Dynamic folding modulation generates FGF21 variant against diabetes

Article9 December 2020free access Transparent process Dynamic folding modulation generates FGF21 variant against diabetes Lei Zhu Lei Zhu orcid.org/0000-0001-5003-9059 High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, ChinaThese authors contributed equally to this work Search for more papers by this author Hongxin Zhao Hongxin Zhao High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, ChinaThese authors contributed equally to this work Search for more papers by this author Juanjuan Liu Juanjuan Liu High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, ChinaThese authors contributed equally to this work Search for more papers by this author Hao Cai Hao Cai School of Biotechnology & Food Engineering, Hefei University of Technology, Hefei, China Search for more papers by this author Bo Wu Bo Wu High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, China Search for more papers by this author Zhijun Liu Zhijun Liu High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, China Search for more papers by this author Shu Zhou Shu Zhou High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, China Search for more papers by this author Qingsong Liu Qingsong Liu High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, China Search for more papers by this author Xiaokun Li Xiaokun Li Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China Search for more papers by this author Bin Bao Bin Bao Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China Search for more papers by this author Jian Liu Jian Liu orcid.org/0000-0002-3220-1240 University of Science and Technology of China, Hefei, China Search for more papers by this author Han Dai Corresponding Author Han Dai [email protected] orcid.org/0000-0001-9234-8386 High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, China Search for more papers by this author Junfeng Wang Corresponding Author Junfeng Wang [email protected] orcid.org/0000-0002-9608-6851 High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, China Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China Institute of Physical Science and Information Technology, Anhui University, Hefei, China Search for more papers by this author Lei Zhu Lei Zhu orcid.org/0000-0001-5003-9059 High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, ChinaThese authors contributed equally to this work Search for more papers by this author Hongxin Zhao Hongxin Zhao High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, ChinaThese authors contributed equally to this work Search for more papers by this author Juanjuan Liu Juanjuan Liu High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, ChinaThese authors contributed equally to this work Search for more papers by this author Hao Cai Hao Cai School of Biotechnology & Food Engineering, Hefei University of Technology, Hefei, China Search for more papers by this author Bo Wu Bo Wu High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, China Search for more papers by this author Zhijun Liu Zhijun Liu High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, China Search for more papers by this author Shu Zhou Shu Zhou High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, China Search for more papers by this author Qingsong Liu Qingsong Liu High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, China Search for more papers by this author Xiaokun Li Xiaokun Li Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China Search for more papers by this author Bin Bao Bin Bao Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China Search for more papers by this author Jian Liu Jian Liu orcid.org/0000-0002-3220-1240 University of Science and Technology of China, Hefei, China Search for more papers by this author Han Dai Corresponding Author Han Dai [email protected] orcid.org/0000-0001-9234-8386 High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, China Search for more papers by this author Junfeng Wang Corresponding Author Junfeng Wang [email protected] orcid.org/0000-0002-9608-6851 High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, China Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China Institute of Physical Science and Information Technology, Anhui University, Hefei, China Search for more papers by this author Author Information Lei Zhu1, Hongxin Zhao1, Juanjuan Liu1, Hao Cai2, Bo Wu1, Zhijun Liu1, Shu Zhou1, Qingsong Liu1, Xiaokun Li3, Bin Bao3, Jian Liu4, Han Dai *,1 and Junfeng Wang *,1,3,5 1High Magnetic Field Laboratory, CAS Key Laboratory of High Magnetic Field and Ion Beam Physical Biology, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei, China 2School of Biotechnology & Food Engineering, Hefei University of Technology, Hefei, China 3Chemical Biology Research Center, School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, China 4University of Science and Technology of China, Hefei, China 5Institute of Physical Science and Information Technology, Anhui University, Hefei, China *Corresponding author. Tel: +86 18210120401; E-mail: [email protected] *Corresponding author. Tel: +86 55165591878; E-mail: [email protected] EMBO Reports (2021)22:e51352https://doi.org/10.15252/embr.202051352 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Fibroblast growth factor 21 (FGF21) is a regulator of glucose and lipid metabolism. It has been widely considered as a promising candidate for the treatment of type 2 diabetes mellitus (T2DM) and other related metabolic disorders. However, lack of structural and dynamic information has limited FGF21-based drug development. Here, using nuclear magnetic resonance (NMR) spectroscopy, we determine the structure of FGF21 and find that its non-canonical flexible β-trefoil conformation affects the folding of β2-β3 hairpin and further overall protein stability. To modulate folding dynamics, we designed an FGF21-FGF19 chimera, FGF21SS. As expected, FGF21SS shows better thermostability without inducing hepatocyte proliferation. Functional characterization of FGF21SS shows its better insulin sensitivity, reduced inflammation in 3T3-L1 adipocytes, and lower blood glucose and insulin levels in ob/ob mice compared with wild type. Our dynamics-based rational design provides a promising approach for FGF21-based therapeutic development against T2DM. Synopsis NMR structural analysis of FGF21 reveals a flexible conformation that affects heparan sulfate binding and β2-β3 hairpin folding dynamics. A folding dynamic modulated variant, FGF21SS, improves protein thermostability and anti-diabetic activity. FGF21 structure reveals a non-canonical flexible β-trefoil conformation that is unfavorable for heparan sulfate and receptor interaction. The untypical β10-β11 region of FGF21 affects the folding dynamics of the β2-β3 hairpin and overall protein stability. Dynamic folding modulation of the β2-β3 hairpin by SS bond mutation and loop replacement improves FGF21 variant’s thermostability and anti-diabetic activity. Introduction FGF21, a member of the FGF family, exerts diverse pharmacological effects on the regulation of glucose homeostasis, lipid metabolism, and insulin sensitivity. Administration of FGF21 leads to reduction of circulating glucose, triglycerides, insulin levels and body weight, as well as improvement of insulin sensitivity, energy metabolism, and amelioration of hepatic steatosis in diabetic rodents and non-human primate (Kharitonenkov et al, 2005; Badman et al, 2007; Kharitonenkov et al, 2007; Dutchak et al, 2012; Schlein et al, 2016; BonDurant et al, 2017; Li et al, 2018). Besides, FGF21 prevents the apoptosis of insulin-producing INS1E cells induced by glucolipotoxicity and cytokine and promotes the survival of pancreatic β-cell from diabetic rodents (Wente et al, 2006). Extensive researches have suggested FGF21 as a promising therapy for T2DM, obesity, non-alcoholic steatohepatitis (NASH), and related metabolic disorders (Degirolamo et al, 2016; Potthoff, 2017; Tucker et al, 2019; Geng et al, 2020). The human FGF21 is a 181-amino-acid secreted FGF19 subfamily protein. Unlike classical paracrine-acting FGFs, which signal by interacting with FGF receptors (FGFRs) in the presence of heparan sulfate (HS) proteoglycan, the FGF19 subfamily members achieve their functions in an endocrine fashion. FGF21 requires β-klotho, a single transmembrane glycoprotein, as a scaffold for its binding to FGFR (Chen et al, 2018; Lee et al, 2018). Therefore, FGF21 performs its metabolic activities in liver, pancreas, adipocytes, and neurons, where the expression level of β-klotho is high (Fon Tacer et al, 2010). The native human FGF21 protein exhibits poor pharmacokinetics (PK) properties, resulting in a great challenge for clinical application (Degirolamo et al, 2016; Potthoff, 2017; Tucker et al, 2019; Geng et al, 2020). To improve the protein activity, stability, and solubility, different engineering approaches have been developed. For example, Xu et al described an Fc tag fused variant, Fc-FGF21(RG), to improve PK, in which they mutated Leu98Arg to resist aggregation and Pro171Gly to impede the C-terminal proteolytic cleavage at Pro171 (Hecht et al, 2012). Kharitonenkov et al designed LY2405319, in which an additional disulfide bond (SS bond) was added at Leu118Cys-Ala134Cys to stabilize the predicted loop between β10 and β12 (Kharitonenkov et al, 2013). Lee et al designed Arg185Trp/Leu166Phe double mutation based on the structure of β-klotho and FGF21 C-terminus complex, to enhance the binding of FGF21 with β-klotho but no in vivo activity result was reported (Lee et al, 2018). Besides, a variety of modifications have been utilized to extend the FGF21 half-life, including Fc fusion (Hecht et al, 2012; Stanislaus et al, 2017), scaffold antibody conjugation (Talukdar et al, 2016), pegylation (Huang et al, 2011; Mu et al, 2012; Xu et al, 2013; Song et al, 2014; Ye et al, 2014), and glycosylation (Weng et al, 2018). However, most of the designing strategies of FGF21 variants so far were limited in protein fusion or modification combined with empirical mutations. Although some variants enhance the protein PK property, they showed limited anti-diabetic therapeutic improvement in vivo. The main obstacle of FGF21 drug development is the lack of structural information of FGF21. Here, a FGF21 structure was determined using NMR spectroscopy and the dynamic property was also characterized. We found that the flexible non-canonical β-trefoil conformation in the β10-β12 region of FGF21 severely affects the folding stability of the neighboring β2-β3 hairpin. The understanding of structural and folding dynamic property of FGF21 enables a rational design of a novel FGF21 variant, with significantly increased anti-diabetic activities. Results Solution structure of FGF21 core region FGF21 belongs to the endocrine FGF19 subfamily. Structural prediction based on homologues reveals that FGF21 contains about 13 N-terminal and 40 C-terminal residues extended random coil regions (FGF21CT) flanking a β-trefoil core domain (FGF21core, residues 14–141), which shares only 38% and 33% sequence identity with other subfamily members FGF19 and FGF23, respectively (Fig EV1). NMR spectral comparison shows that FGF21core has a similar overall fold compared with full-length protein, but less spectral overlap and better T2 relaxation (Fig EV2). Therefore, we determined FGF21core structure using standard multidimensional heteronuclear solution NMR spectroscopy (Fig 1A and B, and Table EV1). The FGF21core structure ensemble shows a converged non-canonical β-trefoil fold, which contains 11 β-strands (residues 17–22 (β1), 32–36 (β2), 40–44 (β3), 53–58 (β4), 62–67 (β5), 72–77 (β6), 81–85 (β7), 95–100 (β8), 104–109 (β9), 114–117 (β10), and 136–139 (β12)). Strand β11 of canonical β-trefoil conformation is missing in FGF21, and β10 and β12 are linked by an 18-residue proline-rich disordered random coil (residues 118–135), for which few inter-residue Nuclear Overhauser Effect (NOE) restraints are available. Click here to expand this figure. Figure EV1. Structural-based sequence alignment of FGF21, FGF19, FGF23, and selected paracrine-acting FGFs Predicted signal peptide sequences were omitted. Residue numbers indicates the sequence of mature FGF21. The locations and lengths of the secondary structure elements are indicated by boxes in the sequences. Cysteine residues that form disulfide bonds are highlighted on a yellow background. Glycine and threonine residues of the GXXXXGXX(T/S) motif are highlighted on an orange background. Residues strictly conserved are highlighted on a red background. Similar residues are rendered as red characters with blue frames. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. 2D 1H-15N Heteronuclear Single Quantum Correlation (HSQC) spectra of FGF21 (blue) and FGF21core (red) Backbone amide resonance assignments were indicated with one-letter amino acid code and sequence number. Arrows indicate the peak shifts upon the truncation of N- and C-termini in FGF21core. Download figure Download PowerPoint Figure 1. Solution structure of FGF21core Ribbon representation of 10 energy-refined structure ensemble of FGF21core. Cartoon representation of a representative structure of FGF21core. β sheets for the barrel and triangular array regions were colored pink and cyan, respectively. Download figure Download PowerPoint Structural features of FGF21 for low HS-binding affinity Unlike classical paracrine-acting FGFs, which interact with FGF receptors (FGFRs) in the presence of HS, FGF19 subfamily members have weak binding affinity for both HS and their cognate FGFRs. For FGF21, which diffuses from the HS-rich secretory tissues and performs its function as an endocrine hormone, it interacts with cognate receptor in the assistance of β-klotho (Goetz et al, 2007; Ogawa et al, 2007; Kharitonenkov et al, 2008; Suzuki et al, 2008; Beenken & Mohammadi, 2012; Ding et al, 2012; Yie et al, 2012; Chen et al, 2018; Lee et al, 2018). The low HS-binding affinity of FGF21 results from the aberrant conformation of FGF21 β10-β12 region. For details, a typical FGF molecule like FGF2 consists of a six-stranded antiparallel β-barrel closed off on one end by a triangular array, where the arrangement of three β-hairpins (the edges are named clock-wisely as β2-β3 hairpin, β6-β7 hairpin and β10-β11 hairpin) gives the molecule a pseudo 3-fold symmetry (Fig 2A, left panel). HS binds to the positively charged groove formed by β10-β11 hairpin and β1-β2 loop (Fig 2A, left panel, and Fig 3). A conserved GXXXXGXX(T/S) motif termed glycine box within β10-β11 hairpin (Fig EV1) interacts with HS by forming tight hydrogen bonds using the sidechains of Arg/Lys and backbone atoms (Luo et al, 1998; Plotnikov et al, 2000). In the case of FGF21, instead of a β-hairpin and/or glycine box, it forms a disordered loop stretching out of the triangular array (Fig 2A, middle panel). The steric hindrance therefore reduces the HS binding to the disrupted FGF21 β10-β11 segment (similar to the cases in FGF19 and FGF23; Goetz et al, 2007; Fig EV3). Moreover, the HS-binding region of FGF21 is primarily more negatively charged than other FGFs; thus, electrostatic force will further repel HS containing highly acidic sulfate groups (Fig 3). The low receptor and HS-binding affinity of FGF21 are of great significance to understand the endocrine properties of FGF21 and the β-klotho-dependent signal transduction mechanism. Figure 2. Dynamics caused folding instability of FGF21 in the triangular array region Triangular array region of FGF2 (PDB ID: 2FGF), FGF21core (β conformation) and FGF21core (loop conformation). The edges of the triangular formed by hairpins or missed (colored magenta) are indicated by solid or dashed lines, respectively. 2D 1H-15N Heteronuclear Single Quantum Correlation (HSQC) spectrum showing the two sets of assignments for residue Tyr22 to Glu50 (labeled by one-letter amino acid code and sequence number). Labels for the loop conformation are colored red and marked with asterisks. 15N T1 and T2 relaxation times of the residues in β2-β3 region. The secondary structure of FGF21core (β conformation) is shown on the top. Error bars show the fitting SDs of ten different experiments. Secondary structure prediction of residues in β2-β3 region using TALOS + program. The probability of β-strand and random coil of the residues are shown by histogram. 1H-1H slices of 15N-edited 3D NOESY-HSQC spectrum for β2-β3 residues. Residue codes are labeled as in (B). Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Superimposition of the FGF21core structure (cyan) with that of FGF2 (blue, PDB ID: 2FGF), FGF19 (orange, PDB ID: 2P23), and FGF23 (yellow, PDB ID: 2P39) shown as a ribbon representation The predicted heparin binding domain is illustrated as a cartoon representation and is enlarged in the right panel separately. Download figure Download PowerPoint Figure 3. Heparin binding sites of FGF21 differ from that of paracrine-acting and other endocrine-acting FGFs Electrostatic potential surface of different FGFs for representing the (HS) binding sites. HS is placed by superimposition of FGF21core, FGF9 (PDB ID: 1IHK), FGF10 (PDB ID: 1NUN), FGF19 (PDB ID: 2P23), and FGF23 (PDB ID: 2P39) onto the FGF2/FGFR1c/HS ternary complex structure (PDB ID: 1FQ9). HS is shown as a stick representation. The positive and negative charges on the protein surface are colored blue and red, respectively. Download figure Download PowerPoint Highly dynamic folding property of β2-β3 hairpin The untypical structural feature of FGF21 is not limited in the β10-β11 region. Our data also revealed that adjacent β2-β3 hairpin and the neighboring loop segments (residues Tyr22 to Glu50) exhibit highly dynamic folding characteristic. For both FGF21core and full-length FGF21, a second set of resonances were observed for the residues in this region, whose 1H chemical shifts distribute mostly in the range of 7.7–8.6 ppm (Fig 2B and Appendix Fig S1), indicating the existence of a second conformation that is dynamically transformed on a slow time scale. Structural modeling based on the TALOS + derived dihedral angles (Fig 2D) and NOE restraints (Fig 2E) yielded a disordered loop conformation (Fig 2A, right panel). Compared with β conformation, the loop conformation displays shorter 15N T1 and longer 15N T2 relaxation times, indicating its higher flexibility (Fig 2C). The destruction of β2-β3 hairpin will further decrease the stability of the triangular array architecture of FGF21 and obviously has a significant impact on the overall protein stability in vitro and in vivo. More importantly, the binding of FGF21 to receptor would be disturbed because the loop conformation affects the β1-β2 loop, which directly contacts with D2 domain of FGFR1c and HS as shown in the receptor interaction model (Fig 4). Therefore, we anticipate that this highly dynamic folding property of FGF21 is unfavorable for the interaction with receptor and would help its escape from local extracellular matrix as an endocrine hormone. Figure 4. Interaction model of 2:2:2:2 FGF21/FGFR1c/β-klotho/HS complex This model is obtained by superimposition of FGF21core structure with the crystal structures of β-klotho/FGF21-CT (PDB ID: 5VAQ), FGF23/FGFR1c/α-klotho (PDB ID: 5W21) and FGF2/FGFR1c/HS dimer (PDB ID: 1FQ9), and is shown as surface representation. FGF21core is colored orange. β1-β2 loop of FGF21 (Asp24 to Ala31) directly interacting with FGFR1c is colored magenta. FGF21CT binding to β-klotho is colored blue. FGFR1c is colored green. D1, D2 domain, and the receptor binding arm of β-klotho are colored cyan, lightblue, and pink, respectively. Download figure Download PowerPoint Design and structural characterization of a stable FGF21 variant The dynamic folding property and associated protein instability of FGF21 may be important for the regulation of hormone release and degradation in the body; nevertheless, the resulted poor PK property of native human FGF21 leads to a great challenge for its clinical application (Degirolamo et al, 2016; Potthoff, 2017; Tucker et al, 2019; Geng et al, 2020). Among protein engineering strategies, SS bonds between strands have turned out to be effective to stabilize β hairpin conformation (Zhang et al, 1994; Lee & Blaber, 2009; Moore et al, 2017). SS bonds appear naturally in the triangular array region of endocrine FGFs. For example, a conserved SS bond in FGF19 subfamily (formed by Cys75 and Cys93 in FGF21) helps the tether of β6-β7 hairpin to the barrel, and loss of this SS bond greatly impaired the activity of FGF21 (Luo et al, 2019). FGF19 has an additional SS bond between strands of β2-β3 hairpin (Goetz et al, 2007). Attempts to introduce SS bond to FGF21 in the β10-β12 loop were also carried out by other researchers. Unfortunately, these efforts did not significantly improve FGF21 stability and function (Kharitonenkov et al, 2013). The availability of FGF21 structure makes it more feasible to design a better therapeutic FGF21 variant. Here, we constructed a novel FGF21-FGF19 chimera—FGF21SS to enhance the protein stability while preserving its major binding feature to FGFR, β-Klotho and HS. First, to stabilize β2-β3 hairpin, Gly43 in β3 was mutated to cysteine (Cys43#, residues in FGF21SS with different sequence compared with that in FGF21 are marked with #) to form SS bond with Ala31Cys (Cys31#) in β2 strand. Secondly, considering that the conformation of HS/receptor binding β1-β2 loop would be disturbed upon the SS bond formation, β1-β2 loop (DDAQQTEA) was replaced by the longer FGF19 loop (SGPHGLSSC) to retain its binding to FGFR (Fig 5A). Figure 5. Dynamic folding modulation design and structural analysis of FGF21SS variant Sequence alignment of FGF21, FGF21SS, and FGF19 in the mutated region. The mutation sites are indicated by boxes. Cysteines for disulfide bond formation are highlighted on a yellow background. The secondary structure of FGF21 is shown on the top. The N-terminal residue IDs of FGF21SS are indicated under the sequence. # is used to identify the mutated residues (codes are colored blue) and the same residues having different sequence number in FGF21SS from wild type. 2D 1H-15N HSQC spectra comparison of FGF21core (black) and FGF21SS/core (red) revealing the elimination of the loop conformation in FGF21SS. Assignments for FGF21core and FGF21SS/core are labeled as in Figs 2B and 5A, respectively. Temperature denaturation CD experiments indicating the significant increase of thermostability. CD value was recorded and normalized at 222 nm. Tm value of the proteins is fitted using Boltzmann function. Superimposition of FGF21SS/core (skyblue) and FGF21core (green) structure ensembles (10 energy-refined structures each) in ribbon representation showing the structure conservation upon SS bond mutation. Cartoon representation of FGF21SS/core. Cys31#-Cys43# (between β2 and β3) and Cys75-Cys93 (between β6 and β9) SS bonds are highlighted and shown as stick representations. Download figure Download PowerPoint The conformation of FGF21SS after mutagenesis was analyzed using NMR spectroscopy. 2D 1H-15N HSQC spectra and backbone atom assignments showed that FGF21SS exists in a single conformation (Figs 5B and EV4). 13Cβ chemical shifts of Cys31# and Cys43# were identified to 44.266 ppm and 44.427 ppm, respectively, indicating the oxidized form of cysteines. Liquid chromatography-mass spectrometry (LC-MS) also demonstrated the existence of SS bond between Cys31# and Cys43# (Appendix Fig S2). We further determined the solution structure of FGF21SS/core and found that the structure of FGF21SS/core is virtually identical to that of wild-type FGF21core, with an overall Cα root mean square deviation (r.m.s.d.) value of 0.198 Å excluding the disordered regions (β1-β2 loop and β10-β12 loop) (Fig 5D and E, and Table EV1). The sidechain orientation and inter-thiol distance of Cys31# and Cys43# support the SS bond formation (Fig 5E). As expected, FGF21SS showed significantly higher melting temperature (Tm) than wild type in temperature denaturation circular dichroism (CD) experiments (98.2 ± 0.3°C vs. 69.3 ± 0.3°C), indicating higher protein thermostability (Fig 5C). Click here to expand this figure. Figure EV4. Backbone assignments of FGF21SS/core specifying one set of resonances # is used to identify the mutated residues (whose labels are colored blue) and the same residues having different sequence number in FGF21SS compared with FGF21. Download figure Download PowerPoint Functional characterization of FGF21SS The carcinogenic risk of the chimera protein needs to be evaluated first, as administration of FGF19 has been reported to increase hepatocyte proliferation (Nicholes et al, 2002). After a 6-day injection of vehicle or 0.6 mg/kg/day proteins in ICR mice, the hepatic cell proliferation was detected by immunohistochemical analysis using BrdU assay. As shown in Fig EV5, while FGF19 obviously induced the mitosis of hepatocyte, both FGF21 and FGF21SS did not show any mitogenic activity, indicating excellent safety profile. Click here to expand this figure. Figure EV5. Representative histological analysis of livers indicating the nontumorigenic feature of FGF21SS and FGF21 ICR mice were injected every day with vehicle or 0.6 mg/kg FGF21, FGF21SS and FGF19 for 6 days, and the hepatocyte proliferation was estimated using nuclear labeling with 5-bromo-2-deoxyuridine (BrdU) (arrows and dotted circles), which was constantly infused simultaneously by osmotic minipump. Tests were repeated as five independent experiments. Download figure Download PowerPoint Recent reports revealed that the anti-inflammatory effect of FGF21 is involved in the improvement of insulin resistance (Li et al, 2018; Wang et al, 2018). Thus, we next tested the anti-insulin-resistant activity of FGF21SS in 3T3-L1 adipocytes. Cells were treated for 24 h with vehicle, FGF21 or FGF21SS in RAW264.7 conditioned medium (RAW-CM, containing several macrophages secreted cytokines, e.g., TNF-α and IL-6, which are responsible for the development of adipocytes inflammation and insulin resistance (Dandona et al, 2004; Permana et al, 2006; Zatterale et al, 2019)). AKT phosphorylation upon insulin stimulation through PI3K/Akt/mTOR signaling pathway was used here as a marker of insulin sensitivity (Haeusler et al, 2018). As shown in Fig 6A, treatment of RAW-CM greatly inhibited insulin sensitivity in 3T3-L1 adipocytes, while the addition of FGF21s effectively antagonized the effect of macrophage-secreted inflammatory cytokines and significantly alleviated insulin resistance, and FGF21SS showed more significant activity than FGF21. Figure 6. Anti-diabetic activity characterization of FGF21SS A. FGF21