An experimental and molecular-modeling study of the binding of linked sulfated tetracyclitols to FGF-1 and FGF-2

The experimental binding affinities of a series of linked sulfated tetracyclitols [Cyc(2)N-R-NCyc(2), where Cyc=C6H6(OSO3Na)(3) and R= (CH2)(n) (n=2-10), p-xylyl or (C2H4),-Ncyc] for the fibroblast growth factors FGF-1 and FGF-2 have been measured by using a surface plasmon resonance assay. The K, values range from 7.0 nM to 1.1 mu M 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 showed that all of the sulfated formers of the FGF-bound ligands showed 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 kcal 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.