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The coronavirus disease 2019 (COVID-19) pandemic has necessitated the development of antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). 3C-like protease (3CL

The CH2 domain is a critical element of the human Immunoglobulin G (IgG) constant region. Although the CH2 domain is the least stable domain in IgG, it is also a promising scaffold candidate for developing novel therapeutic approaches. Recently, we succeeded in preparing glycosylated and non-glycosylated CH2 domain in the host organism Pichia pastoris. Herein, we verified that glycosylation of the CH2 domain decreased both, its tendency to aggregate and its immunogenicity in mice, suggesting that aggregation and immunogenicity are related. In addition, we have produced in P. pastoris a stabilized version of the CH2 domain with and without glycan, and their propensity to aggregate evaluated. We found that stabilization alone significantly decreased the aggregation of the CH2 domain. Moreover, the combination of glycosylation and stabilization completely suppressed its aggregation behavior. Since protein aggregation is related to immunogenicity, the combination of glycosylation and stabilization to eliminate the aggregation behavior of a protein could be a fruitful strategy to generate promising immunoglobulin scaffolds.

Diacylglycerol kinases (DGKs) are multi‐domain lipid kinases that modulate the levels of lipid messengers, diacylglycerol, and phosphatidic acid. Recently, increasing attention has been paid to its α isozyme (DGKα) as a potential target for cancer immunotherapy. However, little progress has been made on the structural biology of DGKs, and a detailed understanding of the Ca(2+)‐triggered activation of DGKα, for which the N‐terminal domains likely play a critical role, remains unclear. We have recently shown that Ca(2+) binding to DGKα‐EF induces conformational changes from a protease‐susceptible “open” conformation in the apo state to a well‐folded one in its holo state. Here, we further studied the structural properties of DGKα N‐terminal (RVH and EF) domains using a series of biophysical techniques. We first revealed that the N‐terminal RVH domain is a novel Ca(2+)‐binding domain, but the Ca(2+)‐induced conformational changes mainly occur in the EF domain. This was corroborated by NMR experiments showing that the EF domain adopts a molten‐globule like structure in the apo state. Further analyses using SEC‐SAXS and NMR indicate that the partially unfolded EF domain interacts with RVH domain, likely via hydrophobic interactions in the absence of Ca(2+), and this interaction is modified in the presence of Ca(2+). Taken together, these results present novel insights into the structural rearrangement of DGKα N‐terminal domains upon binding to Ca(2+), which is essential for the activation of the enzyme.

The pandemic of coronavirus disease 2019 (COVID-19) has urgently necessitated the development of antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The 3C-like protease (3CLpro) is a promising target for COVID-19 treatment. Here, we report the new class of covalent inhibitors for 3CLpro possessing chlorofluoroacetamide (CFA) as a cysteine reactive warhead. Based on the aza-peptide scaffold, we synthesized the series of CFA derivatives in enantiopure form and evaluated their biochemical efficiencies. The data revealed that 8a (YH-6) with R configuration at the CFA unit strongly blocks the SARS-CoV-2 replication in the infected cells and this potency is comparable to that of nirmatrelvir. The X-ray structural analysis shows that 8a (YH-6) forms a covalent bond with Cys145 at the catalytic center of 3CLpro. The strong antiviral activity and sufficient pharmacokinetics property of 8a (YH-6) suggest its potential as a lead compound for treatment of COVID-19.

Glycosylation is one of the major post-translational modifications in eukaryotic cells and has been reported to affect the amyloid fibril formation in several amyloidogenic proteins and peptides. In this study, we expressed a Vλ6 light chain mutant, Wil, which is an amyloidogenic mutant in AL amyloidosis, by the yeast Pichia pastoris. After separation by cation exchange chromatography, we obtained the O-glycosylated and non-glycosylated Wil mutants in high yield. The structures of these Wil mutants were identical except with respect to glycosylation, and the stabilities were also identical. On the other hand, the O-glycosylation retarded the amyloid fibril formation in a sugar size-dependent manner. From these results, we discussed the role of covalently attached glycan in the retardation of amyloid fibril formation.

In Escherichia coli, DNA replication is restarted following DNA repair by the PriA-dependent pathway, in which the binding and dissociation of proteins such as PriA, PriB, and DnaT on ssDNA lead to the formation of a protein-DNA complex for recruiting the DnaB-DnaC replication protein complex. However, the structure of the PriB-DnaT complex, which is an essential step in the PriA-dependent pathway, remains elusive. In this study, the importance of His26 in PriB for replication restart was reconfirmed using plasmid complementation. Furthermore, we used NMR to examine the DnaT interaction sites on PriB. We also evaluated the PriB-DnaT peptide complex model, which was prepared by in silico docking, using molecular dynamics simulation. From these data, we propose a structural model that provides insight into the PriB-DnaT interaction.

P2X4 receptor is known to be involved in neuropathic pain. In order to detect the expression of P2X4 receptor on microglia at the time of onset of neuropathic pain, one approach consists on the preparation of the monoclonal antibodies with both selective binding and high affinity. We have recently established a monoclonal antibody (named 12-10H) which had high affinity to rat P2X4 receptor expressed in 1321N1 cells. The dissociation constants of the complex between the monoclonal antibodies obtained so far and the head domain (HD) in the rat P2X4 receptor were in the nanomolar range. To improve the affinity by rational mutations, we need to know the precious location of the binding site in these monoclonal antibodies. Here, we have analysed and identified the binding residues in the monoclonal antibody (12-10H) with high affinity for the HD of the rat P2X4 receptor by site-directed mutagenesis.

Glycoengineering of therapeutic proteins has been applied to improve the clinical efficacy of several therapeutics. Here, we examined the effect of glycosylation on the properties of the Fab of the therapeutic antibody, adalimumab. An N-glycosylation site was introduced at position 178 of the H chain constant region of adalimumab Fab through site-directed mutagenesis (H:L178N Fab), and the H:L178N Fab was produced in Pichia pastoris. Expressed mutant Fab contained long and short glycan chains (L-glyco Fab and S-glyco Fab, respectively). Under the condition of aggregation of Fab upon pH shift-induced stress, both of L-glyco Fab and S-glyco Fab were less prone to aggregation, with L-glyco Fab suppressing aggregation more effectively than the S-glyco Fab. Moreover, the comparison of the antigenicity of glycosylated and wild-type Fabs in mice revealed that glycosylation resulted in the suppression of antigenicity. Analysis of the pharmacokinetic behaviour of the Fab, L-glyco Fab and S-glyco Fab indicated that the half-lives of glycosylated Fabs in the rats were shorter than that of wild-type Fab, with L-glyco Fab having a shorter half-life than S-glyco Fab. Thus, we demonstrated that the glycan chain influences Fab aggregation and immunogenicity, and glycosylation reduces the elimination half-life in vivo.

Conjugation with polyethylene glycol (PEG) is performed to increase serum half-life of the Fab for clinical applications. However, current designs for recombinant Fab only allow PEGylation at the interchain SS bond (disulfide bond) at the C-terminal end of the heavy chain and light chain of the Fab, which the decrease of thermostability occurred by partial reduction of the interchain SS bond. An adalimumab Fab mutant with a novel interchain SS bond (CH1 : C177-CL : C160) and one cysteine at the C-terminal end (mutSS FabSH) was designed to maintain Fab thermostability and for site-specific PEGylation. MutSS FabSH was expressed in Pichia pastoris and purified mutSS FabSH was conjugated with 20-kDa PEG targeted at the free cysteine. Based on enzyme-linked immunosorbent assay (ELISA), PEGylation did not affect the binding capacity of the mutSS FabSH. To confirm the influence of PEGylation on the pharmacokinetic behavior of the Fab, PEGylated mutSS FabSH was administered to rats via tail vein injection. Analysis of the mean serum concentration of the PEGylated mutSS FabSH versus time through ELISA indicated an increase in half-life compared to that of non-PEGylated wild-type Fab. Consequently, we have successfully demonstrated that a Fab mutant with a novel interchain SS bond and one free cysteine at the C-terminal end can be PEGylated without changes in functionality. This design can potentially be used as a platform for modification of other recombinant Fabs.

The introduction of intermolecular disulfide bonds by amino acid mutations is an effective method for stabilizing dimeric proteins. X-ray crystal structure of Fab of a therapeutic antibody, adalimumab, revealed the first loop of the CH1 domain to be partially unsolved at position 135–141. To find new sites for the introduction of intermolecular disulfide bonds in adalimumab Fab, Fab mutants targeting the unsolved region were predicted using molecular simulation software. Four Fab mutants, H:K137C-L:I117C, H:K137C-L:F209C, H:S138C-L:F116C and H:S140C-L:S114C, were expressed in the methylotrophic yeast Pichia pastoris. SDS-PAGE analysis of these mutants indicated that H:K137C-L:F209C, H:S138C-L:F116C and H:S140C-L:S114C mutants mostly formed intermolecular disulfide bonds, whereas some H:K137C-L:I117C mutants formed intermolecular disulfide bonds and some did not. Differential scanning calorimetry measurements showed increased thermal stability in all Fab mutants with engineered disulfide bonds. The bio-layer interferometry measurements, for binding of the antigen tumor necrotic factor α, indicated that Fab mutants had less antigen-binding activity than wild-type Fab. In particular, the KD value of H:K137C-L:F209C was ~17 times higher than that of wild-type Fab. Thus, we successfully introduced intermolecular disulfide bonds between the first loop region of the CH1 and CL domains and observed that it increases the thermostability of Fab and affects the antigen-binding activity.

AL amyloidosis is a life-threatening disease characterized by the deposition of amyloidogenic immunoglobulin light chain secreted from clonal plasma cells. Here we established an in-vitro screening system of amyloid inhibition of a variable domain in λ6 light chain mutant (Vλ6), Wil, and screened a food-additive compound library to identify compounds inhibiting the fibril formation. We found gossypetin and isoquercitrin as novel inhibitors. NMR analysis showed that both compounds directly interacted with natively-folded Wil, and proteolysis experiments demonstrated that these compounds conferred proteolytic resistance, suggesting that the compounds enhance the kinetic stability of Wil. Since gossypetin and isoquercitrin specifically interacted with the protein at micromolar concentrations, these compounds could be used as lead to further develop inhibitors against AL amyloidosis.

Minimalist photo-reactive probes, which consist of a photo-reactive group and a tag for detection of target proteins, are useful tools in chemical biology. Although several diazirine-based and aryl azide-based minimalist probes are available, no keto-based minimalist probe has yet been reported. Here we describe minimalist probes based on a 2-thienyl-substituted α-ketoamide bearing an alkyne group on the thiophene ring. The 3-alkyne probe showed the highest photo-affinity labeling efficiency.

When the replisome collapses at a DNA damage site, a sequence-independent replication restart system is required. In Escherichia coli, PriA, PriB, and DnaT assemble in an orderly fashion at the stalled replication fork and achieve the reloading of the replisome. PriB-DnaT interaction is considered a significant step in the replication restart. In this study, we examined the contribution of the residues Ser20, His26 and Ser55, which are located on the PriB dimer interface. These residues are proximal to Glu39 and Arg44, which are important for PriB-DnaT interaction. Mutational analyses revealed that His26 and Ser20 of PriB are important for the interaction with DnaT, and that the Ser55 residue of PriB might have a role in negatively regulating the DnaT binding. These residues are involved in not only the interaction between PriB and DnaT but also the dissociation of single-stranded DNA (ssDNA) from the PriB−ssDNA complex due to DnaT binding. Moreover, NMR study indicates that the region Asp66−Glu76 on the linker between DnaT domains is involved in the interaction with wild-type PriB. These findings provide significant information about the molecular mechanism underlying replication restart in bacteria.

Cardiovascular disease (CVD) is a lifestyle-related disease (LSRD) and one of the largest public health issues. Risk factors for CVD correlate with an excessive intake of glucose and/or fructose, which has been shown to induce the production of advanced glycation end-products (AGEs). We previously identified AGEs derived from glyceraldehyde and named them toxic AGEs (TAGE) due to their cytotoxicities and relationship with LSRD. We also reported that extracellular TAGE in the vascular system may promote CVD and that serum TAGE levels are associated with risk factors for CVD. The mechanisms responsible for the onset and/or progression of CVD by extracellular TAGE or the above risk factors involve vascular disorders. In the present study, we revealed that rat primary cultured cardiomyocytes generated intracellular TAGE, which decreased beating rates and induced cell death. LC3-II/LC3-I, a factor of autophagy, also decreased. Although intracellular TAGE may be targets of degradation as cytotoxic proteins via autophagy, they may inhibit autophagy. Furthermore, the mechanisms by which intracellular TAGE decrease beating rates and induce cell death may involve the suppression of autophagy. The present results suggest that intracellular TAGE are generated in cardiomyocytes and directly damage them, resulting in CVD.

Generally, intermolecular disulfide bond contribute to the conformational protein stability. To identify sites where intermolecular disulfide bond can be introduced into the Fab’s constant domain of the therapeutic IgG, Fab mutants were predicted using the MOE software, a molecular simulator, and expressed in Pichia pastoris. SDS-PAGE analysis of the prepared Fab mutants from P. pastoris indicated that among the nine analyzed Fab mutants, the F130C(H):Q124C(L), F174C(H):S176C(L), V177C(H):Q160C(L), F174C(H):S162C(L), F130C(H):S121C(L), and A145C(H):F116C(L) mutants mostly formed intermolecular disulfide bond. All these mutants showed increased thermal stability compared to that of Fab without intermolecular disulfide bond. In the other mutants, the intermolecular disulfide bond could not be completely formed, and the L132C(H):F118C(L) mutant showed only a slight decrease in binding activity and β-helix content, owing to the exertion of adverse intermolecular disulfide bond effects. Thus, our comprehensive analysis reveals that the introduction of intermolecular disulfide bond in the Fab’s constant domain is possible at various locations. These findings provide important insights for accomplishing human Fab stabilization.

Membrane proteins are critical elements of numerous therapeutic approaches ranging from cancer to bacterial infections. MsbA is a bacterial membrane protein that has received increasing attention as an antibacterial target for its role in the processing of Lipid A, a key precursor of lipopolysaccharide that is essential for bacterial growth. When employing nanodiscs it is possible to stabilize MsbA by providing a membrane-like environment that enhances its enzymatic activity. Taking advantage of this property we have carried out a fragment screening using the biophysical method of surface plasmon resonance. This approach identified several compounds that bind specifically to MsbA. In particular, one of these fragment molecules not only binds to the target, but also inhibits the ATPase activity of the MsbA protein. The similarity of this fragment to the adenine moiety of ATP points at a route to generate stronger and more potent inhibitors for MsbA and even other proteins of its family of ABC transporters. Collectively, our study reveals biophysical approaches that facilitate the identification of fragment candidates inhibiting the activity of membrane proteins.

Pichia pastoris is a popular eukaryotic system employed for the fast, simple and inexpensive production of recombinant protein including biotherapeutics such as human albumin. The CH2 domain of human Immunoglobulin G (IgG) is a promising scaffold for developing novel therapeutics. To accelerate the research of CH2 domain, we have established a procedure to highly express human CH2 domain (∼150 mg/l) as well as human Fc (∼30 mg/l) in yeast P. pastoris. The procedure yields, simultaneously, a major glycosylated (∼70%) and non-glycosylated (∼30%) fractions. They can be easily separated with high purity. Although both forms of CH2 domain have essentially the same secondary structure, the presence of the glycan increased the thermal stability of the CH2 domain by about 5°C as determined from calorimetry. The purified glycosylated CH2 domain elicited polyclonal antibodies in mouse, recognizing not only the CH2 domain, but also recombinant human Fc and the commercial IgG1 antibody Rituxan. Protein A and Protein G binding to the kink region between CH2 domain and CH3 domain of human Fc are used to purify therapeutic proteins. Therefore, these antibodies are candidates to develop a novel affinity material to purify human antibodies using their CH2 domain.

P2X purinergic receptors are ATP-driven ionic channels expressed as trimers and showing various functions. A subtype, the P2X4 receptor present on microglial cells is highly involved in neuropathic pain. In this study, in order to prepare antibodies recognizing the native structure of rat P2X4 (rP2X4) receptor, we immunized mice with rP2X4’s head domain (rHD, Gln111–Val167), which possesses an intact structure stabilized by S-S bond formation (Igawa and Abe et al. FEBS Lett. 2015), as an antigen. We generated five monoclonal antibodies with the ability to recognize the native structure of its head domain, stabilized by S-S bond formation. Site-directed mutagenesis revealed that Asn127 and Asp131 of the rHD, in which combination of these amino acid residues is only conserved in P2X4 receptor among P2X family, were closely involved in the interaction between rHD and these antibodies. We also demonstrated the antibodies obtained here could detect rP2X4 receptor expressed in 1321N1 human astrocytoma cells. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11302-019-09646-5) contains supplementary material, which is available to authorized users.

Irreversible inhibition of disease-associated proteins with small molecules is a powerful approach for achieving increased and sustained pharmacological potency. Here, we introduce α-chlorofluoroacetamide (CFA) as a novel warhead of targeted covalent inhibitor (TCI). Despite weak intrinsic reactivity, CFA-appended quinazoline showed high reactivity toward Cys797 of epidermal growth factor receptor (EGFR). In cells, CFA-quinazoline showed higher target specificity for EGFR than the corresponding Michael acceptors in a wide concentration range (0.1–10 μM). The cysteine adduct of the CFA derivative was susceptible to hydrolysis and reversibly yielded intact thiol but was stable in solvent-sequestered ATP-binding pocket of EGFR. This environment-dependent hydrolysis can potentially reduce off-target protein modification by CFA-based drugs. Oral administration of CFA quinazoline NS-062 significantly suppressed tumor growth in a mouse xenograft model. Further, CFA-appended pyrazolopyrimidine irreversibly inhibited Bruton’s tyrosine kinase with higher target specificity. These results demonstrate the utility of CFA as a new class warheads for TCI. Discovery and exploitation of inherent reaction features of chlorofluoroacetamide (CFA) as a warhead such as low off-target activity and reversible reactivity with cysteine enable specific covalent inhibition of targeted kinases.

Rheumatoid factors (RFs) are autoantibodies against the fragment-crystallizable (Fc) region of IgG. In individuals with hematological diseases such as cryoglobulinemia and certain B cell lymphoma forms, the RFs derived from specific heavy- and light-chain germline pairs, so-called “stereotypic RFs,” are frequently produced in copious amounts and form immune complexes with IgG in serum. Of note, many structural details of the antigen recognition mechanisms in RFs are unclear. Here we report the crystal structure of the RF YES8c derived from the IGHV1-69/IGKV3-20 germline pair, the most common of the stereotypic RFs, in complex with human IgG1-Fc at 2.8 Å resolution. We observed that YES8c binds to the CH2–CH3 elbow in the canonical antigen-binding manner involving a large antigen–antibody interface. On the basis of this observation, combined with mutational analyses, we propose a recognition mechanism common to IGHV1-69/IGKV3-20 RFs: (1) the interaction of the Leu(432)–His(435) region of Fc enables the highly variable complementarity-determining region (CDR)-H3 to participate in the binding, (2) the hydrophobic tip in the CDR-H2 typical of IGHV1-69 antibodies recognizes the hydrophobic patch on Fc, and (3) the interaction of the highly conserved RF light chain with Fc is important for RF activity. These features may determine the putative epitope common to the IGHV1-69/IGKV3-20 RFs. We also showed that some mutations in the binding site of RF increase the affinity to Fc, which may aggravate hematological diseases. Our findings unravel the structural basis for germline-encoded antibody autoreactivity.

We constructed a system for expressing the Fab of the therapeutic human monoclonal antibody adalimumab at a yield of 20 mg/L in the methylotrophic yeast Pichia pastoris. To examine the contribution of interchain disulfide bonds to conformational stability, we prepared adalimumab Fab from which the interchain disulfide bond at the C-terminal region at both the CH1 and CL domains was deleted by substitution of Cys with Ala (FabΔSS). DSC measurements showed that the Tm values of FabΔSS were approximately 5 °C lower than those of wild-type Fab, suggesting that the interchain disulfide bond contributes to conformational thermostability. Using computer simulations, we designed a novel interchain disulfide bond outside the C-terminal region to increase the stability of FabΔSS. The resulting Fab (mutSS FabΔSS) had the mutations H:V177C and L:Q160C in FabΔSS, confirming the formation of the disulfide bond between CH1 and CL. The thermostability of mutSS FabΔSS was approximately 5 °C higher than that of FabΔSS. Therefore, the introduction of the designed interchain disulfide bond enhanced the thermostability of FabΔSS and mitigated the destabilization caused by partial reduction of the interchain disulfide bond at the C-terminal region, which occurs in site-specific modification such as PEGylation.

The archaeal exo-β-d-glucosaminidase (GlmA) is a dimeric enzyme that hydrolyzes chitosan oligosaccharides into monomer glucosamines. GlmA is a member of the glycosidase hydrolase (GH)-A superfamily-subfamily 35 and is a novel enzyme in terms of its primary structure. Here, we present the crystal structure of GlmA in complex with glucosamine at 1.27 A resolution. The structure reveals that a monomeric form of GlmA shares structural homology with GH42 β-galactosidases, whereas most of the spatial positions of the active site residues are identical to those of GH35 β-galactosidases. We found that upon dimerization, the active site of GlmA changes shape, enhancing its ability to hydrolyze the smaller substrate in a manner similar to that of homotrimeric GH42 β-galactosidase. However, GlmA can differentiate glucosamine from galactose based on one charged residue while using the “evolutionary heritage residue” it shares with GH35 β-galactosidase. Our study suggests that GH35 and GH42 β-galactosidases evolved by exploiting the structural features of GlmA.

Loading the bacterial replicative helicase DnaB onto DNA requires a specific loader protein, DnaC/DnaI, which creates the loading-competent state by opening the DnaB hexameric ring. To understand the molecular mechanism by which DnaC/DnaI opens the DnaB ring, we solved 3.1-Å co-crystal structure of the interaction domains of Escherichia coli DnaB–DnaC. The structure reveals that one N-terminal domain (NTD) of DnaC interacts with both the linker helix of a DnaB molecule and the C-terminal domain (CTD) of the adjacent DnaB molecule by forming a three α-helix bundle, which fixes the relative orientation of the two adjacent DnaB CTDs. The importance of the intermolecular interface in the crystal structure was supported by the mutational data of DnaB and DnaC. Based on the crystal structure and other available information on DnaB–DnaC structures, we constructed a molecular model of the hexameric DnaB CTDs bound by six DnaC NTDs. This model suggested that the binding of a DnaC would cause a distortion in the hexameric ring of DnaB. This distortion of the DnaB ring might accumulate by the binding of up to six DnaC molecules, resulting in the DnaB ring to open.

Background Protein antigens are degraded by endosomal protease in antigen presentation cell. T cells recognize peptides derived from antigen proteins bound to class II major histocompatibility complex molecules. We previously reported that an increase in the conformational stability of an antigen depressed its immunogenicity. However, there is little information on antigens with differences in molecular properties such as net charges and molecular weight. Methods Denaturation experiments against guanidine hydrochloride. The serum IgE levels to protein antigens at 35 days after the first immunization analyzed using ELISA. Results The Der p 2 mutations in which Ile13 is mutated to Ala (I13A) and Ala122 is mutated to Ile (A122I) were shown to have lower and higher conformational stability than the wild-type, respectively, by denaturation experiments. The amount of IgE production by the less stable I13A mutant was higher and that of the stable A122I mutant was lower than that of the wild-type. Conclusion Our results suggest that the increased conformational stability of Der p 2 depressed the IgE production in mice. General significance These findings should provide a milestone for the engineering of allergen vaccines.

Purification of milligram quantities of target proteins is required for structural and biophysical studies. However, mammalian membrane proteins, many of which are important therapeutic targets, are too unstable to be expressed in heterologous hosts and to be solubilized by detergents. One of the most promising ways to overcome these limitations is to stabilize the membrane proteins by generating variants via introduction of truncated flexible regions, fusion partners, and site-directed mutagenesis. Therefore, an effective screening strategy is a key to obtaining successful protein stabilization. Herein, we report the micro-scale and high-throughput screening of stabilized membrane protein variants using Saccharomyces cerevisiae as a host. All steps of the screening, including cultivation and disruption of cells, solubilization of the target protein, and the pretreatment for fluorescence-detected size exclusion chromatography (FSEC), could be performed in a 96-well microplate format. We demonstrated that the dispersion among wells was small, enabling detection of a small but important improvement in the protein stability. We also demonstrated that the thermally stable mutants of a human G protein-coupled receptor could be distinguished based on an increase of the peak height in the FSEC profile, which was well correlated with increased ligand binding activity of the protein. This strategy represents a significant platform for handling numerous mutants, similar to alanine scanning.

Asn46Asp/Asp52Ser or Asn46Glu/Asp52Ser hen egg white lysozyme (HEL) mutant was designed by introducing the substituted catalytic residue Asp46 or Glu46, respectively, based on Venerupis philippinarum (Vp) lysozyme structure as a representative of invertebrate-type (i-type) lyzozyme. These mutations restored the bell-shaped pH-dependency of the enzyme activity from the sigmoidal pH-dependency observed for the Asp52Ser mutant. Furthermore both lysozyme mutants possessed retaining mechanisms like Vp lysozyme and HEL. The Asn46Glu/Asp52Ser mutant, which has a shorter distance between two catalytic residues, formed a glycosyl adduct in the reaction with the N-acetylglucosamine oligomer. Furthermore, we found the accelerated turnover through its glycosyl adduct formation and decomposition. The turnover rate estimated from the glycosyl formation and decomposition rates was only 20% of the observed hydrolysis rate of the substrate. Based on these results, we discussed the catalytic mechanism of lysozymes.

Objective Regulator of calcineurin 1 (RCAN1) controls plasticity of the nervous system and depressive conditions by regulating brain-derived neurotropic factor (BDNF) and plays a crucial role in neural and cardiac pathways. The apolipoprotein E gene ( ApoE) is a robust risk factor for progression of Alzheimer’s disease. A fatty diet is considered detrimental for metabolic disorders, such as obesity and cardiovascular diseases. Methods We examined the neuronal and cardiac protective roles of RCAN1 in ApoE−/− mice that were fed a high- or low-fat diet with and without voluntary movement for 3 months. Organ weights, laboratory data, histology, RNA expression, and behavior were examined. Results A high-fat diet with exercise improved depressive function, as examined by the forced swimming test, and RCAN1 mRNA expression was induced in the hippocampus. A low-fat diet with exercise resulted in a reduced body weight, higher heart weight/body weight ratio, and lower circulating triglyceride levels compared with a low-fat diet without exercise. RCAN1 mRNA expression was increased in cardiomyocytes in ApoE−/− mice. Conclusions The combination of a high-fat diet and exercise might reduce depressive function, whereas a low-fat diet with exercise leads to cardioprotection. Induction of RCAN1 expression might affect neuroplasticity and cardiac function.

Protein tyrosine sulfation (PTS) is a post-translational modification regulating numerous biological events. PTS generally occurs at flexible regions of proteins, enhancing intermolecular interactions between proteins. Because of the high flexibility associated with the regions where PTS is generally encountered, an atomic-level understanding has been difficult to achieve by X-ray crystallography or nuclear magnetic resonance techniques. In this study, we focused on the conformational behavior of a flexible sulfated peptide and its interaction with an antibody. Molecular dynamics simulations and thermodynamic analysis indicated that PTS reduced the main-chain fluctuations upon the appearance of sulfate-mediated intramolecular H-bonds. Collectively, our data suggested that one of the mechanisms by which PTS may enhance protein–protein interactions consists of the limitation of conformational dynamics in the unbound state, thus reducing the loss of entropy upon binding and boosting the affinity for its partner.

The production of therapeutic monoclonal antibodies is costly; therefore, antigen-binding fragments (Fabs) can be used instead. However, their tendency toward aggregation can reduce the half-life in the plasma and the therapeutic effectiveness. To examine the effect of glycosylation on the properties of the Fab of a therapeutic antibody, an N-glycosylation site was introduced at position 178 of the H-chain constant region of adalimumab Fab through site-directed mutagenesis of L178 N (H:L178 N Fab), and then H:L178 N Fab was expressed in Pichia pastoris. SDS-PAGE analysis with treatment of N-glycosidase F or periodic acid–Schiff reagent showed that H:L178 N Fab contained a relatively low glycan level. Moreover, the H:L178 N mutation did not decrease the binding activity and thermal stability of Fab, and H:L178 N Fab was more resistant to protease digestion than wild-type Fab. The aggregation of Fab induced by pH-shift stress was measured by monitoring the optical density at 350 nm. Although the wild-type Fab showed a large increase in optical density with an increase of protein concentration, no such increase of turbidity during aggregation was found in H:L178 N Fab. These results demonstrated that glycosylation at position 178 of the H-chain constant region of adalimumab Fab can prevent protein aggregation, and therefore serve as a potentially effective platform for drug development.

Nonalcoholic fatty liver disease (NAFLD) ranges from simple steatosis to nonalcoholic steatohepatitis (NASH), leads to fibrosis and potentially cirrhosis, liver failure, and hepatocellular carcinoma, and is one of the most common causes of liver disease worldwide. NAFLD has also been implicated in other medical conditions such as insulin resistance, obesity, metabolic syndrome, hyperlipemia, hypertension, cardiovascular disease, and diabetes. Continuous hyperglycemia has been implicated in the pathogenesis of diabetic micro- and macro-vascular complications via various metabolic pathways, and numerous hyperglycemia-induced metabolic and hemodynamic conditions exist, including the increased generation of various types of advanced glycation end-products (AGEs). We recently demonstrated that glyceraldehyde-derived AGEs (Glycer-AGEs), the predominant components of toxic AGEs (TAGE), played an important role in the pathogenesis of angiopathy in diabetic patients. Moreover, a growing body of evidence suggests that the interaction between TAGE and the receptor for AGEs may alter intracellular signaling, gene expression, and the release of pro-inflammatory molecules and also elicits the generation of oxidative stress in numerous types of cells including hepatocytes and hepatic stellate cells. Serum levels of TAGE were significantly higher in NASH patients than in those with simple steatosis and healthy controls. Moreover, serum levels of TAGE inversely correlated with adiponectin (adiponectin is produced by adipose tissue and is an anti-inflammatory adipokine that can increase insulin sensitivity). Furthermore, immunohistochemical staining of TAGE showed intense staining in the livers of patients with NASH. Serum levels of TAGE may be a useful biomarker for discriminating NASH from simple steatosis. The administration of atorvastatin (10 mg daily) for 12 months significantly improved NASH-related metabolic parameters and significantly decreased serum levels of TAGE. The steatosis grade and NAFLD activity score were also significantly improved. These results demonstrated that atorvastatin decreased the serum levels of TAGE in NASH patients with dyslipidemia and suggest the usefulness of TAGE as a biomarker for the attenuation of NASH. Serum levels of TAGE were significantly higher in non-B or non-C hepatocellular carcinoma (NBNC-HCC) patients than in NASH subjects without HCC or control subjects. TAGE may be involved in the pathogenesis of NBNC-HCC, and could, therefore, be a biomarker that could discriminate NBNC-HCC from NASH. We propose that serum levels of TAGE are promising novel targets for the diagnosis of and therapeutic interventions against NASH.

In bacterial organisms, the oriC-independent primosome plays an essential role in replication restart after dissociation of the replication DNA-protein complex following DNA damage. PriC is a key protein component in the oriC-independent replication restart primosome. Our previous study suggested that PriC was divided into an N-terminal domain and a C-terminal domain, with the latter domain being the major contributor to single-stranded DNA (ssDNA) binding capacity. In this study, we prepared several PriC mutants in which basic and aromatic amino acid residues were mutated to alanine. Five of these residues, Arg107, Lys111, Phe118, Arg121 and Lys165 in the C-terminal domain, were shown to be involved in ssDNA binding. Moreover, we evaluated the binding of the PriC mutants to the ssDNA-binding protein (SSB) complex. Five residues, Phe118, Arg121, Arg129, Tyr152 and Arg155 in the C-terminal domain of PriC, were shown to be involved in SSB binding in the presence of ssDNA. On the basis of these results, we propose a structural model of the C-terminal domain of PriC and discuss how the interactions of PriC with SSB and ssDNA may contribute to the regulation of PriC-dependent replication restart.

Taurine is one of the osmolytes that maintain the structure of proteins in cells exposed to denaturing environmental stressors. Recently, cryoelectron tomographic analysis of eukaryotic cells has revealed that their cytoplasms are crowded with proteins. Such crowding conditions would be expected to hinder the efficient folding of nascent polypeptide chains. Therefore, we examined the role of taurine on the folding of denatured and reduced lysozyme, as a model protein, under a crowding condition. The results confirmed that taurine had a better effect on protein folding than did β-alanine, which has a similar chemical structure, when the protein to be folded was present at submillimolar concentration. NMR analyses further revealed that under the crowding condition, taurine had more interactions than did β-alanine with the lysozyme molecule in both the folded and denatured states. We concluded that taurine improves the folding of the reduced lysozyme at submillimolar concentration to allow it to interact more favorably with the lysozyme molecule. Thus, the role of taurine, as an osmolyte in vivo, may be to assist in the efficient folding of proteins.

The heat shock protein HspQ (YccV) of Escherichia coli has been proposed to participate in the retardation of replication initiation in cells with the dnaA508 allele. In this study, we have determined the 2.5-A-resolution X-ray structure of the trimer of HspQ, which is also the first structure of a member of the YccV superfamily. The acidic character of the HspQ trimer suggests an interaction surface with basic proteins. From these results, we discuss the cellular function of HspQ, including its relationship with the DnaA508 protein. This article is protected by copyright. All rights reserved.

Biotherapeutics have been clinically used since the 1990s. Recently, next-generation optimized biotherapeutics, which are expected to act on the same molecular target as their predecessors with further properties by antibody-drug conjugation, radiolabeling, PEGylation and glycoconjugation, are on the market. This article reviews recent next-generation optimized biotherapeutics. Moreover, since trials of protein engineering for biotherapeutics have been conducted, these preclinical approaches are also described. This article is part of a Special Issue entitled: Recent advances in molecular engineering of antibody.

The β-N-acetylglucosaminidase from the hyperthermophilic bacteria Thermotoga maritima (NagA) hydrolyzes chitooligomers into monomer β-N-acetylglucosamine. Although NagA contains a highly conserved sequence motif found in glycoside hydrolase (GH) family 3, it can be distinguished from other GH family 3 β-N-acetylglucosaminidases by its substrate specificity and biological assembly. To investigate its unique structure around the active site, we determined the crystal structure of NagA at a resolution of 2.43 A. The NagA forms a dimer structure in which the monomer structure consists of an N- and a C-terminal domain. The dimer structure exhibits high solvation free energy for dimer formation. From mutagenesis analyses, the catalytic nucleophile and general acid–base residues were supposed to be Asp245 and His173, respectively. The most striking characteristic of NagA was that it forms the active site cleft from the N-terminal domain and the C-terminal domain of the next polypeptide chain, whereas the other two-domain GH family 3 enzymes form the site within the same molecule. Another striking feature is that the loops located around the active site show high flexibility. One of the flexible loops contains the general acid–base His173 and was thought to be involved in substrate distortion during catalysis. In addition, a loop in close contact with the active site, which comes from the C-terminal domain of the next polypeptide chain, contains a region of high B-factor values, indicating the possibility that the C-terminal domain is involved in catalysis. These results suggest that the dimer structure of NagA is important for its activity and thermostability. Database Structural data are available in the Protein Data Bank under accession number 3WO8. Structured digital abstract NagA and NagA bind by x-ray crystallography (View interaction)

PriB is a basic 10-kDa protein that acts as a facilitator in PriA-dependent replication restart in Escherichia coli. PriB has an OB-fold dimer structure and exhibits single-stranded DNA (ssDNA)-binding activities similar to single-stranded binding protein (SSB). In this study, we examined PriB's interaction with ssDNA (oligo-dT35, -dT15, and -dT7) using heteronuclear NMR analysis. Interestingly, (1)H or (15)N chemical shift changes of the PriB main-chain showed two distinct modes using oligo-dT35. The chemical shift perturbation sites in the primary mode were consistent with the main contact site in PriB-ssDNA, which was previously determined by crystal structure analysis. The results also suggested that approximately 8nt in ssDNA was the main contact site to PriB. In the secondary mode, residues in the α-helix region (His57-Ser65) and in β4-loop3-β5 were mainly perturbed. On the other hand, we examined the state of ssDNA by FRET using 5'-Cy3- and 3'-Cy5-modified oligo-dT35. As the PriB concentration increased, two-step saturation curves were observed in the FRET assay, suggesting a compact structure of ssDNA. Moreover, we confirmed two-step PriB binding to oligo-dT35 using EMSA. The pH dependence of FRET suggested contribution of the His residues. Therefore, we prepared His mutants of PriB and found that His64 in the α-helix region contributed to the second interaction between PriB and ssDNA using FRET and EMSA. Thus, from a structural standpoint, we suggested the role of His64 on the compactness of the PriB-ssDNA complex and on the positive cooperativity of PriB.