Refinement of the structure of human basic fibroblast growth factor at 1.6 Å resolution and analysis of presumed heparin binding sites by selenate substitution

Abstract

The three‐dimensional structure of human basic fibroblast growth factor has been refined to a crystallographic residual of 16.1% at 1.6 Å resolution. The structure has a Kunitz‐type fold and is composed of 12 antiparallel β‐strands, 6 of which form a β‐barrel. One bound sulfate ion has been identified in the model, hydrogen bonded to the side chains of Asn 27, Arg 120, and Lys 125. The side chain of Arg 120 has two conformations, both of which permit hydrogen bonds to the sulfate. This sulfate binding site has been suggested as the binding site for heparin (Eriksson, A.E., Cousens, L.S., Weaver, L.H., & Matthews, B.W., 1991, Proc. Natl. Acad. Sci. USA 88, 3441–3445). Two β‐mercaptoethanol (BME) molecules are also included in the model, each forming a disulfide bond to the Sγ atoms of Cys 69 and Cys 92, respectively. The side chain of Cys 92 has two conformations of which only one can bind BME. Therefore the BME molecule is half occupied at this site. The locations of possible sulfate binding sites on the protein were examined by replacing the ammonium sulfate in the crystallization medium with ammonium selenate. Diffraction data were measured to 2.2 Å resolution and the structure refined to an R‐factor of 13.8%. The binding of the more electron‐dense selenate ion was identified at two positions. One position was identical to the sulfate binding site identified previously. The second selenate binding site, which is of lower occupancy, is situated 5.6 Å from the first. This ion is hydrogen bonded by the side chain of Lys 135 and Arg 120. Thus the side chain of Arg 120 binds two selenate ions simultaneously. It is suggested that the observed second selenate binding site should also be considered as a possible binding site for heparin, or that both selenate binding sites might simultaneously contribute to the binding of heparin.

Glu-96 of Basic Fibroblast Growth Factor Is Essential for High Affinity Receptor Binding

“…Peptide mapping studies (Baird et al, 1988) have shown that a peptide related to residues 106 -115 of bFGF can inhibit the binding of 125 I-bFGF to its receptor. Subsequently, the threedimensional structure determination of bFGF (Zhang et al, 1991;Eriksson et al, 1993) suggested that these residues form an antiparallel ␤-turn on the surface of the molecule. To identify residues on bFGF for the receptor binding, the crystal structure is first subjected to molecular dynamics treatment.…”

“…Alignment of amino acid sequences in aFGF and bFGF reveals that the residues comprising the heparin binding site 2Ј on bFGF correspond to Gln-63, Asp-68, Leu-72, and Gln-77 on aFGF, none of which are either basic or conserved residues (Eriksson et al, 1993). This argues against the possibility that these particular residues on aFGF interact with trisaccharide or heparin and likewise makes it difficult to reconcile the Ornitz model with the induction of FGF receptor dimerization by aFGF.…”

“…An early peptide mapping approach by Baird et al (1988) suggested that residues 106 -115 are involved in receptor binding. Later, studies of the three-dimensional structure of bFGF revealed that part of this putative receptor binding peptide forms a loop consisting of residues 109 -114 on the surface of the molecule (Eriksson et al, 1993). In addition, Seno et al (1990) studied the action of truncated N-and C-terminal forms of bFGF on mitogenesis and heparin binding.…”

A two-dimensional histogram-matching method for protein phase refinement and extension

“…The consensus histograms used in the test cases were generated from the atomic coordinates of ®broblast growth factor (4fgf; Eriksson et al, 1993) after removing the overall temperature factor from the electron-density map. Since 2D histograms are resolution dependent, several ideal histograms were generated for a range of resolutions from 4.0 to 1.5 A Ê using the method described by Goldstein & Zhang (1998).…”

Examination of the mechanism by which heparin antagonizes activation of a model endothelium by interferon-gamma (IFN-γ)

“…HS-related GAG are known to interact with a wide range of growth factors and immunomodulatory cytokines, including the chemokines [10], IL-1, IL-2, IL-3, IL-6 [11], tumour necrosis factor (TNF) [12] and IFN-g [13]. The nature of this interaction is best understood for the basic fibroblast growth factor (bFGF) family [14], whose heparin-binding domain has been characterized [15]. In common with other heparin-binding proteins, bFGF binds to the GAG by electrostatic bonding to characteristic short clusters of basic amino acids [16].…”