Molecule Inhibitors

Abstract Two separate conformational changes have been proposed to accompany activation of platelet αIIbβ3: 1) leg separation leading to extension of the head region composed of the αIIb propeller and β3 βA (I-like) domains, and 2) a swing-out motion at the junction of the β3 βA (I-like) and hybrid domains. Small molecule inhibitors of αIIbβ3 competitively block the RGD ligand binding site and variably induce conformational changes in αIIbβ3 as judged by the binding of ligand-induced binding site (LIBS)-specific monoclonal antibodies. In an attempt to identify molecules that may inhibit αIIbβ3 activation without initiating the conformational changes associated with ligand binding, we screened 33,264 compounds from four different chemical libraries (Prestwick, Chembridge, Cerep and ChemDiv) for their ability to inhibit the adhesion of washed platelets in HEPES-modified Tyrode’s buffer with 1 mM Ca2+/0.5 mM Mg2+ to immobilized fibrinogen adsorbed from a 50 μg/ml solution. When tested at a final concentration of 16 μM, a total of 102 compounds (0.31%) demonstrated greater than 50% inhibition of platelet adhesion, and two of these (Figure 1) demonstrated >30% inhibition of the initial wave of ADP-induced aggregation of platelets in citrated platelet-rich plasma. IC50s for inhibition of ADP (5 μM)-induced platelet aggregation for compounds 1 and 2 were 13 ± 4.5 and 17 ± 5 μM (n=3), respectively. Compounds 1 and 2 also inhibited fibrinogen binding to platelets induced by the activating LIBS antibody AP5 with IC50s of 27 and 30 μM, and 20 and 27 μM, respectively, in two experiments. Since AP5 binds to and directly activates αIIbβ3, it is likely that the compounds’ inhibitory effects are due to direct binding to αIIbβ3 rather than inhibition of signal transduction. In two separate experiments, compound 1 at 15 - 20 μM produced variable increases in the binding of LIBS mAbs AP5, PMI-1 or LIBS1 to unactivated and ADP-activated platelets, whereas tirofiban (20 μM) consistently increased the binding of each mAb. Compound 2 did not increase the binding of any of the mAbs. Neither compound contains a negatively charged carboxyl group, which mediates the interaction of the Asp group in RGD ligands with the β3 MIDAS metal ion, but compound 1 has a carbonyl group that may potentially interact with the MIDAS metal ion. Compound 1 resembles 1,2-fused pyrimidine derivatives that have previously been demonstrated to inhibit platelet aggregation (Roma et al., Bioorg. Med. Chem. 2003, 11, 123). We conclude that high throughput screening of molecular libraries can identify novel compounds that inhibit αIIbβ3 and that one of them appears to inhibit αIIbβ3 without inducing conformational changes in the receptor. Figure Figure

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