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Direct and allosteric inhibition of the FGF2/HSPGs/FGFR1 ternary complex formation by an antiangiogenic, thrombospondin-1-mimic small molecule.

Pagano K, Torella R, Foglieni C, Bugatti A, Tomaselli S, Zetta L, Presta M, Rusnati M, Taraboletti G, Colombo G, Ragona L - PLoS ONE (2012)

Bottom Line: The formation of a ternary complex with the transmembrane tyrosine kinase receptors (FGFRs), and heparan sulphate proteoglycans (HSPGs) is required for FGF2 pro-angiogenic activity.NMR and MD data demonstrate that sm27 engages the heparin-binding site of FGF2 and induces long-range dynamics perturbations along FGF2/FGFR1 interface regions.We propose that sm27 antiangiogenic activity is based on a twofold-direct and allosteric-mechanism, inhibiting FGF2 binding to both its receptors.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio NMR, Istituto per lo Studio delle Macromolecole, Consiglio Nazionale delle Ricerche, Milano, Italy.

ABSTRACT
Fibroblast growth factors (FGFs) are recognized targets for the development of therapies against angiogenesis-driven diseases, including cancer. The formation of a ternary complex with the transmembrane tyrosine kinase receptors (FGFRs), and heparan sulphate proteoglycans (HSPGs) is required for FGF2 pro-angiogenic activity. Here by using a combination of techniques including Nuclear Magnetic Resonance, Molecular Dynamics, Surface Plasmon Resonance and cell-based binding assays we clarify the molecular mechanism of inhibition of an angiostatic small molecule, sm27, mimicking the endogenous inhibitor of angiogenesis, thrombospondin-1. NMR and MD data demonstrate that sm27 engages the heparin-binding site of FGF2 and induces long-range dynamics perturbations along FGF2/FGFR1 interface regions. The functional consequence of the inhibitor binding is an impaired FGF2 interaction with both its receptors, as demonstrated by SPR and cell-based binding assays. We propose that sm27 antiangiogenic activity is based on a twofold-direct and allosteric-mechanism, inhibiting FGF2 binding to both its receptors.

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Internal dynamics of apo and holo FGF2.A) Difference matrix obtained by subtracting the distance fluctuations of the apo protein from those of the holo protein. B) Time evolution of the geometrical strain for the apo FGF2; C. Time evolution of the geometrical strain for the holo FGF2. The magnitude of the relative fluctuations, expressed in Å2 units, is color coded according to the legend on the right of each panel.
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pone-0036990-g004: Internal dynamics of apo and holo FGF2.A) Difference matrix obtained by subtracting the distance fluctuations of the apo protein from those of the holo protein. B) Time evolution of the geometrical strain for the apo FGF2; C. Time evolution of the geometrical strain for the holo FGF2. The magnitude of the relative fluctuations, expressed in Å2 units, is color coded according to the legend on the right of each panel.

Mentions: The mean squared fluctuations (see Experimental Procedures) of all pair wise distances in the apo and in the simulation of the FGF2/sm27 complex are shown in Figure S5A, B. Due to its high degree of disorder and flexibility, the N-terminal tail was excluded from the analysis and the results refer to the region 25–155. The matrices showed patterns reflecting the alternation of substructures with small and large fluctuations of inter-residue distances. The difference matrix, reporting on the variation of mean squared fluctuations between apo and holo states, is shown in Figure 4A. The presence of the ligand appeared to significantly affect the degree of fluctuations of different groups of FGF2 aminoacids. Specifically, the regions displaying quenched conformational freedom upon binding (represented by gray to black shadings in the matrix) correspond to part of the binding site region (residues 128–131) of b10–b12 loop, and to b4 strand and b4–b5 loop (residues 66–71). Another striking feature of holo protein simulation is the enhancement in the conformational freedom (blue shading in Figure 4A) of residues 139–143 of the binding site, and of residues 107 to 112, corresponding to the b8–b9 turn region. This analysis is unable to catch the differences in the motions observed for loops b3–b4, b7–b8 and b9–b10, identified by NMR analysis. This inconsistency may be due to two main causes: 1) insufficient or limited sampling of the MD-simulations, which limits the conformational statistics that is used for the analysis compared to the experimental situation. This may affect particularly the evaluation of the motional properties of long and flexible loops; 2) the fact that this specific analysis aims at identifying coordinated motions between residue-pairs, while NMR-based indicators mainly refer to single-residue motions. In this framework, in order to analyze the mobility properties of single residues in the context of a dynamic protein environment, we calculated the time-dependent variation of the geometric deformation (also named geometric strain, see [29]) experienced by the various aminoacids in the course of the simulations.


Direct and allosteric inhibition of the FGF2/HSPGs/FGFR1 ternary complex formation by an antiangiogenic, thrombospondin-1-mimic small molecule.

Pagano K, Torella R, Foglieni C, Bugatti A, Tomaselli S, Zetta L, Presta M, Rusnati M, Taraboletti G, Colombo G, Ragona L - PLoS ONE (2012)

Internal dynamics of apo and holo FGF2.A) Difference matrix obtained by subtracting the distance fluctuations of the apo protein from those of the holo protein. B) Time evolution of the geometrical strain for the apo FGF2; C. Time evolution of the geometrical strain for the holo FGF2. The magnitude of the relative fluctuations, expressed in Å2 units, is color coded according to the legend on the right of each panel.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3351436&req=5

pone-0036990-g004: Internal dynamics of apo and holo FGF2.A) Difference matrix obtained by subtracting the distance fluctuations of the apo protein from those of the holo protein. B) Time evolution of the geometrical strain for the apo FGF2; C. Time evolution of the geometrical strain for the holo FGF2. The magnitude of the relative fluctuations, expressed in Å2 units, is color coded according to the legend on the right of each panel.
Mentions: The mean squared fluctuations (see Experimental Procedures) of all pair wise distances in the apo and in the simulation of the FGF2/sm27 complex are shown in Figure S5A, B. Due to its high degree of disorder and flexibility, the N-terminal tail was excluded from the analysis and the results refer to the region 25–155. The matrices showed patterns reflecting the alternation of substructures with small and large fluctuations of inter-residue distances. The difference matrix, reporting on the variation of mean squared fluctuations between apo and holo states, is shown in Figure 4A. The presence of the ligand appeared to significantly affect the degree of fluctuations of different groups of FGF2 aminoacids. Specifically, the regions displaying quenched conformational freedom upon binding (represented by gray to black shadings in the matrix) correspond to part of the binding site region (residues 128–131) of b10–b12 loop, and to b4 strand and b4–b5 loop (residues 66–71). Another striking feature of holo protein simulation is the enhancement in the conformational freedom (blue shading in Figure 4A) of residues 139–143 of the binding site, and of residues 107 to 112, corresponding to the b8–b9 turn region. This analysis is unable to catch the differences in the motions observed for loops b3–b4, b7–b8 and b9–b10, identified by NMR analysis. This inconsistency may be due to two main causes: 1) insufficient or limited sampling of the MD-simulations, which limits the conformational statistics that is used for the analysis compared to the experimental situation. This may affect particularly the evaluation of the motional properties of long and flexible loops; 2) the fact that this specific analysis aims at identifying coordinated motions between residue-pairs, while NMR-based indicators mainly refer to single-residue motions. In this framework, in order to analyze the mobility properties of single residues in the context of a dynamic protein environment, we calculated the time-dependent variation of the geometric deformation (also named geometric strain, see [29]) experienced by the various aminoacids in the course of the simulations.

Bottom Line: The formation of a ternary complex with the transmembrane tyrosine kinase receptors (FGFRs), and heparan sulphate proteoglycans (HSPGs) is required for FGF2 pro-angiogenic activity.NMR and MD data demonstrate that sm27 engages the heparin-binding site of FGF2 and induces long-range dynamics perturbations along FGF2/FGFR1 interface regions.We propose that sm27 antiangiogenic activity is based on a twofold-direct and allosteric-mechanism, inhibiting FGF2 binding to both its receptors.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio NMR, Istituto per lo Studio delle Macromolecole, Consiglio Nazionale delle Ricerche, Milano, Italy.

ABSTRACT
Fibroblast growth factors (FGFs) are recognized targets for the development of therapies against angiogenesis-driven diseases, including cancer. The formation of a ternary complex with the transmembrane tyrosine kinase receptors (FGFRs), and heparan sulphate proteoglycans (HSPGs) is required for FGF2 pro-angiogenic activity. Here by using a combination of techniques including Nuclear Magnetic Resonance, Molecular Dynamics, Surface Plasmon Resonance and cell-based binding assays we clarify the molecular mechanism of inhibition of an angiostatic small molecule, sm27, mimicking the endogenous inhibitor of angiogenesis, thrombospondin-1. NMR and MD data demonstrate that sm27 engages the heparin-binding site of FGF2 and induces long-range dynamics perturbations along FGF2/FGFR1 interface regions. The functional consequence of the inhibitor binding is an impaired FGF2 interaction with both its receptors, as demonstrated by SPR and cell-based binding assays. We propose that sm27 antiangiogenic activity is based on a twofold-direct and allosteric-mechanism, inhibiting FGF2 binding to both its receptors.

Show MeSH
Related in: MedlinePlus