An Experimental and Molecular-Modeling Study of the Binding of Linked Sulfated Tetracyclitols to FGF-1 and FGF-2

摘要

The experimental binding affinities of a series of linked sulfated tetracyclitols [Cyc_2N-R-NCyc_2,where Cyc=C_6H_6(OSO_3Na)_3 and R=(CH_2)_n (n=2-10),p-xylyl or (C_2H_4)_2-Ncyc] for the fibroblast growth factors FGF-1 and FGF-2 have been measured by using a surface plasmon resonance assay.The K_D values range from 7.0 nM fo 7,1 muM for the alkyl-linked ligands.The binding affinity is independent of the flexibility of the linker,as replacement of the alkyl linker with a rigid p-xylyl group did not affect the K_D.Calculations suggest that binding modes for the p-xylyl-linked ligand are similar to those calculated for the flexible alkyl-linked tetracyclitols.The possible formation of cross-linked FGF:cyclitol complexes was examined by determining K_D values at increasing protein concentrations.No changes in K_D were observed;this suggesting that only 1:1 complexes are formed under these assay conditions.Monte Carlo multiple-minima calculations of low-energy con-formers of the FGF-bound ligands showed that all of the sulfated tetracyclitol ligands can bind effectively in the heparan sulfate-binding sites of FGF-1 and FGF-2.Binding affinities of these complexes were estimated by the Linear Interaction Energy (LIE) method to within a root-mean-square deviation of 1 kca/mol~(-1) of the observed values.The effect of incorporating cations to balance the overall charge of the complexes during the LIE calculations was also explored.