Structure-function relationship in FGF signaling: rational design of highly stable FGF2 variants and their effect on cell growth, metabolism, and survival.

Abstract

Background: Fibroblast growth factor 2 (FGF2) is a canonical member of the fibroblast growth factor family with essential roles in embryonic development, tissue regeneration, and stem cell biology, where it supports pluripotency and high proliferative capacity. Consequently, FGF2 is widely used in experimental models of regeneration and stem cell maintenance; however, its rapid thermal degradation at physiological temperatures limits its practical utility and therapeutic potential. Despite the availability of stabilized variants, developing FGF2 analogs that combine exceptional thermodynamic resilience with favorable pharmacokinetic properties remains a critical challenge for stem cell research and regenerative medicine.

Results: Using a combination of consensus sequence analysis and structure-guided protein engineering, we generated novel FGF2 variants with unprecedented thermodynamic stability and optimized extracellular matrix interactions. To enhance bioavailability, we introduced mutations that reduced affinity for heparan sulfate proteoglycans, thereby limiting sequestration within the extracellular matrix. The most stable variant exhibited a denaturation temperature increase of more than 27 °C relative to wild-type FGF2 while fully retaining mitogenic and migratory activity after 5 days of incubation at 70 °C. This exceptional thermal stability was accompanied by markedly increased resistance to proteolytic degradation. In functional cellular models, including adipocyte differentiation and stem cell culture, an optimized long-acting variants demonstrated superior metabolic efficacy and sustained signaling, enabling a reduction in dosing frequency from daily administration to once every three days.

Conclusions: We report the development of highly stable, highly bioactive FGF2 variants that retain full receptor specificity and binding affinity even under extreme conditions. By overcoming the intrinsic instability of the wild-type protein, these engineered FGF2s may enable future stem cell expansion and regenerative therapy applications.