Fibroblast growth factor (FGF)21 improves insulin sensitivity, reduces body weight, and reverses hepatic steatosis in preclinical species. We generated long-acting FGF21 mimetics by site-specific conjugation of the protein to a scaffold antibody. Linking FGF21 through the C terminus decreased bioactivity, whereas bioactivity was maintained by linkage to selected internal positions. In mice, these CovX-Bodies retain efficacy while increasing half-life up to 70-fold compared with wild-type FGF21. A preferred midlinked CovX-Body, CVX-343, demonstrated enhanced in vivo stability in preclinical species, and a single injection improved glucose tolerance for 6 days in ob/ob mice. In diet-induced obese mice, weekly doses of CVX-343 reduced body weight, blood glucose, and lipids levels. In db/db mice, CVX-343 increased glucose tolerance, pancreatic b-cell mass, and proliferation. CVX-343, created by linkage of the CovX scaffold antibody to the engineered residue A129C of FGF21 protein, demonstrated superior preclinical pharmacodynamics by extending serum half-life of FGF21 while preserving full therapeutic functionality.

Human interleukin 5 receptor ␣ (IL5R␣) comprises three fibronectin type III domains (D1, D2, and D3) in the extracellular region. Previous results have indicated that residues in the D1D2 domains are crucial for high affinity interaction with human interleukin 5 (IL5). Yet, it is the D2D3 domains that have sequence homology with the classic cytokine recognition motif that is generally assumed to be the minimum cytokinerecognizing unit. In the present study, we used kinetic interaction analysis of alanine-scanning mutational variants of IL5R␣ to define the residues involved in IL5 recognition. Soluble forms of IL5R␣ variants were expressed in S2 cells, selectively captured via their C-terminal V5 tag by anti-V5 tag antibody immobilized onto the sensor chip and examined for IL5 interaction by using a sandwich surface plasmon resonance biosensor method. Marked effects on the interaction kinetics were observed not only in D1 (Asp 55 , Asp 56 , and Glu 58 ) and D2 (Lys 186 and Arg 188 ) domains, but also in the D3 (Arg 297 ) domain. Modeling of the tertiary structure of IL5R␣ indicated that these binding residues fell into two clusters. The first cluster consists of D1 domain residues that form a negatively charged patch, whereas the second cluster consists of residues that form a positively charged patch at the interface of D2 and D3 domains. These results suggest that the IL5⅐IL5R␣ system adopts a unique binding topology, in which the cytokine is recognized by a D2D3 tandem domain combined with a D1 domain, to form an extended cytokine recognition interface. Interleukin 5 (IL5)1 is a T cell-derived cytokine that plays a central role in maturation and proliferation of eosinophils (1). IL5 exerts biological functions through recruitment of a cell surface receptor composed of two polypeptide chains (2), ␣ and ␤. The ␣ chain is IL5-specific and is called IL5 receptor ␣ (IL5R␣), whereas the ␤ chain is shared with IL3 and GM-CSF (3, 4) and is called common ␤ chain (␤c). The ␣ chains for IL3, GM-CSF, and IL5R␣ also share a high degree of amino acid sequence similarity and constitute a distinct subgroup within the cytokine receptor family (5). Because IL5 has been implicated in the pathology of eosinophil-related inflammatory diseases, designing specific antagonists for IL5R␣ may offer therapeutic benefits in the treatment of such diseases (6, 7).IL5 initially binds to IL5R␣ with high affinity, and the resulting complex recruits ␤c to induce cytoplasmic signal transduction. Human IL5R␣ alone binds IL5 with an equilibrium dissociation constant (K d ) of 0.3-0.6 nM when expressed in COS cells (8). The binding affinity is increased only 2-to 5-fold when human ␣ and ␤ chains are co-expressed (3). In other words, IL5R␣ provides most of the IL5 binding energy, whereas ␤c is essentially for signaling. IL5R␣ consists of an extracellular region, a single transmembrane region and a cytoplasmic region. A soluble form of IL5R␣ (sIL5R␣) containing only the extracellular region was cloned and expressed (9). This form has provided a conv...

Escherichia coli MutT hydrolyzes 8-oxo-dGTP to 8-oxodGMP, an event that can prevent the misincorporation of 8-oxoguanine opposite adenine in DNA. Of the several enzymes that recognize 8-oxoguanine, MutT exhibits high substrate specificity for 8-oxoguanine nucleotides; however, the structural basis for this specificity is unknown. The crystal structures of MutT in the apo and holo forms and in the binary and ternary forms complexed with the product 8-oxodGMP and 8-oxo-dGMP plus Mn 2؉ , respectively, were determined. MutT strictly recognizes the overall conformation of 8-oxo-dGMP through a number of hydrogen bonds. This recognition mode revealed that 8-oxoguanine nucleotides are discriminated from guanine nucleotides by not only the hydrogen bond between the N7-H and O␦ (N119) atoms but also by the syn glycosidic conformation that 8-oxoguanine nucleotides prefer. Nevertheless, these discrimination factors cannot by themselves explain the roughly 34,000-fold difference between the affinity of MutT for 8-oxo-dGMP and dGMP. When the binary complex of MutT with 8-oxo-dGMP is compared with the ligand-free form, ordering and considerable movement of the flexible loops surrounding 8-oxodGMP in the binary complex are observed. These results indicate that MutT specifically recognizes 8-oxoguanine nucleotides by the ligand-induced conformational change.Although spontaneous mutations are indispensable to the evolutionary process of living organisms, they can also be lethal to the organism. Among the various modified bases in DNA, RNA, and nucleotides, 8-oxoguanine (8-oxoG), 2 a damaged form of guanine (G) generated by reactive oxygen species, is known to have highly mutagenic potency because of its mispairing with adenine. Therefore, organisms have an error avoidance pathway for preventing mutations caused by 8-oxoG. The Escherichia coli MutT protein (129 amino acids, M r ϭ 14,900) hydrolyzes 8-oxo-dGTP and 8-oxo-GTP to their corresponding nucleoside monophosphates and inorganic pyrophosphate in the presence of Mg 2ϩ (1, 2). Because 8-oxodGTP and 8-oxo-GTP can be misincorporated opposite adenine by DNA and RNA polymerases, the hydrolysis of the damaged nucleotides by MutT can avoid replicational and transcriptional errors. In DNA, 8-oxoG paired with cytosine is excised by MutM, an 8-oxoG DNA glycosylase, whereas MutY, an adenine DNA glycosylase, removes adenine paired with 8-oxoG (3-6).The substrate specificities of enzymes that recognize 8-oxoG are quite varied. MutT exhibits high substrate specificity for 8-oxoG nucleotides; that is, the K m for 8-oxo-dGTP is 14,000-fold lower than that for dGTP (7). In contrast, human MutT homologue 1 (hMTH1) hydrolyzes not only 8-oxo-dGTP but also several oxidized purine nucleotides such as 2-oxo-dATP, 2-oxo-ATP, 8-oxo-dATP, and 8-oxo-ATP. In terms of the hydrolysis of 8-oxo-dGTP, the K m of hMTH1 for 8-oxo-dGTP is only 17-fold lower than that for dGTP (8, 9). The solution structure of hMTH1 as determined by NMR has revealed its overall architecture and possible substrate-binding region ...

The solution structure of ribosome recycling factor (RRF) from hyperthermophilic bacterium, Aquifex aeolicus, was determined by heteronuclear multidimensional NMR spectroscopy. Fifteen structures were calculated using restraints derived from NOE, J-coupling, and T1/T2 anisotropies. The resulting structure has an overall L-shaped conformation with two domains and is similar to that of a tRNA molecule. The domain I (corresponding to the anticodon stem of tRNA) is a rigid three alpha-helix bundle. Being slightly different from usual coiled-coil arrangements, each helix of domain I is not twisted but straight and parallel to the main axis. The domain II (corresponding to the portion with the CCA end of tRNA) is an alpha/beta domain with an alpha-helix and two beta-sheets, that has some flexible regions. The backbone atomic root-mean-square deviation (rmsd) values of both domains were 0.7 A when calculated separately, which is smaller than that of the molecule as a whole (1.4 A). Measurement of 15N-[1H] NOE values show that the residues in the corner of the L-shaped molecule are undergoing fast internal motion. These results indicate that the joint region between two domains contributes to the fluctuation in the orientation of two domains. Thus, it was shown that RRF remains the tRNA mimicry in solution where it functions.

PEG8-(NTA)(8) is the first derivative able to associate with native proteins and form soluble complexes with a nanomolar K (D). The study highlights the need of a multivalent and flexible coordination and encourages further investigations to increase the stability of PEG8-(NTA)(8) complexes in vivo either through the use of protein mutants or His-tag proteins.

Background:The bivalency of IgG and Fc fusion could cause undesired therapeutic properties. Results: We developed a stable monomeric Fc modality by N-glycosylation engineering, enabling the generation of crystal structure. Conclusion:The monomeric Fc prolonged the half-life of Fab domain through the interaction with neonatal Fc receptor. Significance: The monomeric Fc will be used for pharmacokinetics enhancement of biotherapeutics that require monovalent properties.

Holo-neocarzinostatin (holo-NCS) is a complex protein carrying the anti-tumor active enediyne ring chromophore by a scaffold consisting of an immunoglobulinlike seven-stranded anti-parallel ␤-barrel. Because of the labile chromophore reflecting its extremely strong DNA cleavage activity and complete stabilization in the complex, holo-NCS has attracted much attention in clinical use as well as for drug delivery systems. Despite many structural analyses for holo-NCS, the chromophore-releasing mechanism to trigger prompt attacks on the target DNA is still unclear. We determined the three-dimensional structure of the protein and the internal motion by multinuclear NMR to investigate the releasing mechanism. The internal motion studied by 13 C NMR methine relaxation experiments showed that the complex has a rigid structure for its loops as well as the ␤-barrel in aqueous solution. This agrees with the refined NMR solution structure, which has good convergence in the loop regions. We also showed that the chromophore displayed a similar internal motion as the protein moiety. The structural comparison between the refined solution structure and x-ray crystal structure indicated characteristic differences. Based on the findings, we proposed the chromophore-releasing mechanism by a three-state equilibrium, which sufficiently describes both the strong binding and the prompt releasing of the chromophore. We demonstrated that we could bridge the dynamic properties and the static structure features with simple kinetic assumptions to solve the biochemical function.Holo-neocarzinostatin (holo-NCS) 1 (molecular mass 12 kDa, 113 amino acid residues ϩ chromophore) is a prominent member of the strongest anti-tumor reagent chromoprotein family (Fig. 1, B and D) (1). It is a non-covalent complex of an enediyne ring chromophore and its carrier protein isolated from Streptomyces carzinostaticus (2-4). The chromophore, which has a selective bulged DNA-cleaving activity (5), is easily inactivated by light, heat, and molecular oxygen because of its labile structure. Despite its intrinsic instability in the free state, the chromophore in the complex is stable even in vivo. It is thought that holo-NCS penetrates into the target cell carrying the chromophore and promptly releases it at the cell nucleus to kill the cell. Therefore, the holo-NCS family has attracted much attention in clinical use as well as for use in targeting drug delivery systems (6 -14). The three-dimensional structures of holo-NCS and the chromoprotein family have been investigated by solution NMR (12, 15-19) and x-ray diffraction (20), elucidating an immunoglobulin-like sevenstranded anti-parallel ␤-barrel structure (Fig. 1B) (21).Based on the previous NMR and x-ray studies for holo-NCS, several mechanisms of the chromophore stabilization have been proposed. These mechanisms were dominated by hydrophobic interactions with the binding pocket. However, the mechanism of releasing and binding of the chromophore remains unclear, even though the process has attracted much atte...

The fortuitously discovered antiaging membrane protein αKlotho (Klotho) is highly expressed in the kidney, and deletion of the Klotho gene in mice causes a phenotype strikingly similar to that of chronic kidney disease (CKD). Klotho functions as a co-receptor for fibroblast growth factor 23 (FGF23) signaling, whereas its shed extracellular domain, soluble Klotho (sKlotho), carrying glycosidase activity, is a humoral factor that regulates renal health. Low sKlotho in CKD is associated with disease progression, and sKlotho supplementation has emerged as a potential therapeutic strategy for managing CKD. Here, we explored the structure-function relationship and post-translational modifications of sKlotho variants to guide the future design of sKlotho-based therapeutics. Chinese hamster ovary (CHO)- and human embryonic kidney (HEK)-derived WT sKlotho proteins had varied activities in FGF23 co-receptor and β-glucuronidase assays in vitro and distinct properties in vivo. Sialidase treatment of heavily sialylated CHO-sKlotho increased its co-receptor activity 3-fold, yet it remained less active than hyposialylated HEK-sKlotho. MS and glycopeptide-mapping analyses revealed that HEK-sKlotho is uniquely modified with an unusual N-glycan structure consisting of N,N′-di-N-acetyllactose diamine at multiple N-linked sites, one of which at Asn-126 was adjacent to a putative GalNAc transfer motif. Site-directed mutagenesis and structural modeling analyses directly implicated N-glycans in Klotho's protein folding and function. Moreover, the introduction of two catalytic glutamate residues conserved across glycosidases into sKlotho enhanced its glucuronidase activity but decreased its FGF23 co-receptor activity, suggesting that these two functions might be structurally divergent. These findings open up opportunities for rational engineering of pharmacologically enhanced sKlotho therapeutics for managing kidney disease.