Engineering a Cysteine-Free Form of Human Fibroblast Growth Factor-1 for "Second Generation" Therapeutic Application

Abstract

Human fibroblast growth factor-1 (FGF-1) has broad therapeutic potential in regenerative medicine but has undesirable biophysical properties of low thermostability and 3 buried cysteine (Cys) residues (at positions 16, 83, and 117) that interact to promote irreversible protein unfolding under oxidizing conditions. Mutational substitution of such Cys residues eliminates reactive buried thiols but cannot be accomplished simultaneously at all 3 positions without also introducing further substantial instability. The mutational introduction of a novel Cys residue (Ala66Cys) that forms a stabilizing disulfide bond (i.e., cystine) with one of the extant Cys residues (Cys83) effectively eliminates one Cys while increasing overall stability. This increase in stability offsets the associated instability of remaining Cys substitution mutations and permits production of a Cys-free form of FGF-1 (Cys16Ser/Ala66Cys/Cys117Ala) with only minor overall instability. The addition of a further stabilizing mutation (Pro134Ala) creates a Cys-free FGF-1 mutant with essentially wild-type biophysical properties. The elimination of buried free thiols in FGF-1 can substantially increase the protein half-life in cell culture. Here, we show that the effective cell survival/mitogenic functional activity of a fully Cys-free form is also substantially increased and is equivalent to wild-type FGF-1 formulated in the presence of heparin sulfate as a stabilizing agent. The results identify this Cys-free FGF-1 mutant as an advantageous "second generation" form of FGF-1 for therapeutic application.

Keywords: FGF-1; X-ray crystallography; cysteine-free mutant; cystine; disulfide; empirical phase diagram; protein engineering; protein stability.

Figure 1

Relaxed stereo ribbon diagram overlay of WT FGF-1 and C16S/A66C/C117A/P134A mutant X-ray structures. The C16S/A66C/C117A/P134A mutant structure (light gray) is overlaid onto the WT FGF-1 structure (black). The images include a “side” view (top panel) and a “top” view (parallel to the pseudo 3-fold axis of cyclic symmetry; bottom panel). Also shown are space-filling representations of the mutant residues Cys16Ser, Ala66Cys, Cys117Ala, and Pro134Ala.

Figure 2

X-ray structure B-factor comparison of WT FGF-1 and C16S/A66C/C117A/P134A mutant. Left panel: an overlay of Cα B-factors for WT FGF-1 (1JQZ, molecule A; black line) and C16S/A66C/C117A/P134A (molecule A; gray line). The location of the mutation sites (as well as the oxidized half-cystine at Cys83) is indicated. Right panel: a ΔB-factor plot scaled by the WT FGF-1 B-factor ((mutant–WT FGF-1)/WT FGF-1). The 2 graphs demonstrate broad conservation of Cα B-factors in response to mutation, with the exception of a demonstrable increase in B-factors for the region 75–80 in the C16S/A66C/C117A/P134A mutant.

Figure 3

pH versus temperature EPDs of WT FGF-1 and Cys-free disulfide mutants. EPDs were determined for WT FGF-1, C16S/A66C/C117A, C16S/A66C/C117A/P134A, and C16S/A66C/C117A/P134V Cys-free mutants. EPDs as a function of pH and temperature were constructed from FL, ANS dye binding, and SLS data in citrate-phosphate buffer for the indicated proteins (see Methods). In this tricolor diagram, the red color indicates the regime of temperature and pH stability of the native state.

Figure 4

Biological activity of WT FGF-1 and Cys-free mutants in BaF3/FGFR1c cell survival/mitogenic and NIH 3T3 fibroblast mitogenic assays. The induced cell survival/proliferation in the BaF3/FGFR-1c cell assay is quantified by radioactive ³H-thimidine incorporation (CPM). The mitogenic activity in the NIH 3T3 cell assay is determined by cell count and normalized to maximum mitogenic activity. The protein concentration is plotted as log (pg/mL) in both assays. The BaF3/FGFR-1c cell system lacks HS proteoglycan, and the assay is performed in the presence of heparin sulfate (see Methods). The NIH 3T3 cell system expresses HS proteoglycan, and the assay for WT FGF-1 was performed in the presence and absence of heparin sulfate (see Methods). The single letter amino acid code is used in this figure. Error bars are standard deviation values.