Structural basis of the specific interaction of SMRT corepressor with histone deacetylase 4.

Modification of chromatin and related transcription factors by histone deacetylases (HDACs) is one of the major strategies for controlling gene expression in eukaryotes. The HDAC domains of class IIa HDACs repress the respective target genes by interacting with the C-terminal region of the silencing mediator for retinoid and thyroid receptor (SMRT) repression domain 3 (SRD3c). However, latent catalytic activity suggests that their roles as deacetylases in gene regulation are unclear. Here, we found that two conserved GSI-containing motifs of SRD3c are critical for HDAC4 binding. Two SMRT peptides including these motifs commonly form a ß-hairpin structure in the cleft and block the catalytic entry site of HDAC4. They interact mainly with class IIa HDAC-specific residues of HDAC4 in a closed conformation. Structure-guided mutagenesis confirmed critical interactions between the SMRT peptides and HDAC4 and -5 as well as the contribution of the Arg1369 residue in the first motif for optimal binding to the two HDACs. These results indicate that SMRT binding does not activate the cryptic deacetylase activity of HDAC4 and explain how class IIa HDACs and the SMRT-HDAC3 complex are coordinated during gene regulation.

Identification of BR101549 as a lead candidate of non-TZD PPARγ agonist for the treatment of type 2 diabetes: Proof-of-concept evaluation and SAR.

The new class of PPARgamma non-TZD agonist originally derived from the backbone of anti-hypertensive Fimasartan, BR101549, was identified as a potential lead for anti-diabetic drug development. The X-ray crystallography of BR101549 with PPARgamma ligand binding domain (LBD) revealed unique binding characteristics versus traditional TZD full agonists. The lead candidate, BR101549, has been found activating PPARgamma to the level of Pioglitazone in vitro and indeed has demonstrated its effects on blood glucose control in mouse proof-of-concept evaluation. The attempts to improve its metabolic stability profile through follow-up SAR including deuterium incorporation have been also described.

Discovery of BR102375, a new class of non-TZD PPARγ full agonist for the treatment of type 2 diabetes.

As a potential treatment of type 2 diabetes, a novel PPARγ non-TZD full agonist, compound 18 (BR102375) was identified from the original lead BR101549 by the SAR efforts of the labile metabolite control through bioisosteres approach. In vitro assessments of BR102375 demonstrated its activating potential of PPARγ comparable to Pioglitazone as well as the induction of related gene expressions. Further in vivo evaluation of BR102375 in diabetic rodent models successfully proved its glucose lowering effect as a potential antidiabetic agent, but the anticipated suppression of weight gain was not evident. The X-ray co-crystal analysis of BR102375-PPARγ LBD unexpectedly revealed binding modes totally different from those of BR101549, which was found, instead, closely resembled to those of TZD full agonists.

Effects of acid etching and calcium chloride immersion on removal torque and bone-cutting ability of orthodontic mini-implants.

INTRODUCTION: The 2-fold purpose of this study was to evaluate the effects of acid etching and calcium chloride immersion on removal torque and the bone-cutting ability of orthodontic mini-implants (OMIs). METHODS: For the removal torque part of the study, 3 types of OMIs (titanium alloy) were evaluated in a rabbit model: OMIs with acid surface etching with and without calcium chloride immersion (ECG and EG, respectively) and a control group (CG), in which the OMIs had an untreated, machined surface. We inserted 126 OMIs (42 OMIs per type) into both tibias of 21 male rabbits (5 months of age) with body weights of 3.0 to 3.5 kg. Removal torque was evaluated after 1, 4, and 7 weeks. To determine the OMIs' bone-cutting ability, total insertion time to place an OMI 6 mm into artificial bone was measured (6 OMIs per group). RESULTS: Removal torque values for the EG (3.97 ± 0.52 Ncm) and ECG (4.21 ± 0.44 Ncm) were statistically and significantly higher than those of the CG (3.02 ± 0.53 Ncm) 1 week after implantation (P <0.05). The ECG (6.54 ± 0.50, 6.61 ± 0.66 Ncm) showed the highest removal torque value followed by the EG (5.68 ± 0.58, 5.89 ± 0.70 Ncm) and CG (3.43 ± 0.62, 3.38 ± 0.54 Ncm) at 4 and 7 weeks after implantation (P <0.05). Removal torque did not change over time with the CG, but with the ECG and EG, it was significantly higher in weeks 4 and 7 than in week 1 (P <0.05). Total insertion time was significantly greater for the EG than for the ECG and CG (P <0.05). CONCLUSIONS: Treating OMIs with a calcium chloride solution improved the initial bone reaction by preventing contamination of the implant surface, and increasing the surface roughness of OMIs by acid etching enhanced their stability without decreasing the bone-cutting ability compared with OMIs without surface treatment.

Crystal structure of human POP1 and its distinct structural feature for PYD domain.

Inflammatory caspases, such as caspase-1, which is critical for the innate immune response, are activated upon the formation of a molecular complex called the inflammasome. The inflammasome is composed of three proteins, the Nod-like receptor (NLRP, NLRC or AIM2), apoptosis associated speck-loke protein containing a caspase-recruitment domain (ASC), and caspase-1. ASC is an adaptor molecule that contains an N-terminal PYD domain and a C-terminal CARD domain for interaction with other proteins. Upon activation, the N-terminal PYD of ASC homotypically interacts with the PYD domain of the Nod-like receptor, while its C-terminal CARD homotypically interacts with the CARD domain of caspase-1. PYD only protein 1 (POP1) negatively regulates inflammatory response by blocking the formation of the inflammasome. POP1 directly binds to ASC via a PYD:PYD interaction, thereby preventing ASC recruitment to Nod-like receptor NLRPs. POP1-mediated regulation of inflammation is of great biological importance. Here, we report the crystal structure of human POP1 and speculate about the inhibitory mechanism of POP1-mediated inflammasome formation based on the current structure.

Structural study of the RIPoptosome core reveals a helical assembly for kinase recruitment.

Receptor interaction protein kinase 1 (RIP1) is a molecular cell-fate switch. RIP1, together with Fas-associated protein with death domain (FADD) and caspase-8, forms the RIPoptosome that activates apoptosis. RIP1 also associates with RIP3 to form the necrosome that triggers necroptosis. The RIPoptosome assembles through interactions between the death domains (DDs) of RIP1 and FADD and between death effector domains (DEDs) of FADD and caspase-8. In this study, we analyzed the overall structure of the RIP1 DD/FADD DD complex, the core of the RIPoptosome, by negative-stain electron microscopy and modeling. The results show that RIP1 DD and FADD DD form a stable complex in vitro similar to the previously described Fas DD/FADD DD complex, suggesting that the RIPoptosome and the Fas death-inducing signaling complex share a common assembly mechanism. Both complexes adopt a helical conformation that requires type I, II, and III interactions between the death domains.

A comprehensive manually curated protein-protein interaction database for the Death Domain superfamily.

The Death Domain (DD) superfamily, which is one of the largest classes of protein interaction modules, plays a pivotal role in apoptosis, inflammation, necrosis and immune cell signaling pathways. Because aberrant or inappropriate DD superfamily-mediated signaling events are associated with various human diseases, such as cancers, neurodegenerative diseases and immunological disorders, the studies in these fields are of great biological and clinical importance. To facilitate the understanding of the molecular mechanisms by which the DD superfamily is associated with biological and disease processes, we have developed the DD database (http://www.deathdomain.org), a manually curated database that aims to offer comprehensive information on protein-protein interactions (PPIs) of the DD superfamily. The DD database was created by manually curating 295 peer-reviewed studies that were published in the literature; the current version documents 175 PPI pairs among the 99 DD superfamily proteins. The DD database provides a detailed summary of the DD superfamily proteins and their PPI data. Users can find in-depth information that is specified in the literature on relevant analytical methods, experimental resources and domain structures. Our database provides a definitive and valuable tool that assists researchers in understanding the signaling network that is mediated by the DD superfamily.

Molecular basis for homo-dimerization of the CIDE domain revealed by the crystal structure of the CIDE-N domain of FSP27.

FSP27 (CIDE-3 in humans) plays critical roles in lipid metabolism and apoptosis and is known to be involved in regulation of lipid droplet (LD) size and lipid storage and apoptotic DNA fragmentation. Given that CIDE-containing proteins including FSP27 are associated with many human diseases including cancer, aging, diabetes, and obesity, studies of FSP27 and other CIDE-containing proteins are of great biological importance. As a first step toward elucidating the molecular mechanisms of FSP27-mediated lipid droplet growth and apoptosis, we report the crystal structure of the CIDE-N domain of FSP27 at a resolution of 2.0 Å. The structure revealed a possible biologically important homo-dimeric interface similar to that formed by the hetero-dimeric complex, CAD/ICAD. Comparison with other structural homologues revealed that the PB1 domain of BEM1P, ubiquitin-like domain of BAG6 and ubiquitin are structurally similar proteins. Our homo-dimeric structure of the CIDE-N domain of FSP27 will provide important information that will enable better understanding of the function of FSP27.

Crystal structure of Rab6A'(Q72L) mutant reveals unexpected GDP/Mg²âº binding with opened GTP-binding domain.

The Ras small G protein-superfamily is a family of GTP hydrolases whose activity is regulated by GTP/GDP binding states. Rab6A, a member of the Ras superfamily, is involved in the regulation of vesicle trafficking, which is critical for endocytosis, biosynthesis, secretion, cell differentiation and cell growth. Rab6A exists in two isoforms, termed RabA and Rab6A'. Substitution of Gln72 to Leu72 (Q72L) at Rab6 family blocks GTP hydrolysis activity and this mutation usually causes the Rab6 protein to be constitutively in an active form. Here, we report the crystal structure of the human Rab6A'(Q72L) mutant form at 1.9Å resolution. Unexpectedly, we found that Rab6A'(Q72L) possesses GDP/Mg(2+) in the GTP binding pockets, which is formed by a flexible switch I and switch II. Large conformational changes were also detected in the switch I and switch II regions. Our structure revealed that the non-hydrolysable, constitutively active form of Rab6A' can accommodate GDP/Mg(2+) in the open conformation.

Crystallization and preliminary X-ray crystallographic studies of Rab6A'(Q72L): a GTP-locked form.

Rab6A, a member of the Ras superfamily of small G proteins, is involved in the regulation of vesicle trafficking, which is critical for endocytosis, cell differentiation and cell growth. Rab6A can exist in two isoforms termed Rab6A and Rab6A'. The substitution of Gln72 by Leu (Q72L) in the Rab6A family blocks GTP-hydrolysis activity, and this mutation usually causes the Rab6A protein to be in a constitutively active form. In this study, in order to understand the functional uniqueness of Rab6A' and the molecular mechanism of the control of activity by GTP and GDP from the crystal structure, a Rab6A'(Q72L) mutant form was overexpressed in Escherichia coli with an engineered N-terminal His tag. Rab6A'(Q72L) was then purified to homogeneity and crystallized at 293 K. X-ray diffraction data were collected to a resolution of 1.9 Å from a crystal belonging to space group P22(1)2(1) with unit-cell parameters a = 36.84, b = 96.78, c = 109.99 Å. The asymmetric unit was estimated to contain two molecules.

The structure of a novel antibody against the spike protein inhibits Middle East respiratory syndrome coronavirus infections.

Middle East respiratory syndrome coronavirus (MERS-CoV) is a zoonotic virus, responsible for outbreaks of a severe respiratory illness in humans with a fatality rate of 30%. Currently, there are no vaccines or United States food and drug administration (FDA)-approved therapeutics for humans. The spike protein displayed on the surface of MERS-CoV functions in the attachment and fusion of virions to host cellular membranes and is the target of the host antibody response. Here, we provide a molecular method for neutralizing MERS-CoV through potent antibody-mediated targeting of the receptor-binding subdomain (RBD) of the spike protein. The structural characterization of the neutralizing antibody (KNIH90-F1) complexed with RBD using X-ray crystallography revealed three critical epitopes (D509, R511, and E513) in the RBD region of the spike protein. Further investigation of MERS-CoV mutants that escaped neutralization by the antibody supported the identification of these epitopes in the RBD region. The neutralizing activity of this antibody is solely provided by these specific molecular structures. This work should contribute to the development of vaccines or therapeutic antibodies for MERS-CoV.

Identification and analysis of dominant negative mutants of RAIDD and PIDD.

Caspases are cysteine proteases that are essential during the initiation and execution of apoptosis and inflammation. The formation of large oligomeric protein complexes is critical to the activation of caspases in apoptotic and inflammatory signaling pathways. These oligomeric protein complexes function as a platform to recruit caspases, which leads to caspase activation via a proximity-induced mechanism. One well-known oligomeric caspase-activating complex is the PIDDosome for caspase-2 activation, which is composed of 3 protein components, PIDD, RAIDD and Caspase-2. Despite the significant role that caspase-2 activated by PIDDosome plays during genotoxic stress-induced apoptosis, the oligomerization mechanism and the method by which the caspase-activating process is mediated by the formation of PIDDosome is currently not well understood. Here, we show that the assembly mechanism of the core of PIDDosome is time-dependent and salt concentration-dependent. In addition, we demonstrate that point mutations on RAIDD (R147E) and on PIDD (Y814A) exert a dominant negative effect on the formation of the PIDDosome, and that this effect cannot be applied after the PIDDosome has been formed.

Crystallization and preliminary X-ray crystallographic studies of ß-transaminase from Mesorhizobium sp. strain LUK.

ß-Transaminase (ß-TA) catalyzes the transamination reaction between ß-aminocarboxylic acids and keto acids. This enzyme is a particularly suitable candidate for use as a biocatalyst for the asymmetric synthesis of enantiochemically pure ß-amino acids for pharmaceutical purposes. The ß-TA from Mesorhizobium sp. strain LUK (ß-TAMs) belongs to a novel class in that it shows ß-transaminase activity with a broad and unique substrate specificity. In this study, ß-TAMs was overexpressed in Escherichia coli with an engineered C-terminal His tag. ß-TAMs was then purified to homogeneity and crystallized at 293 K. X-ray diffraction data were collected to a resolution of 2.5 Šfrom a crystal that belonged to the orthorhombic space group C222(1), with unit-cell parameters a = 90.91, b = 192.17, c = 52.75 Å.

Crystallization and preliminary X-ray crystallographic studies of the N-terminal domain of human ribosomal protein L7a (RPL7a).

Ribosomal proteins are a major component of ribosomes, which catalyze protein synthesis. One ribosomal protein, L7a (RPL7a), which is a component of the 60S large ribosomal subunit, has additional functions involved in cell growth and differentiation that occur via interaction with human thyroid hormone receptor (THR) and retinoic acid receptor (RAR) and in turn inhibit the activities of the two nuclear hormone receptors. In this study, the N-terminal domain of human RPL7a was overexpressed in Escherichia coli using an engineered C-terminal His tag. The N-terminal domain of human RPL7a was then purified to homogeneity and crystallized at 293 K. X-ray diffraction data were collected to a resolution of 3.5 Šfrom a crystal belonging to the tetragonal space group P4(1)22 or P4(3)22 with unit-cell parameters a = 92.28, b = 92.28, c = 236.59 Å.

Discovery of highly potent human glutaminyl cyclase (QC) inhibitors as anti-Alzheimer's agents by the combination of pharmacophore-based and structure-based design.

The inhibition of glutaminyl cyclase (QC) may provide a promising strategy for the treatment of early Alzheimer's disease (AD) by reducing the amount of the toxic pyroform of ß-amyloid (AßΝ3pE) in the brains of AD patients. In this work, we identified potent QC inhibitors with subnanomolar IC50 values that were up to 290-fold higher than that of PQ912, which is currently being tested in Phase II clinical trials. Among the tested compounds, the cyclopentylmethyl derivative (214) exhibited the most potent in vitro activity (IC50 = 0.1 nM), while benzimidazole (227) showed the most promising in vivo efficacy, selectivity and druggable profile. 227 significantly reduced the concentration of pyroform Aß and total Aß in the brain of an AD animal model and improved the alternation behavior of mice during Y-maze tests. The crystal structure of human QC (hQC) in complex with 214 indicated tight binding at the active site, supporting that the specific inhibition of QC results in potent in vitro and in vivo activity. Considering the recent clinical success of donanemab, which targets AßΝ3pE, small molecule-based QC inhibitors may also provide potential therapeutic options for early-stage AD treatment.

In vitro reconstitution of the interactions in the PIDDosome.

Caspase-2 is critical for genotoxic stress induced apoptosis and is activated by formation of the PIDDosome, an oligomeric caspase-2 activating complex. The PIDDosome comprises three protein components, PIDD, RAIDD, and caspase-2. RAIDD contains both a death domain (DD) and a caspase recruitment domain (CARD). It acts as the bridge to recruit PIDD using the DD: DD interaction and to recruit caspase-2 via the CARD: CARD interaction. Here we report biochemical characterization and in vitro reconstitution of the core interactions in the PIDDosome. We show that RAIDD CARD and RAIDD DD interact with their binding partners, caspase-2 CARD and PIDD DD, respectively. However, full-length RAIDD fails to interact with either caspase-2 CARD or PIDD DD under a physiological buffer condition. We reveal that this lack of interaction of full-length RAIDD is not due to its tendency to aggregate under the physiological buffer condition, as decreasing full-length RAIDD aggregation using high salt or high pH is not able to promote complex formation. Instead, full-length RAIDD forms both binary and ternary complexes under a low salt condition. Successful in vitro reconstitution of the ternary complex provides a basis for further structural studies of the PIDDosome.

Crystallization and preliminary X-ray crystallographic studies of ω-transaminase from Vibrio fluvialis JS17.

Omega-transaminase (ω-TA) catalyzes the transfer of an amino group from a non-alpha-position amino acid or an amine compound with no carboxylic group to an amino acceptor. ω-TA from Vibrio fluvialis JS17 (ω-TAVf) is a novel amine:pyruvate transaminase that is capable of stereoselective transamination of aryl chiral amines. In this study, omega-TAVf was overexpressed in Escherichia coli with engineered C-terminal His tags. ω-TAVf was then purified to homogeneity and crystallized at 292 K. X-ray diffraction data were collected to a resolution of 2.5 A from a crystal belonging to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a=78.43, b=95.95, c=122.89 A.

Purification, crystallization and preliminary x-ray crystallographic studies of RAIDD Death-Domain (DD).

Caspase-2 activation by formation of PIDDosome is critical for genotoxic stress induced apoptosis. PIDDosome is composed of three proteins, RAIDD, PIDD, and Caspase-2. RAIDD is an adaptor protein containing an N-terminal Caspase-Recruiting-Domain (CARD) and a C-terminal Death-Domain (DD). Its interactions with Caspase-2 and PIDD through CARD and DD respectively and formation of PIDDosome are important for the activation of Caspase-2. RAIDD DD cloned into pET26b vector was expressed in E. coli cells and purified by nickel affinity chromatography and gel filtration. Although it has been known that the most DDs are not soluble in physiological condition, RAIDD DD was soluble and interacts tightly with PIDD DD in physiological condition. The purified RAIDD DD alone has been crystallized. Crystals are trigonal and belong to space group P3(1)21 (or its enantiomorph P3(2)21) with unit-cell parameters a = 56.3, b = 56.3, c = 64.9 A and gamma = 120 degrees . The crystals were obtained at room temperature and diffracted to 2.0 A resolution.

Molecular basis of maintaining an oxidizing environment under anaerobiosis by soluble fumarate reductase.

Osm1 and Frd1 are soluble fumarate reductases from yeast that are critical for allowing survival under anaerobic conditions. Although they maintain redox balance during anaerobiosis, the underlying mechanism is not understood. Here, we report the crystal structure of a eukaryotic soluble fumarate reductase, which is unique among soluble fumarate reductases as it lacks a heme domain. Structural and enzymatic analyses indicate that Osm1 has a specific binding pocket for flavin molecules, including FAD, FMN, and riboflavin, catalyzing their oxidation while reducing fumarate to succinate. Moreover, ER-resident Osm1 can transfer electrons from the Ero1 FAD cofactor to fumarate either by free FAD or by a direct interaction, allowing de novo disulfide bond formation in the absence of oxygen. We conclude that soluble eukaryotic fumarate reductases can maintain an oxidizing environment under anaerobic conditions, either by oxidizing cellular flavin cofactors or by a direct interaction with flavoenzymes such as Ero1.

The mechanism of folding robustness revealed by the crystal structure of extra-superfolder GFP.

Stability of green fluorescent protein (GFP) is sometimes important for a proper practical application of this protein. Random mutagenesis and targeted mutagenesis have been used to create better-folded variants of GFP, including recently reported extra-superfolder GFP. Our aim was to determine the crystal structure of extra-superfolder GFP, which is more robustly folded and stable than GFP and superfolder GFP. The structural and structure-based mutagenesis analyses revealed that some of the mutations that created extra-superfolder GFP (F46L, E126K, N149K, and S208L) contribute to folding robustness by stabilizing extra-superfolder GFP with various noncovalent bonds.