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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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Summary of change in FGF2 dynamics upon sm27 binding. The representative structure of FGF2-sm27 complex is shown in grey. Dynamics variations are mapped on the FGF2 structure. Blu and red colors code for decreased and increased backbone motions upon inhibitor binding. A) NMR data: colored ribbon represents S2 variations, sticks describe slow conformational exchange variations (R1rho) and spheres indicate residues affected by HSQC intensity changes upon sm27 binding. B) MD data: mean squared fluctuations differences of all pair wise distances between the apo and holo form. For clarity purposes the complex was superimposed to the X-ray structure of FGFR1-FGF1 dimer (PDB id: 1FQ9) and the FGFR1 regions making contacts with FGF2 are shown. The two FGFR1 units are shown as green and light green cartoons.
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pone-0036990-g006: Summary of change in FGF2 dynamics upon sm27 binding. The representative structure of FGF2-sm27 complex is shown in grey. Dynamics variations are mapped on the FGF2 structure. Blu and red colors code for decreased and increased backbone motions upon inhibitor binding. A) NMR data: colored ribbon represents S2 variations, sticks describe slow conformational exchange variations (R1rho) and spheres indicate residues affected by HSQC intensity changes upon sm27 binding. B) MD data: mean squared fluctuations differences of all pair wise distances between the apo and holo form. For clarity purposes the complex was superimposed to the X-ray structure of FGFR1-FGF1 dimer (PDB id: 1FQ9) and the FGFR1 regions making contacts with FGF2 are shown. The two FGFR1 units are shown as green and light green cartoons.

Mentions: Direct interference of sm27 with HSPGs recognition of FGF2 is however only one side of sm27 inhibitory mechanism. Indeed dynamics analysis of the apo and holo FGF2 states performed by NMR and MD, which allows the characterization of motion changes in a timescale range from picosecond to milliseconds, showed that the effects of sm27 binding extended far beyond the interaction site. Figure 6 provides the structural representation of residues affected by dynamical changes upon binding, as deduced by data analysis. Three protein regions could be identified involving residues: i) close to the binding site; ii) distal from the binding site; iii) belonging to N and C-terminal tails. NMR hydration studies detected as well changes of hydration pattern at the level of the binding site and in the flexible N-terminal region upon binding (Figure 3B). Overall, the combined results from NMR and MD investigations revealed common perturbation patterns that impact on functionally relevant regions of FGF2 in response to ligand binding. As shown in the results, the employed MD-derived parameters show variable levels of agreement with NMR-derived data. Specifically, coordination analysis of residue pairs, based on the calculation of mean-square fluctuations, catches motional variations that involve set of residues in part different from the ones revealed by strain analysis. On this basis, we speculate that the observed differences may arise from distinct perturbation mechanisms. The coordination analysis may indicate sets of residues that respond in a cooperative, concerted manner to the presence of the ligand in the binding site. The aminoacids that are highlighted by the strain analysis, on the other hand, may indicate protein regions that respond by simply changing their contacts with the perturbed local environment. At this stage, however, this discussion must be considered as a qualitative one, and further and more quantitative coordination analysis of MD simulations are required [38].


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)

Summary of change in FGF2 dynamics upon sm27 binding. The representative structure of FGF2-sm27 complex is shown in grey. Dynamics variations are mapped on the FGF2 structure. Blu and red colors code for decreased and increased backbone motions upon inhibitor binding. A) NMR data: colored ribbon represents S2 variations, sticks describe slow conformational exchange variations (R1rho) and spheres indicate residues affected by HSQC intensity changes upon sm27 binding. B) MD data: mean squared fluctuations differences of all pair wise distances between the apo and holo form. For clarity purposes the complex was superimposed to the X-ray structure of FGFR1-FGF1 dimer (PDB id: 1FQ9) and the FGFR1 regions making contacts with FGF2 are shown. The two FGFR1 units are shown as green and light green cartoons.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0036990-g006: Summary of change in FGF2 dynamics upon sm27 binding. The representative structure of FGF2-sm27 complex is shown in grey. Dynamics variations are mapped on the FGF2 structure. Blu and red colors code for decreased and increased backbone motions upon inhibitor binding. A) NMR data: colored ribbon represents S2 variations, sticks describe slow conformational exchange variations (R1rho) and spheres indicate residues affected by HSQC intensity changes upon sm27 binding. B) MD data: mean squared fluctuations differences of all pair wise distances between the apo and holo form. For clarity purposes the complex was superimposed to the X-ray structure of FGFR1-FGF1 dimer (PDB id: 1FQ9) and the FGFR1 regions making contacts with FGF2 are shown. The two FGFR1 units are shown as green and light green cartoons.
Mentions: Direct interference of sm27 with HSPGs recognition of FGF2 is however only one side of sm27 inhibitory mechanism. Indeed dynamics analysis of the apo and holo FGF2 states performed by NMR and MD, which allows the characterization of motion changes in a timescale range from picosecond to milliseconds, showed that the effects of sm27 binding extended far beyond the interaction site. Figure 6 provides the structural representation of residues affected by dynamical changes upon binding, as deduced by data analysis. Three protein regions could be identified involving residues: i) close to the binding site; ii) distal from the binding site; iii) belonging to N and C-terminal tails. NMR hydration studies detected as well changes of hydration pattern at the level of the binding site and in the flexible N-terminal region upon binding (Figure 3B). Overall, the combined results from NMR and MD investigations revealed common perturbation patterns that impact on functionally relevant regions of FGF2 in response to ligand binding. As shown in the results, the employed MD-derived parameters show variable levels of agreement with NMR-derived data. Specifically, coordination analysis of residue pairs, based on the calculation of mean-square fluctuations, catches motional variations that involve set of residues in part different from the ones revealed by strain analysis. On this basis, we speculate that the observed differences may arise from distinct perturbation mechanisms. The coordination analysis may indicate sets of residues that respond in a cooperative, concerted manner to the presence of the ligand in the binding site. The aminoacids that are highlighted by the strain analysis, on the other hand, may indicate protein regions that respond by simply changing their contacts with the perturbed local environment. At this stage, however, this discussion must be considered as a qualitative one, and further and more quantitative coordination analysis of MD simulations are required [38].

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