Improvement of FGF7 thermal stability by introduction of mutations in close vicinity to disulfide bond and surface salt bridge

Abstract

Fibroblast growth factor 7 (FGF7) or exogenous keratinocyte growth factor (KGF1) is one of the 22 FGF families (Eswarakumar et al. 2005; Harmer et al. 2004; Hui et al. 2018; Imamura 2014; Ornitz and Itoh 2001) and belongs to subfamily of FGF3/7/10/22. FGF7 plays an important role in regulation of embryonic development, cell differentiation and cell proliferation by binding to epithelial cellspecific FGFR 2IIIb with heparin or heparan sulfate (Jang et al. 1997). Due to its cell proliferative activity, FGF7 has been developed as therapeutics for wound healing and tissue regeneration. Currently, recombinant FGF7 in its N-terminally truncated form, called Palifermin, is prescribed to remedy oral mucositis caused by chemotherapy and radiation therapy used to treat cancers of blood or bone marrow (Spielberger et al. 2004). Palifermin has been reported to effectively reduce the incidence and duration of oral mucositis (Henke et al. 2011) and increase thermal stability and activity than wildtype FGF7 (Hsu et al. 2006). However, the biological half-life of Palifermin is not long enough yet, so its stability improvement is required for better usefulness. All FGFs protein have core FGF domain that contains b-trefoil fold consisting of 12-stranded b-sheet structure, therefore FGFs share similar structure (Bellosta et al. 2001; Eriksson et al. 1991; Osslund et al. 1998; Plotnikov et al. 2001; Zhu et al. 1991). This structural similarity of FGFs helps to study and improve the stability and activity by comparing differences between FGFs. Generally, introduction of disulfide bond in protein can increase thermal stability (Betz 1993). FGF8 (2FDB PDB), FGF19 (2p23 PDB), and FGF23 (2p39 PDB) have a disulfide-bonded protein crystal structure between C109 and C127 residue in FGF8. However, the FGF1, FGF2, FGF4, FGF9, FGF10 and FGE12 protein including FGF7 do not form a complete disulfide bond in the same location of FGF8, because the amino acid of these FGFs corresponding to 109th residue of FGF8 is not cysteine (Lee and Blaber 2009). The substitution of Ala 66 (120 for FGF7) of FGF1 into Cys 66 yielded to a disulfide bond between 66th and 83th amino acid, which increased stability (Lee and Blaber 2010; Lee and Blaber 2013). Even though A120 (FGF1 mutation A66C) of FGF7 is not cysteine, a disulfide bond between C133 and C137 (FGF1 C83) was already formed (Hsu et al. 1998), which was unusual to the structure of typical FGF family. However, the crystal structures of FGF7 (PDB: 1qql, 1qqk) exhibited no disulfide bond between C133 and C137, which was due to the presence of reducing agent. Since FGF7 can be utilized in various fields but has low stability, there have been several results to overcome the problem. Most of FGFs have positive charge clusters that can bind to heparin (Kan et al. 1993; Plotnikov et al. 1999). Addition of negatively-charged heparin to FGFs with positively-charged clusters brings electrostatic stability through the binding between the two molecules. And the additives such as heparin, osmolyte, and salt increased the melting temperature and elongated half-life of native FGF7 (Chen and Arakawa 1996; Chen et al. 1994). The replacement of R175 in FGF7 with Glu or Gln increased protein stability. The improvement of protein stability by R175 mutation may result from ionic interaction around the residue . However, as a result of analyzing the 3D structure, it is expected that stability can be further improved through mutations around R175.