Fibroblast growth factor 2 (FGF2) is a signaling protein that plays a significant role in tissue development and repair. FGF2 binds to fibroblast growth factor receptors (FGFRs) alongside its co-factor heparin, which protects FGF2 from degradation. The binding between FGF2 and FGFRs induces intracellular signaling pathways such as RAS-MAPK, PI3K-AKT, and STAT. FGF2 has strong potential for application in cell culturing, wound healing, and cosmetics but the potential is severely limited by its low protein stability. The thermostable variant FGF2-STAB was constructed by computer-assisted protein engineering to overcome the natural limitation of FGF2. Previously reported characterization of FGF2-STAB revealed an enhanced ability to induce MAP/ERK signaling while having a lower dependence on heparin when compared with FGF2-wt. Here we report the crystal structure of FGF2-STAB solved at 1.3 Å resolution. Protein stabilization is achieved by newly formed hydrophobic interactions, polar contacts, and one additional hydrogen bond. The overall structure of FGF2-STAB is similar to FGF2-wt and does not reveal information on the experimentally observed lower dependence on heparin. A noticeable difference in flexibility in the receptor binding region can explain the differences in signaling between FGF2-STAB and its wild-type counterpart. Our structural analysis provided molecular insights into the stabilization and unique biological properties of FGF2-STAB.

Enantis Ltd Biotechnology Incubator INBIT Brno Czech Republic

Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic Prague Czech Republic

International Clinical Research Center St Anne's University Hospital Brno Czech Republic

Loschmidt Laboratories Department of Experimental Biology and RECETOX Faculty of Science Masaryk University Brno Czech Republic

See more in PubMed

Adams P.D., Afonine P.V., Bunkóczi G., Chen V.B., Davis I.W., Echols N., Headd J.J., Hung L.-W., Kapral G.J., Grosse-Kunstleve R.W., McCoy A.J., Moriarty N.W., Oeffner R., Read R.J., Richardson D.C., Richardson J.S., Terwilliger T.C., Zwart P.H. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr. 2010;66:213–221. doi: 10.1107/S0907444909052925. PubMed DOI PMC

Amit M., Carpenter M.K., Inokuma M.S., Chiu C.-P., Harris C.P., Waknitz M.A., Itskovitz-Eldor J., Thomson J.A. Clonally derived human embryonic stem cell lines maintain pluripotency and proliferative potential for prolonged periods of culture. Dev. Biol. 2000;227:271–278. doi: 10.1006/dbio.2000.9912. PubMed DOI

Anandakrishnan R., Aguilar B., Onufriev A.V. H++ 3.0: automating pK prediction and the preparation of biomolecular structures for atomistic molecular modeling and simulations. Nucleic Acids Res. 2012;40:W537–W541. doi: 10.1093/nar/gks375. PubMed DOI PMC

Barrientos S., Brem H., Stojadinovic O., Tomic-Canic M. Clinical application of growth factors and cytokines in wound healing. Wound Repair Regen. 2014;22:569–578. doi: 10.1111/wrr.12205. PubMed DOI PMC

Bednar D., Beerens K., Sebestova E., Bendl J., Khare S., Chaloupkova R., Prokop Z., Brezovsky J., Baker D., Damborsky J. FireProt: energy- and evolution-based computational design of thermostable multiple-point mutants. PLoS Comput. Biol. 2015;11 doi: 10.1371/journal.pcbi.1004556. PubMed DOI PMC

Beerens K., Mazurenko S., Kunka A., Marques S.M., Hansen N., Musil M., Chaloupkova R., Waterman J., Brezovsky J., Bednar D., Prokop Z., Damborsky J. Evolutionary analysis as a powerful complement to energy calculations for protein stabilization. ACS Catal. 2018;8:9420–9428. doi: 10.1021/acscatal.8b01677. DOI

Belov A.A., Mohammadi M. Molecular mechanisms of fibroblast growth factor signaling in physiology and pathology. Cold Spring Harb Perspect Biol. 2013;5 doi: 10.1101/cshperspect.a015958. PubMed DOI PMC

Bikfalvi A., Klein S., Pintucci G., Rifkin D.B. Biological roles of fibroblast growth factor-2. Endocr. Rev. 1997;18:26–45. doi: 10.1210/edrv.18.1.0292. PubMed DOI

Buchtova M., Chaloupkova R., Zakrzewska M., Vesela I., Cela P., Barathova J., Gudernova I., Zajickova R., Trantirek L., Martin J., Kostas M., Otlewski J., Damborsky J., Kozubik A., Wiedlocha A., Krejci P. Instability restricts signaling of multiple fibroblast growth factors. Cell Mol Life Sci. 2015;72:2445–2459. doi: 10.1007/s00018-015-1856-8. PubMed DOI PMC

Caldararu O., Kumar R., Oksanen E., Logan D.T., Ryde U. Are crystallographic B-factors suitable for calculating protein conformational entropy? Phys Chem Chem Phys. 2019;21:18149–18160. doi: 10.1039/c9cp02504a. PubMed DOI

Caldwell M.A., Garcion E., terBorg M.G., He X., Svendsen C.N. Heparin stabilizes FGF-2 and modulates striatal precursor cell behavior in response to EGF. Exp Neurol. 2004;188:408–420. doi: 10.1016/j.expneurol.2004.05.007. PubMed DOI

Case D.A., Aktulga H.M., Belfon K., Cerutti D.S., Cisneros G.A., Cruzeiro V.W.D., Forouzesh N., Giese T.J., Götz A.W., Gohlke H., Izadi S., Kasavajhala K., Kaymak M.C., King E., Kurtzman T., Lee T.-S., Li P., Liu J., Luchko T., Luo R., Manathunga M., Machado M.R., Nguyen H.M., O’Hearn K.A., Onufriev A.V., Pan F., Pantano S., Qi R., Rahnamoun A., Risheh A., Schott-Verdugo S., Shajan A., Swails J., Wang J., Wei H., Wu X., Wu Y., Zhang S., Zhao S., Zhu Q., Cheatham T.E.I., Roe D.R., Roitberg A., Simmerling C., York D.M., Nagan M.C., Merz K.M., Jr. Ambertools. J Chem Inf Model. 2023;63:6183–6191. doi: 10.1021/acs.jcim.3c01153. PubMed DOI PMC

Cerutti D.S., Swope W.C., Rice J.E., Case D.A. ff14ipq: A self-consistent force field for condensed-phase simulations of proteins. J Chem Theory Comput. 2014;10:4515–4534. doi: 10.1021/ct500643c. PubMed DOI PMC

Chen V.B., Arendall W.B., Headd J.J., Keedy D.A., Immormino R.M., Kapral G.J., Murray L.W., Richardson J.S., Richardson D.C. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D Biol Crystallogr. 2010;66:12–21. doi: 10.1107/S0907444909042073. PubMed DOI PMC

Chen G., Gulbranson D.R., Yu P., Hou Z., Thomson J.A. Thermal stability of fibroblast growth factor protein is a determinant factor in regulating self-renewal, differentiation, and reprogramming in human pluripotent stem cells. Stem Cells. 2012;30:623–630. doi: 10.1002/stem.1021. PubMed DOI PMC

Delehedde M., Lyon M., Gallagher J., Rudland P.S., Fernig D.G. Fibroblast growth factor-2 binds to small heparin-derived oligosaccharides and stimulates a sustained phosphorylation of p42/44 mitogen-activated protein kinase and proliferation of rat mammary fibroblasts. Biochem J. 2002;366:235–244. doi: 10.1042/bj20011718. PubMed DOI PMC

Dvorak P., Bednar D., Vanacek P., Balek L., Eiselleova L., Stepankova V., Sebestova E., Kunova Bosakova M., Konecna Z., Mazurenko S., Kunka A., Vanova T., Zoufalova K., Chaloupkova R., Brezovsky J., Krejci P., Prokop Z., Dvorak P., Damborsky J. Computer-assisted engineering of hyperstable fibroblast growth factor 2. Biotechnol Bioeng. 2018;115:850–862. doi: 10.1002/bit.26531. PubMed DOI

Dvorak, Petr, Krejci, P., Balek, L., Eiselleova, L., Konecna, Z., Dvorak, Pavel, Bednar, D., Brezovsky, J., Sebestova, E., Chaloupkova, R., Stepankova, V., Vanacek, P., Prokop, Z., Damborsky, J., Bosakova, M, M., 2017. Thermostable fgf2 polypeptide, use thereof. WO2017089016A1.

Elber R., Ruymgaart A.P., Hess B. SHAKE parallelization. Eur Phys J Spec Top. 2011;200:211–223. doi: 10.1140/epjst/e2011-01525-9. PubMed DOI PMC

Emsley P., Lohkamp B., Scott W.G., Cowtan K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr. 2010;66:486–501. doi: 10.1107/S0907444910007493. PubMed DOI PMC

Eriksson A.E., Cousens L.S., Matthews B.W. Refinement of the structure of human basic fibroblast growth factor at 1.6 A resolution and analysis of presumed heparin binding sites by selenate substitution. Protein Sci. 1993;2:1274–1284. doi: 10.1002/pro.5560020810. PubMed DOI PMC

Fannon M., Nugent M.A. Basic fibroblast growth factor binds its receptors, is internalized, and stimulates DNA synthesis in Balb/c3T3 cells in the absence of heparan sulfate. J Biol Chem. 1996;271:17949–17956. doi: 10.1074/jbc.271.30.17949. PubMed DOI

Götz A.W., Williamson M.J., Xu D., Poole D., Le Grand S., Walker R.C. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 1. Generalized Born. J. Chem. Theory Comput. 2012;8:1542–1555. doi: 10.1021/ct200909j. PubMed DOI PMC

Harvey M.J., De Fabritiis G. An implementation of the smooth particle mesh ewald method on GPU hardware. J Chem Theory Comput. 2009;5:2371–2377. doi: 10.1021/ct900275y. PubMed DOI

Hui Q., Jin Z., Li X., Liu C., Wang X. FGF family: from drug development to clinical application. Int J Mol Sci. 2018;19:1875. doi: 10.3390/ijms19071875. PubMed DOI PMC

Ibrahimi O.A., Zhang F., Lang Hrstka S.C., Mohammadi M., Linhardt R.J. Kinetic model for FGF, FGFR, and proteoglycan signal transduction complex assembly. Biochemistry. 2004;43:4724–4730. doi: 10.1021/bi0352320. PubMed DOI

Javerzat S., Auguste P., Bikfalvi A. The role of fibroblast growth factors in vascular development. Trends Mol Med. 2002;8:483–489. doi: 10.1016/s1471-4914(02)02394-8. PubMed DOI

Karplus P.A., Diederichs K. Linking crystallographic model and data quality. Science. 2012;336:1030–1033. doi: 10.1126/science.1218231. PubMed DOI PMC

Koledova Z., Sumbal J., Rabata A., Bourdonnaye G.de.L., Chaloupkova R., Hrdlickova B., Damborsky J., Stepankova V. Fibroblast growth factor 2 protein stability provides decreased dependence on heparin for induction of FGFR signaling and alters ERK signaling dynamics. Front Cell Dev Biol. 2019;7:331. doi: 10.3389/fcell.2019.00331. PubMed DOI PMC

La Venuta G., Zeitler M., Steringer J.P., Müller H.-M., Nickel W. The startling properties of fibroblast growth factor 2: how to exit mammalian cells without a signal peptide at hand. J Biol Chem. 2015;290:27015–27020. doi: 10.1074/jbc.R115.689257. PubMed DOI PMC

Levenstein M.E., Ludwig T.E., Xu R.-H., Llanas R.A., VanDenHeuvel-Kramer K., Manning D., Thomson J.A. Basic fibroblast growth factor support of human embryonic stem cell self-renewal. Stem Cells. 2006;24:568–574. doi: 10.1634/stemcells.2005-0247. PubMed DOI PMC

Lotz S., Goderie S., Tokas N., Hirsch S.E., Ahmad F., Corneo B., Le S., Banerjee A., Kane R.S., Stern J.H., Temple S., Fasano C.A. Sustained levels of FGF2 maintain undifferentiated stem cell cultures with biweekly feeding. PLoS One. 2013;8 doi: 10.1371/journal.pone.0056289. PubMed DOI PMC

Mueller U., Förster R., Hellmig M., Huschmann F.U., Kastner A., Malecki P., Pühringer S., Röwer M., Sparta K., Steffien M., Ühlein M., Wilk P., Weiss M.S. The macromolecular crystallography beamlines at BESSY II of the Helmholtz-Zentrum Berlin: Current status and perspectives. Eur Phys J Plus. 2015;130:141–150. doi: 10.1140/epjp/i2015-15141-2. DOI

Murshudov G.N., Skubák P., Lebedev A.A., Pannu N.S., Steiner R.A., Nicholls R.A., Winn M.D., Long F., Vagin A.A. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr D Biol Crystallogr. 2011;67:355–367. doi: 10.1107/S0907444911001314. PubMed DOI PMC

Murzin A.G., Lesk A.M., Chothia C. beta-Trefoil fold. Patterns of structure and sequence in the Kunitz inhibitors interleukins-1 beta and 1 alpha and fibroblast growth factors. J Mol Biol. 1992;223:531–543. doi: 10.1016/0022-2836(92)90668-a. PubMed DOI

Nugent M.A., Edelman E.R. Kinetics of basic fibroblast growth factor binding to its receptor and heparan sulfate proteoglycan: a mechanism for cooperactivity. Biochemistry. 1992;31:8876–8883. doi: 10.1021/bi00152a026. PubMed DOI

Nugent M.A., Iozzo R.V. Fibroblast growth factor-2. Int J Biochem Cell Biol. 2000;32:115–120. doi: 10.1016/S1357-2725(99)00123-5. PubMed DOI

Ornitz D.M., Itoh N. The fibroblast growth factor signaling pathway. Wiley Interdiscip Rev Dev Biol. 2015;4:215–266. doi: 10.1002/wdev.176. PubMed DOI PMC

Paluck S.J., Nguyen T.H., Lee J.P., Maynard H.D. A Heparin-mimicking block copolymer both stabilizes and increases the activity of fibroblast growth factor 2 (FGF2) Biomacromolecules. 2016;17:3386–3395. doi: 10.1021/acs.biomac.6b01182. PubMed DOI PMC

Plotnikov A.N., Schlessinger J., Hubbard S.R., Mohammadi M. Structural basis for FGF receptor dimerization and activation. Cell. 1999;98:641–650. doi: 10.1016/s0092-8674(00)80051-3. PubMed DOI

Plotnikov A.N., Hubbard S.R., Schlessinger J., Mohammadi M. Crystal structures of two FGF-FGFR complexes reveal the determinants of ligand-receptor specificity. Cell. 2000;101:413–424. doi: 10.1016/s0092-8674(00)80851-x. PubMed DOI

Salomon-Ferrer R., Götz A.W., Poole D., Le Grand S., Walker R.C. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 2. explicit solvent particle mesh ewald. J Chem Theory Comput. 2013;9:3878–3888. doi: 10.1021/ct400314y. PubMed DOI

Vagin A., Teplyakov A. Molecular replacement with MOLREP. Acta Crystallogr D Biol Crystallogr. 2010;66:22–25. doi: 10.1107/S0907444909042589. PubMed DOI

Winn M.D., Ballard C.C., Cowtan K.D., Dodson E.J., Emsley P., Evans P.R., Keegan R.M., Krissinel E.B., Leslie A.G.W., McCoy A., McNicholas S.J., Murshudov G.N., Pannu N.S., Potterton E.A., Powell H.R., Read R.J., Vagin A., Wilson K.S. Overview of the CCP4 suite and current developments. Acta Crystallogr D Biol Crystallogr. 2011;67:235–242. doi: 10.1107/S0907444910045749. PubMed DOI PMC

Xu C., Rosler E., Jiang J., Lebkowski J.S., Gold J.D., O’Sullivan C., Delavan-Boorsma K., Mok M., Bronstein A., Carpenter M.K. Basic fibroblast growth factor supports undifferentiated human embryonic stem cell growth without conditioned medium. Stem Cells. 2005;23:315–323. doi: 10.1634/stemcells.2004-0211. PubMed DOI

Yun Y.-R., Won J.E., Jeon E., Lee S., Kang W., Jo H., Jang J.-H., Shin U.S., Kim H.-W. Fibroblast growth factors: biology, function, and application for tissue regeneration. J Tissue Eng. 2010;2010 doi: 10.4061/2010/218142. PubMed DOI PMC

Zakrzewska M., Krowarsch D., Wiedlocha A., Olsnes S., Otlewski J. Highly stable mutants of human fibroblast growth factor-1 exhibit prolonged biological action. J Mol Biol. 2005;352:860–875. doi: 10.1016/j.jmb.2005.07.066. PubMed DOI

Zehe C., Engling A., Wegehingel S., Schäfer T., Nickel W. Cell-surface heparan sulfate proteoglycans are essential components of the unconventional export machinery of FGF-2. Proc Natl Acad Sci U S A. 2006;103:15479–15484. doi: 10.1073/pnas.0605997103. PubMed DOI PMC

Zhang J.D., Cousens L.S., Barr P.J., Sprang S.R. Three-dimensional structure of human basic fibroblast growth factor, a structural homolog of interleukin 1 beta. Proc Natl Acad Sci U S A. 1991;88:3446–3450. doi: 10.1073/pnas.88.8.3446. PubMed DOI PMC

Zhu H., Duchesne L., Rudland P.S., Fernig D.G. The heparan sulfate co-receptor and the concentration of fibroblast growth factor-2 independently elicit different signalling patterns from the fibroblast growth factor receptor. Cell Commun Signal. 2010;8:14. doi: 10.1186/1478-811X-8-14. PubMed DOI PMC