Structure and Stability of an Acidic Fibroblast Growth Factor from Notophthalmus viridescens

Abstract

The three-dimensional solution structure of an acidic fibroblast growth factor (nFGF-1) from the newt (Notophthalmus viridescens) is determined using multidimensional NMR techniques. Complete assignment of all the atoms ( 1 H, 15 N, and 13 C) has been achieved using a variety of triple resonance experiments. 50 structures were calculated using hybrid distance geometry-dynamical simulated annealing technique with a total of 1359 constraints. The atomic root mean square distribution for the backbone atoms in the structured region is 0.60 Å. The secondary structural elements include 12 ␤-strands arranged antiparallely into a ␤-barrel structure. The protein (nFGF-1) exists in a monomeric state upon binding to the ligand, sucrose octa sulfate (SOS), in a stoichiometric ratio of 1:1. The SOS binding site consists of a dense cluster of positively charged residues located at the C-terminal end of the molecule. The conformational stabilities of nFGF-1 and its structural and functional homologue from the human source (hFGF-1) are drastically different. The differential stabilities of nFGF-1 and hFGF-1 are attributed to the differences in the number of hydrogen bonds and the presence of solvent inaccessible cavities in the two proteins.Mammalian acidic fibroblast growth factors 1 are a family of proteins, which play crucial roles in proliferation, migration, and differentiation of a wide variety of cell lines and also exhibit potent angiogenic activity on endothelial cell lines (1-4). Acidic fibroblast growth factors exert their biological activities through two cell surface receptors (5-8). The high affinity receptors are composed of an extracellular ligand-binding domain consisting of three immunoglobulin (Ig)-like domains, a transmembrane domain, and a cytoplasmic tyrosine kinase domain (9 -12). The low affinity receptors have been identified as heparin sulfate proteoglycans (13)(14)(15)(16)(17)(18)(19). Although the significance of binding to these polyanions is not yet clear, heparin/heparan sulfate are believed to be required for binding of FGF-1 to their high affinity receptor (20 -23).FGF-1 plays an important role in vertebrate development (24,25). The regeneration of the amphibian (newt) limb has been used as a model system to understand the role of FGF-1 in embryonic growth, differentiation, and morphogenesis (26,27). Recently, the isolation and characterization of an acidic fibroblast growth factor, nFGF-1, from the newt, Notophthalmus viridescens, has been reported (28, 29). Amino acid sequence comparison indicates that nFGF-1 exhibits about 80% homology with its mammalian counterparts (Fig. 1). nFGF-1 has been shown to elicit a mitogenic response, which is similar to that exhibited by the human FGF-1 (hFGF-1, Ref. 28). Interestingly, despite the high degree of sequence homology and functional similarity, polyclonal antibodies directed against mammalian FGF-1 failed to recognize nFGF-1 (28). In this context, we determine the three-dimensional structure(s) of nFGF-1 using a variety of multidimensional NMR techn...

Relevance of Partially Structured States in the Non-Classical Secretion of Acidic Fibroblast Growth Factor

“…Fibroblast growth factors (FGF) are ~17 kDa β-sheet proteins that play crucial roles in the regulation of key cellular processes, such as angiogenesis, morphogenesis, differentiation, and tumor growth (1)(2)(3)(4)(5). The biological functions of FGFs are mediated by a family of transmembrane tyrosine kinase receptors, FGFRs (6)(7)(8).…”

Time-dependent Changes in the Denatured State(s) Influence the Folding Mechanism of an All β-Sheet Protein

“…The secondary structural elements in the protein include 12 antiparallel ␤-strands arranged into a ␤-barrel structure. The folding/unfolding pathway(s) of nFGF-1 have been well characterized (22)(23)(24)(25). In the present study we demonstrate that the folding mechanism of nFGF-1 depends on the time of incubation of the protein in the denaturant.…”

NMR characterization of copper and lipid interactions of the C2B domain of synaptotagmin I—relevance to the non-classical secretion of the human acidic fibroblast growth factor (hFGF-1)

“…The concentration of the protein sample used was 0.1 mM in 90% H 2 O and 10 % D 2 O prepared in 50 mM MES buffer containing 150 mM NaCl and 2 mM DTT (pH 6.3). The spectra were processed on a Windows workstation using XWIN-NMR and Sparky software [38]. …”