Oligomeric state of the N-terminal domain of DnaT for replication restart in Escherichia coli.

DNA replication stops when chemical or physical damage occurs to the DNA. Repairing genomic DNA and reloading the replication helicase are crucial steps for restarting DNA replication. The Escherichia coli primosome is a complex of proteins and DNA responsible for reloading the replication helicase DnaB. DnaT, a protein found in the primosome complex, contains two functional domains. The C-terminal domain (89-179) forms an oligomeric complex with single-stranded DNA. Although the N-terminal domain (1-88) forms an oligomer, the specific residues responsible for this oligomeric structure have not yet been identified. In this study, we proposed that the N-terminal domain of DnaT has a dimeric antitoxin structure based on its primary sequence. Based on the proposed model, we confirmed the site of oligomerization in the N-terminal domain of DnaT through site-directed mutagenesis. The molecular masses and thermodynamic stabilities of the site-directed mutants located at the dimer interface, namely Phe42, Tyr43, Leu50, Leu53, and Leu54, were found to be lower than those of the wild-type. Moreover, we observed a decrease in the molecular masses of the V10S and F35S mutants compared to the wild-type DnaT. NMR analysis of the V10S mutant revealed that the secondary structure of the N-terminal domain of DnaT was consistent with the proposed model. Additionally, we have demonstrated that the stability of the oligomer formed by the N-terminal domain of DnaT is crucial for its function. Based on these findings, we propose that the DnaT oligomer plays a role in replication restart in Escherichia coli.

Stability of hen egg-white lysozyme during embryonic development.

It is of interest to determine whether and how egg-white proteins are maintained in fertile eggs. We previously observed that egg-white ovalbumin attained high stability during embryogenesis. Herein, we observed that the total mass of egg white and that of its gross protein content showed a decrease according to the days of incubation. The total bacteriolytic activity also lowered, in accord with previous observations. We purified lysozyme from egg-white samples on several incubation days. These purified lysozyme proteins were observed to have enzymatic and bacteriolytic activities against Micrococcus lysodeikticus as well as growth-inhibition potency against Staphylococcus aureus. As the embryogenesis proceeded, the purified lysozyme showed changes in Km and Vmax, a small decrease in the denaturation temperature, and symptoms of an increase in surface hydrophobicity. These results indicate that the lysozyme protein maintained its enzymatic and antibacterial activities until the late period of incubation while undergoing slight conformational changes.

Compound screening identified gossypetin and isoquercitrin as novel inhibitors for amyloid fibril formations of Vλ6 proteins associated with AL amyloidosis.

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.

Improvement of the affinity of an anti-rat P2X4 receptor antibody by introducing electrostatic interactions.

We have recently developed a mouse monoclonal antibody (12-10H) binding to the head domain region in rat P2X4 receptor (rP2X4R, which is crucial for the pathogenesis of neuropathic pain) expressed on the cell with the highest binding affinity (KD = 20 nM). However, the 12-10H antibody failed to detect endogenously expressed P2X4Rs in microglia isolated from the spinal cord of rats whose spinal nerves were injured. Then, we prepared R5 mutant, in which five arginine residues were introduced into variable regions except for the "hot spot" in the 12-10H antibody to increase electrostatic interactions with the head domain, an anionic region, in rP2X4R. The mutation resulted in an increase of 50-fold in the affinity of the R5 mutant for the head domain with respect to the intact 12-10H antibody. As a result, detection of P2X4Rs endogenously expressed on primary cultured microglial cells originated from the neonatal rat brain and spinal cord microglia isolated from a rat model of neuropathic pain was achieved. These findings suggest a strategy to improve the affinity of a monoclonal antibody for an anionic antigen by the introduction of several arginine residues into variable regions other than the "hot spot" in the paratope.

Structural Analysis of Hen Egg Lysozyme Refolded after Denaturation at Acidic pH.

Protein structures fluctuate in solution; therefore, proteins have multiple stable structures that are slightly different from each other. In this study, we determined the crystal structure of hen egg lysozyme refolded after denaturation at acidic pH (rHEL) and found a structure different from native HEL (nHEL). The different local conformations of the peptide bond between Asp101 and Gly102 found in the crystal structure was supported by the NMR results for nHEL and rHEL. The NMR experiments also showed shifts in the heteronuclear single quantum coherence signals derived from Thr43 and Asp52. The chemical shift change of Asp52 could be explained by the crystal structure of rHEL, showing the conformational change of Tyr53, whose phenol ring directly lies on the main chain of Asp52. The catalytic activity of rHEL was similar to that of nHEL, indicating that the conformational change had little effect on activity. In contrast, conformational changes could be detected by the binding of monoclonal antibodies against HEL. Using multiple methods, we successfully detected the unusual structure of HEL, which might be another stable structure of HEL in solution.

Functional Characterization of Pembrolizumab Produced in Nicotiana benthamiana Using a Rapid Transient Expression System.

The striking innovation and clinical success of immune checkpoint inhibitors (ICIs) have undoubtedly contributed to a breakthrough in cancer immunotherapy. Generally, ICIs produced in mammalian cells requires high investment, production costs, and involves time consuming procedures. Recently, the plants are considered as an emerging protein production platform due to its cost-effectiveness and rapidity for the production of recombinant biopharmaceuticals. This study explored the potential of plant-based system to produce an anti-human PD-1 monoclonal antibody (mAb), Pembrolizumab, in Nicotiana benthamiana. The transient expression of this mAb in wild-type N. benthamiana accumulated up to 344.12 ± 98.23 µg/g fresh leaf weight after 4 days of agroinfiltration. The physicochemical and functional characteristics of plant-produced Pembrolizumab were compared to mammalian cell-produced commercial Pembrolizumab (Keytruda®). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis results demonstrated that the plant-produced Pembrolizumab has the expected molecular weight and is comparable with the Keytruda®. Structural characterization also confirmed that both antibodies have no protein aggregation and similar secondary and tertiary structures. Furthermore, the plant-produced Pembrolizumab displayed no differences in its binding efficacy to PD-1 protein and inhibitory activity between programmed cell death 1 (PD-1) and programmed cell death ligand 1 (PD-L1) interaction with the Keytruda®. In vitro efficacy for T cell activation demonstrated that the plant-produced Pembrolizumab could induce IL-2 and IFN-γ production. Hence, this proof-of-concept study showed that the plant-production platform can be utilized for the rapid production of functional mAbs for immunotherapy.

Expression and Functional Evaluation of Recombinant Anti-receptor Activator of Nuclear Factor Kappa-B Ligand Monoclonal Antibody Produced in Nicotiana benthamiana.

Denosumab, an anti-receptor activator of nuclear factor-kappa B ligand antibody (anti-RANKL), is a fully human monoclonal antibody (mAb) available for the treatment of osteoporosis. In the present study, an anti-RANKL mAb was transiently expressed using the geminiviral expression system in Nicotiana benthamiana, and the functional activity of the plant-produced mAb was determined. The highest expression level of the plant-produced mAb was found at 8 days post-infiltration, and it was estimated to be 0.5 mg/g leaf fresh weight. The recombinant mAb from the plant crude extracts was purified by using Protein A affinity column chromatography. The plant-produced mAb demonstrated good in vitro affinity binding with human RANKL, as determined by RANKL-ELISA binding. The function of the plant-produced mAb was evaluated in vitro. CD14-positive cells isolated from human peripheral blood mononuclear cells (PBMCs) were cultured in vitro in the presence of human RANKL and macrophage-colony-stimulating factor (M-CSF) to stimulate osteoclastogenesis. The results demonstrated that plant-produced mAb could significantly decrease the number of osteoclasts compared to commercial denosumab. These results demonstrated that the plant-produced mAb has the potential to inhibit osteoclast differentiation and that it could be considered for osteoporosis treatment.

Effect of O-glycosylation on amyloid fibril formation of the variable domain in the Vλ6 light chain mutant Wil.

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.

Analysis of binding residues in monoclonal antibody with high affinity for the head domain of the rat P2X4 receptor.

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.

A structural model of the PriB-DnaT complex in Escherichia coli replication restart.

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 dynamic simulation. From these data, we propose a structural model that provides insight into the PriB-DnaT interaction.

Principal component analysis of data from NMR titration experiment of uniformly 15N labeled amyloid beta (1-42) peptide with osmolytes and phenolic compounds.

A simple NMR method to analyze the data obtained by NMR titration experiment of amyloid formation inhibitors against uniformly 15N-labeled amyloid-ß 1-42 peptide (Aß(1-42)) was described. By using solution nuclear magnetic resonance (NMR) measurement, the simplest method for monitoring the effects of Aß fibrilization inhibitors is the NMR chemical shift perturbation (CSP) experiment using 15N-labeled Aß(1-42). However, the flexible and dynamic nature of Aß(1-42) monomer may hamper the interpretation of CSP data. Here we introduced principal component analysis (PCA) for visualizing and analyzing NMR data of Aß(1-42) in the presence of amyloid inhibitors including high concentration osmolytes. We measured 1H-15N 2D spectra of Aß(1-42) at various temperatures as well as of Aß(1-42) with several inhibitors, and subjected all the data to PCA (PCA-HSQC). The PCA diagram succeeded in differentiating the various amyloid inhibitors, including epigallocatechin gallate (EGCg), rosmarinic acid (RA) and curcumin (CUR) from high concentration osmolytes. We hypothesized that the CSPs reflected the conformational equilibrium of intrinsically disordered Aß(1-42) induced by weak inhibitor binding rather than the specific molecular interactions.

DnaB helicase is recruited to the replication initiation complex via binding of DnaA domain I to the lateral surface of the DnaB N-terminal domain.

The DNA replication protein DnaA in Escherichia coli constructs higher-order complexes on the origin, oriC, to unwind this region. DnaB helicase is loaded onto unwound oriC via interactions with the DnaC loader and the DnaA complex. The DnaB-DnaC complex is recruited to the DnaA complex via stable binding of DnaB to DnaA domain I. The DnaB-DnaC complex is then directed to unwound oriC via a weak interaction between DnaB and DnaA domain III. Previously, we showed that Phe46 in DnaA domain I binds to DnaB. Here, we searched for the DnaA domain I-binding site in DnaB. The DnaB L160A variant was impaired in binding to DnaA complex on oriC but retained its DnaC-binding and helicase activities. DnaC binding moderately stimulated DnaA binding of DnaB L160A, and loading of DnaB L160A onto oriC was consistently and moderately inhibited. In a helicase assay with partly single-stranded DNA bearing a DnaA-binding site, DnaA stimulated DnaB loading, which was strongly inhibited in DnaB L160A even in the presence of DnaC. DnaB L160A was functionally impaired in vivo On the basis of these findings, we propose that DnaB Leu160 interacts with DnaA domain I Phe46 DnaB Leu160 is exposed on the lateral surface of the N-terminal domain, which can explain unobstructed interactions of DnaA domain I-bound DnaB with DnaC, DnaG primase, and DnaA domain III. We propose a probable structure for the DnaA-DnaB-DnaC complex, which could be relevant to the process of DnaB loading onto oriC.

Structural and In Vitro Functional Analyses of Novel Plant-Produced Anti-Human PD1 Antibody.

Immunotherapy has emerged as a promising and effective treatment for cancer. The frequently used immunotherapy agents are immune checkpoint inhibitors, such as antibodies specific to PD1, PD-L1, or CTLA-4. However, these drugs are highly expensive, and most people in the world cannot access the treatment. The development of recombinant protein production platforms that are cost-effective, scalable, and safe is needed. Plant platforms are attractive because of their low production cost, speed, scalability, lack of human and animal pathogens, and post-translational modifications that enable them to produce effective monoclonal antibodies. In this study, an anti-PD1 IgG4 monoclonal antibody (mAb) was transiently produced in Nicotiana benthamiana leaves. The plant-produced anti-PD1 mAb was compared to the commercial nivolumab produced in CHO cells. Our results showed that both antibodies have similar protein structures, and the N-glycans on the plant-produced antibody lacks plant-specific structures. The PD1 binding affinity of the plant-produced and commercial nivolumab, determined by two different techniques, that is, enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR), are also comparable. Plant-produced nivolumab binds to human PD1 protein with high affinity and specificity, blocks the PD-1/PD-L1 interaction, and enhances T cell function, comparable to commercial nivolumab. These results confirmed that plant-produced anti-PD1 antibody has the potential to be effective agent for cancer immunotherapy.

Insight into the interaction between PriB and DnaT on bacterial DNA replication restart: Significance of the residues on PriB dimer interface and highly acidic region on DnaT.

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.

Evidence for detection of rat P2X4 receptor expressed on cells by generating monoclonal antibodies recognizing the native structure.

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.

Selective and reversible modification of kinase cysteines with chlorofluoroacetamides.

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.

Inhibition of amyloid fibril formation in the variable domain of λ6 light chain mutant Wil caused by the interaction between its unfolded state and epigallocatechin-3-O-gallate.

BACKGROUND: Light chains are abnormally overexpressed from disordered monoclonal B-cells and form amyloid fibrils, which are then deposited on the affected organ, leading to a form of systemic amyloidosis known as AL (Amyloid Light chain) amyloidosis. A green tea catechin, epigallocatechin-3-O-gallate (EGCG), which is thought to inhibit various amyloidoses, is a potent inhibitor of amyloid fibril formation in AL amyloidosis. METHODS: An amyloidogenic variable domain in λ6 light chain mutant, Wil was incubated in the presence of EGCG. The incubation products were analyzed by SDS-PAGE and reverse-phase HPLC. The interaction between Wil and EGCG was observed by using NMR and tryptophan fluorescence. RESULTS: EGCG inhibited the amyloid fibril formation of Wil at pH 7.5 and 42 °C. Under these conditions, most Wil populations were in the unfolded state and several chemical reactions, i.e., oxidation and/or covalent bond oligomerization could be induced by auto-oxidated EGCG. Moreover, we found that EGCG bound to the unfolded state of Wil with higher affinity (Kd = 7 µM). CONCLUSIONS: Inhibition of amyloid fibril formation of Wil was caused by 1) EGCG binding to unfolded state rather than folded state and 2) chemical modifications of Wil by auto oxidation of EGCG. GENERAL SIGNIFICANCE: In the competitive formation of amyloid fibrils and off-pathway oligomers, EGCG produces the latter immediately after it preferentially binds to the unfolded state. It may be general mechanism of EGCG inhibition for amyloidosis.

Crystallization of Human Erythrocyte Band 3, the anion exchanger, at the International Space Station "KIBO".

Band 3 mediates the Cl- and HCO3- exchange across the red blood cell membrane and plays a pivotal role for delivering oxygen appropriately to metabolically active tissues. For understanding molecular mechanisms, it is essential to know the structure and function relationship. In terrestrial environments, however, nobody could make good quality crystals of Band 3 for the X-ray crystallographic study. In this study, we purified the transmembrane domain of Band 3 from human red blood cells and crystallized the purified Band 3 without the Fab fragment at the International Space Station "KIBO" under microgravity environments.

Tyrosine Sulfation Restricts the Conformational Ensemble of a Flexible Peptide, Strengthening the Binding Affinity for an Antibody.

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.

X-ray crystal structure of Escherichia coli HspQ, a protein involved in the retardation of replication initiation.

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-Å-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.