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.

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.

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.

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.

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.

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.

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.

The Structure of an Archaeal ß-Glucosaminidase Provides Insight into Glycoside Hydrolase Evolution.

The archaeal exo-ß-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 Å resolution. The structure reveals that a monomeric form of GlmA shares structural homology with GH42 ß-galactosidases, whereas most of the spatial positions of the active site residues are identical to those of GH35 ß-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 ß-galactosidase. However, GlmA can differentiate glucosamine from galactose based on one charged residue while using the "evolutionary heritage residue" it shares with GH35 ß-galactosidase. Our study suggests that GH35 and GH42 ß-galactosidases evolved by exploiting the structural features of GlmA.

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.

Involvement of histidine in complex formation of PriB and single-stranded DNA.

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 ß4-loop3-ß5 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.

Role of the osmolyte taurine on the folding of a model protein, hen egg white lysozyme, under a crowding condition.

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 ß-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 ß-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.

Domain separation and characterization of PriC, a replication restart primosome factor in Escherichia coli.

In Escherichia coli the oriC-independent primosome plays an essential role in replication restart after dissociation of the replication DNA-protein complex by DNA damage. Primosome is thought to form via two pathways: one PriA dependent and the other PriA independent. PriC is a key protein in the replication restart of the PriA-independent pathway. In this study, we determined that PriC was divided into two domains. Then, we obtained information that: (i) the C-terminal domain preferentially binds to single-stranded DNA (ssDNA); (ii) the binding of PriC to ssDNA depends on salt concentration; and (iii) the binding site size of PriC is approximately 7-9 nucleotides. The protease digestion of PriC suggested that a possible DNA-binding site is the N-terminus of the C-terminal domain where basic amino acid residues are concentrated. Interestingly, α-helical induction of the C-terminal domain of PriC occurred after the addition of DNAs. Also, we examined the role of heptad repeat of leucine or valine residues in the C-terminal domain and PriC oligomerization. This study describes the structure and function analysis of PriC which forms the primosome complex in replication restart.

Arg 901 in the AE1 C-terminal tail is involved in conformational change but not in substrate binding.

In our previous paper, we demonstrated that Arg 901 in the C-terminal tail of human AE1 (band 3, anion exchanger 1) had a functional role in conformational change during anion exchange. To further examine how Arg 901 is involved in conformational change, we expressed various Arg 901 mutants and alanine mutants of the C-terminal tail (from Leu 886 to Val 911) on the plasma membrane of Saccharomyces cerevisiae and evaluated the kinetic parameters of sulfate ion transport. As a result, Vmax decreased as the hydrophobicities of the 901st and peripheral hydrophilic residues increased, indicating that the hydrophobicity of the C-terminal residue is involved in the conformational change. We also found the alkali and protease resistance of the C-terminal region after Arg 901 modification with hydroxyphenylglyoxal (HPG) or phenylglyoxal (PG), a hydrophobic reagent. These results suggested that the increased hydrophobicity of the C-terminal region around Arg 901 leads to inefficient conformational change by the newly produced hydrophobic interaction.

The DnaA N-terminal domain interacts with Hda to facilitate replicase clamp-mediated inactivation of DnaA.

DnaA activity for replication initiation of the Escherichia coli chromosome is negatively regulated by feedback from the DNA-loaded form of the replicase clamp. In this process, called RIDA (regulatory inactivation of DnaA), ATP-bound DnaA transiently assembles into a complex consisting of Hda and the DNA-clamp, which promotes inter-AAA+ domain association between Hda and DnaA and stimulates hydrolysis of DnaA-bound ATP, producing inactive ADP-DnaA. Using a truncated DnaA mutant, we previously demonstrated that the DnaA N-terminal domain is involved in RIDA. However, the precise role of the N-terminal domain in RIDA has remained largely unclear. Here, we used an in vitro reconstituted system to demonstrate that the Asn-44 residue in the N-terminal domain of DnaA is crucial for RIDA but not for replication initiation. Moreover, an assay termed PDAX (pull-down after cross-linking) revealed an unstable interaction between a DnaA-N44A mutant and Hda. In vivo, this mutant exhibited an increase in the cellular level of ATP-bound DnaA. These results establish a model in which interaction between DnaA Asn-44 and Hda stabilizes the association between the AAA+ domains of DnaA and Hda to facilitate DnaA-ATP hydrolysis during RIDA.

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.

Orally available pyridinylpyrimidine derivatives as novel RANKL-induced osteoclastogenesis inhibitors.

An HTS campaign led to the identification of 4-pyrroldino-2-(pyridin-2-yl)pyrimidine compound 1 as an RANKL-induced osteoclastogenesis inhibitor. The compound 1 showed high clearance values in microsomes, however. Modification of the pyrrolidino group to a benzylamino group improved human microsomal stability with a slight loss of in vitro activity. Substitution at the ortho position of the benzyl group ameliorated in vitro activity, and further fluorination of the benzyl group improved microsomal stability in rodents. Representative members of this series, compounds 20 and 23, exhibited efficacy in RANKL-induced osteopenic mice when administered orally at 0.3 mg/kg.

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.

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.

Mutation of His 834 in human anion exchanger 1 affects substrate binding.

Anion exchanger 1 (AE1 or band 3) is responsible for Cl(-)-HCO3(-) exchange on erythrocyte membrane. Previously, we showed that band 3 is fixed in an inward-facing conformation by specific modification of His 834 with DEPC, resulting in a strong inhibition of its anion transport activity. To clarify the physiological role of His 834, we evaluated the sulfate transport activities of various band 3 mutants: different mutants at His 834 and alanine mutants of peripheral residues around 834 (Lys 829-Phe 836) in yeast cell membranes. The K(m) values of the His 834 mutants were 4-10 times higher than that of the wild type, while their V(max) values were barely lower than that of wild type. Meanwhile, the K(m) values of the peripheral alanine mutants were only slightly increased. These data suggest that His 834 is critically important for the efficient binding of sulfate anion, but not for the conformational change induced by substrate binding.

Factor G utilizes a carbohydrate-binding cleft that is conserved between horseshoe crab and bacteria for the recognition of beta-1,3-D-glucans.

In the horseshoe crab, the recognition of beta-1,3-D-glucans by factor G triggers hemolymph coagulation. Factor G contains a domain of two tandem xylanase Z-like modules (Z1-Z2), each of which recognizes beta-1,3-D-glucans. To gain an insight into the recognition of beta-1,3-D-glucans from a structural view point, recombinants of Z1-Z2, the C-terminal module Z2, Z2 with a Cys to Ala substitution (Z2A), and its tandem repeat Z2A-Z2A were characterized. Z2 and Z1-Z2, but not Z2A and Z2A-Z2A, formed insoluble aggregates at higher concentrations more than approximately 30 and 3 microM, respectively. Z1-Z2 and Z2A-Z2A bound more strongly to an insoluble beta-1,3-D-glucan (curdlan) than Z2A. The affinity of Z2A for a soluble beta-1,3-D-glucan (laminarin) was equivalent to those of Z1-Z2, Z2A-Z2A, and native factor G, suggesting that the binding of a single xylanase Z-like module prevents the subsequent binding of another module to laminarin. Interestingly, Z2A as well as intact factor G exhibited fungal agglutinating activity, and fungi were specifically detected with fluorescently tagged Z2A by microscopy. The chemical shift perturbation of Z2A induced by the interaction with laminaripentaose was analyzed by nuclear magnetic resonance spectroscopy. The ligand-binding site of Z2A was located in a cleft on a beta-sheet in a predicted beta-sandwich structure, which was superimposed onto cleft B in a cellulose-binding module of endoglucanase 5A from the soil bacterium Cellvibrio mixtus. We conclude that the pattern recognition for beta-1,3-D-glucans by factor G is accomplished via a carbohydrate-binding cleft that is evolutionally conserved between horseshoe crab and bacteria.