Molecular cloning, overexpression, and characterization of the ligand-binding D2 domain of fibroblast growth factor receptor.
Fibroblast growth factors (FGFs) regulate a wide range of important cellular processes. The biological activities of FGFs are mediated by cell surface receptors (FGFRs). In the present study for the first time we report the cloning, expression, and characterization of the ligand (FGF)-binding D2 domain of human FGFR2. D2 domain is expressed in Escherichia coli in high yields (10 mg/L) as inclusion bodies. The protein is recovered by dissolving the inclusion bodies in 8 M urea and subsequently refolding on nickel affinity column. The protein is purified (to approximately 97% purity) to homogeneity using heparin-Sepharose affinity column. Far-UV circular dichroism data and chemical shift index plot based on 1H-alpha, 13C-alpha, 13C-beta, and 13carbonyl group chemical shifts suggest that D2 domain is an all beta-sheet protein consisting of 9 beta-strands. Isothermal titration calorimetry and equilibrium urea unfolding experiments show that recombinant D2 domain is in a biologically active conformation and binds strongly to its ligand (FGF) and to the heparin analog, sucrose octasulfate (SOS). Using a variety of triple resonance NMR experiments, complete assignment of 1H, 15N, and 13C resonances in D2 domain has been accomplished. The findings of the present study not only pave way for an in-depth investigation of the molecular mechanism(s) underlying the activation of FGF signaling but also provide avenues for the rational design of potent inhibitors against FGF-mediated pathogenesis.
Hung
Kuo-Wei
KW
Department of Chemistry, National Tsing Hua University, Hsinchu 300, Taiwan, ROC.
Kumar
T K S
TK
Chi
Ya-Hui
YH
Chiu
Ing-Ming
IM
Yu
Chin
C
D000595:Amino Acid Sequence; D002151:Calorimetry / Q000379:methods; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D005346:Fibroblast Growth Factors / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D020673:Protein Renaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051497:Receptor, Fibroblast Growth Factor, Type 2; D017468:Receptors, Fibroblast Growth Factor / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013395:Sucrose / Q000031:analogs & derivatives* / Q000378:metabolism; D014508:Urea / Q000737:chemistry
D017468:Receptors, Fibroblast Growth Factor; D011994:Recombinant Proteins; D013395:Sucrose; D005346:Fibroblast Growth Factors; D014508:Urea; C496353:FGFR2 protein, human; D020794:Receptor Protein-Tyrosine Kinases; D051497:Receptor, Fibroblast Growth Factor, Type 2; C053081:sucrose octasulfate