Abstract
Fibroblast growth factor-2 (basic FGF), a potent inducer of angiogenesis, and the naphthalene sulfonic distamycin A derivative, 7,7-(carbonyl-bis[imino-N-methyl-4,2-pyrrolecarbonylimino[N-methyl-4,2-pyrrole]-carbonylimino])-bis-(1,3-naphtalene disulfonate) (PNU145156E), which exhibits in vivo antiangiogenic activity, form a tight reversible (1:1) complex. PNU145156E binds to the heparin and the selenate-binding sites on bFGF. The cis bFGF-heparin (2:1) complex, essential for the activation of the angiogenic process, is thus prevented. The nature of the forces involved in bFGF:PNU145156E complex, using the wild-type and the K128Q, K138Q, K134Q, and K128Q-K138Q point mutated bFGFs was sought. Based on thermodynamic analysis of the complexation constants, protein temperature stability profiles by ultraviolet absorption, circular dichroism measurements, fluorescence Förster energy-transfer, and anisotropy studies, in harmony with the published x-ray crystallographic structure, the following molecular interactions are proposed: reduced coulombic interactions, hence loosening of the complex by the removal of charged polar groups from the bFGF-heparin binding cleft resulted in decreased binding constants and in a change in the binding mode from polar to nonpolar. Concomitantly, upon mutation, the protein was rendered more compact, less flexible, and less aqueously exposed compared with the wild type. These were further pronounced with the double mutant: weaker dominantly nonpolar protein-drug interactions were accompanied by conspicuous folding. With heparin, however, wild-type bFGF forms a tighter complex with a more compact structure.