3-DIMENSIONAL STRUCTURE OF ACIDIC FIBROBLAST GROWTH-FACTOR IN SOLUTION - EFFECTS OF BINDING TO A HEPARIN FUNCTIONAL ANALOG

Acidic and basic fibroblast growth factors (aFGF and bFGF; FGFs) are p aradigms of a group of nine closely related proteins known as the fibr oblast growth factor family. FGFs induce mitosis in most mesoderm and neuroectoderm-derived cells, and appear to be involved in diseases cau sed by anomalous cell proliferation. In vitro assays show that binding to heparin-like glycosaminoglycans is required to elicit the mitogeni c activity of these proteins. It has been shown that myo-inositol hexa sulfate (MIHS) emulates heparin in the mitogenesis assays of aFGF, and a low-resolution three-dimensional structure in solution of this prot ein bound to MIHS has been reported. Here Mie describe the H-1-NMR thr ee-dimensional structure in solution of the free aFGF. Comparison of t his structure with that of the protein bound to MIHS, upgraded to a le vel of refinement equivalent to that of the free protein, shows that M IHS binding causes some slight conformational changes with an increase in the definition of the structure. In addition, amide exchange H/H-2 rates of the most protected protons, and exchange data of the interme diate and fast-exchanging ones show that the free protein is less stab le (less than or equal to 2 kcal/mol) and more flexible in terms of lo cal unfolding equilibria, respectively, than the MIHS-bound one. Thus, MIHS binding to aFGF causes a decrease of its flexibility, which tran slates into an enhancement of the definition of its three-dimensional structure. The increase of aFGF rigidity affects regions that include those involved in recognizing the cell membrane receptor. Thus, our da ta suggest that enhancement of structural definition may play a kev ro le in the modulation of the affinity of aFGF by its receptor, and, con sequently, or its specific mitogenic activity. (C) 1996 Academic Press limited