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The ribonuclease (RNase) H family of enzymes catalyze the specific cleavage of RNA strands of RNA/DNA hybrid duplexes and play an important role in DNA replication and repair. Since the first report of the crystal structure of RNase HI, its catalytic mechanisms, which require metal ions, have been discussed based on numerous structural and functional analyses, including X-ray crystallography. In contrast, the function of the conserved histidine residue (His124 in Escherichia coli) in the flexible loop around the active site remains poorly understood, although an important role was suggested by NMR analyses. Here, novel high-resolution X-ray crystal structures of E. coli RNase HI are described, with a particular focus on the interactions of divalent cations with His124 oriented towards the active site. The enzyme–Mg2+ complex contains two metal ions in the active site, one of which has previously been observed. The second ion lies alongside the first and binds to His124 in an octahedral coordination scheme. In the enzyme–Zn2+ complex a single metal ion was found to bind to the active site, showing a tetrahedral coordination geometry with the surrounding atoms, including His124. These results provide structural evidence that His124 plays a crucial role in the catalytic activity of RNase HI by interacting weakly and transiently with metal ions in the catalytic center.

Statistical inference with nonresponse is quite challenging, especially when the response mechanism is nonignorable. In this case, the validity of statistical inference depends on untestable correct specification of the response model. To avoid the misspecification, we propose semiparametric Bayesian estimation in which an outcome model is parametric, but the response model is semiparametric in that we do not assume any parametric form for the nonresponse variable. We adopt penalized spline methods to estimate the unknown function. We also consider a fully nonparametric approach to modeling the response mechanism by using radial basis function methods. Using Polya-gamma data augmentation, we developed an efficient posterior computation algorithm via Gibbs sampling in which most full conditional distributions can be obtained in familiar forms. The performance of the proposed method is demonstrated in simulation studies and an application to longitudinal data., 25 pages; The title has been changed from "Bayesian semiparametric estimation under nonignorable nonresponse"

Publication bias is a major concern in conducting systematic reviews and meta-analyses. Various sensitivity analysis or bias-correction methods have been developed based on selection models and they have some advantages over the widely used bias-correction method of the trim-and-fill method. However, likelihood methods based on selection models may have difficulty in obtaining precise estimates and reasonable confidence intervals or require a complicated sensitivity analysis process. In this paper, we develop a simple publication bias adjustment method utilizing information on conducted but still unpublished trials from clinical trial registries. We introduce an estimating equation for parameter estimation in the selection function by regarding the publication bias issue as a missing data problem under missing not at random. With the estimated selection function, we introduce the inverse probability weighting (IPW) method to estimate the overall mean across studies. Furthermore, the IPW versions of heterogeneity measures such as the between-study variance and the I2 measure are proposed. We propose methods to construct asymptotic confidence intervals and suggest intervals based on parametric bootstrapping as an alternative. Through numerical experiments, we observed that the estimators successfully eliminate biases and the confidence intervals had empirical coverage probabilities close to the nominal level. On the other hand, the asymptotic confidence interval is much wider in some scenarios than the bootstrap confidence interval. Therefore, the latter is recommended for practical use., Comment: 50 pages, 3 figures

Facilitates chromatin transcription (FACT) is a histone chaperone, which accomplishes both nucleosome assembly and disassembly. Our combined cryo-electron microscopy (EM) and native mass spectrometry (MS) studies revealed novel key steps of nucleosome reorganization conducted by a Mid domain and its adjacent acidic AID segment of human FACT. We determined three cryo-EM structures of respective octasomes complexed with the Mid-AID and AID regions, and a hexasome alone. We discovered extensive contacts between a FACT region and histones H2A, H2B, and H3, suggesting that FACT is competent to direct functional replacement of a nucleosomal DNA end by its phosphorylated AID segment (pAID). Mutational assays revealed that the aromatic and phosphorylated residues within pAID are essential for octasome binding. The EM structure of the hexasome, generated by the addition of Mid-pAID or pAID, indicated that the dissociation of H2A-H2B dimer causes significant alteration from the canonical path of the nucleosomal DNA.

Background: It is commonly believed that a full moon affects human behavior or the occurrence and outcome of various diseases; thus, the occurrence of out-of-hospital cardiac arrest (OHCA) might increase during full moon nights. Methods and Results: This nationwide, population-based observational study consecutively enrolled OHCA patients in Japan with attempted resuscitation between 2005 and 2016. The primary outcome measure was the occurrence of OHCA. Based on the double-control method, assuming Poisson sampling, we evaluated the average number of OHCA events that occurred on full moon nights compared with that which occurred on control nights, which included events that occurred on the same calendar days 1 week before and after the full moon nights. A total of 29,552 OHCA that occurred on 148 full moon nights and 58,707 OHCA that occurred on 296 control nights were eligible for analysis. The occurrence of OHCA did not differ between full moon and control nights (199.7 vs. 198.3 per night; relative risk [RR], 1.007; 95% CI: 0.993-1.021). On subgroup analysis, compared with control nights, the RR of OHCA occurrence were 1.013 (95% CI: 0.994-1.032, P=0.166) and 0.998 (95% CI: 0.977-1.020, P=0.866) for cardiac and non-cardiac origins, respectively. Conclusions: In this population, there was no significant difference in OHCA occurrence between full moon and control nights.

Aim To assess the impact of the Summit on Financial Markets and the World Economy held in Osaka City, Japan (G20 Osaka Summit) on the emergency medical services (EMS) system. Methods This study used the ORION database with its population-based registry of emergency patients comprising both ambulance and in-hospital records in Osaka Prefecture, Japan. The G20 Osaka Summit was held in Osaka City from 28 to 29 June, 2019. Changes in the EMS system and traffic regulations in Osaka were made during the period from 27 to 30 June, but we focused on the two summit days as the G20 period. The control periods comprised the same calendar days 1 week before and 1 week after the G20 period. We evaluated differences in the number of emergency transports, difficulties in obtaining hospital acceptance of patients, deaths among hospitalized emergency patients, and ambulance transport times between the two periods. Results In total, 2,590 cases in the G20 period and 5,152 cases in the control periods were registered. The relative risk of cases during the G20 versus control periods was 1.01 (0.96-1.05). Significant decreases were observed in the number of traffic accidents as ambulance calls (relative risk = 0.77; 95% confidence interval, 0.64-0.91). There were no significant differences in difficulties in obtaining hospital acceptance or deaths among hospitalized emergency patients between the G20 and control periods. In addition, ambulance transport times during the G20 period were not significantly longer than those in the control periods. Conclusion The G20 Osaka Summit did not adversely impact the provision of emergency medical care in the Osaka area.

Summary The FACT (facilitates chromatin transcription) complex, comprising SPT16 and SSRP1, conducts structural alterations during nucleosome unwrapping. Our previous cryoelectron microscopic (cryo-EM) analysis revealed the first intermediate structure of an unwrapped nucleosome with human FACT, in which 112-bp DNA and the phosphorylated intrinsically disordered (pAID) segment of SPT16 jointly wrapped around the histone core instead of 145-bp DNA. Using NMR, here we clarified that the histone H3 N-terminal tails, unobserved in the cryo-EM structure, adopt two different conformations reflecting their asymmetric locations at entry/exit sites: one corresponds to the original nucleosome site buried in two DNA gyres (DNA side), whereas the other, comprising pAID and DNA, is more exposed to the solvent (pAID side). NMR real-time monitoring showed that H3 acetylation is faster on the pAID side than on the DNA side. Our findings highlight that accessible conformations of H3 tails are created by the replacement of nucleosomal DNA with pAID., Graphical Abstract, Highlights • H3 N-tail, restricted to two DNA gyres of nucleosome, is protected from Gcn5 • H3 N-tail is dynamically exposed by replacement of nucleosomal DNA with pAID of FACT • Gcn5 efficiently acetylates accessible H3 N-tail of nucleosome with FACT • FACT acts as a modulator for dynamic behavior of H3 tails in nucleosome, Biochemistry; Molecular Biology; Structural Biology

Ribonuclease HI, an endoribonuclease, catalyzes the hydrolysis of the RNA strand of an RNA/DNA hybrid and requires divalent metal ions for its enzymatic activity. However, the mechanistic details of the activity of ribonuclease HI and its interaction with divalent metal ions remain unclear. In this study, we performed real-time monitoring of the enzyme–substrate complex in the presence of divalent metal ions (Mn2+ or Zn2+) using electrospray ionization–mass spectrometry (ESI-MS). The findings provide clear evidence that the enzymatic activity of the ternary complex requires the binding of two divalent metal ions. The Zn2+ ions bind to both the enzyme itself and the enzyme:substrate complex more strongly than Mn2+ ions, and gives, in part, the ternary complex, [RNase HI:nicked RNA/DNA hybrid:2Zn2+], suggesting that the ternary complex is retained, even after the hydrolysis of the substrate. The collective results presented herein shed new light on the essential role of divalent metal ions in the activity of ribonuclease HI and demonstrate how Zn2+ ions confer inhibitory properties on the activity of this enzyme by forming a highly stable complex with the substrate.

We consider semiparametric estimation with nonignorable nonresponse data where only a parametric response model is assumed. We clarify the relationship of existing estimators and propose a new estimator which attains the semiparametric efficiency bound and is robust to model misspecification.

Nonresponse is frequently encountered in empirical studies. When the response mechanism is missing not at random (MNAR) statistical inference using the observed data is quite challenging. Handling MNAR data often requires two model assumptions: one for the outcome and the other for the response propensity. Correctly specifying these two model assumptions is challenging and difficult to verify from the responses obtained. In this article we propose a semiparametric maximum likelihood method for MNAR data in the sense that a parametric assumption is used for the response propensity part of the model and a nonparametric model is used for the outcome part. The resulting analysis is more robust than the fully parametric approach. Some asymptotic properties of our estimators are derived. Results from a simulation study are also presented. The Canadian Journal of Statistics xx: 1–17; 2017 © 2017 Statistical Society of Canada

SUMMARYUncovering the distribution of magnitudes and arrival times of aftershocks is a key to comprehending the characteristics of earthquake sequences, which enables us to predict seismic activities and conduct hazard assessments. However, identifying the number of aftershocks immediately after the main shock is practically difficult due to contaminations of arriving seismic waves. To overcome this difficulty, we construct a likelihood based on the detected data, incorporating a detection function to which Gaussian process regression (GPR) is applied. The GPR is capable of estimating not only the parameters of the distribution of aftershocks together with the detection function, but also credible intervals for both the parameters and the detection function. The property that the distributions of both the Gaussian process and aftershocks are exponential functions leads to an efficient Bayesian computational algorithm to estimate hyperparameters. After its validation through numerical tests, the proposed method is retrospectively applied to the catalogue data related to the 2004 Chuetsu earthquake for the early forecasting of the aftershocks. The results show that the proposed method stably and simultaneously estimates distribution parameters and credible intervals, even within t ≤ 3 hr after the main shock.

In Eukarya and Archaea, the lagging strand synthesis is accomplished mainly by three key factors, DNA polymerase (Pol), flap endonuclease (FEN), and DNA ligase (Lig), in the DNA replication process. These three factors form important complexes with proliferating cell nuclear antigen (PCNA), thereby constructing a platform that enable each protein factor to act successively and smoothly on DNA. The structures of the Pol-PCNA-DNA and Lig-PCNA-DNA complexes alone have been visualized by single particle analysis. However, the FEN-PCNA-DNA complex structure remains unknown. In this report, we for the first time present this tertiary structure determined by single particle analysis. We also successfully visualized the structure of the FEN-Lig-PCNA-DNA complex, corresponding to a putative intermediate state between the removal of the DNA flap by FEN and the sealing of the nicked DNA by Lig. This structural study presents the direct visualization of the handing-over action, which proceeds between different replication factors on a single PCNA clamp bound to DNA. We detected a drastic conversion of the DNA from a bent form to a straight form, in addition to the dynamic motions of replication factors in the switching process.

The intrinsically disordered region (IDR) of a protein is an important topic in molecular biology. The functional significance of IDRs typically involves gene-regulation processes and is closely related to posttranslational modifications such as phosphorylation. We previously reported that the Drosophila facilitates chromatin transcription (FACT) protein involved in chromatin remodeling contains an acidic ID fragment (AID) whose phosphorylation modulates FACT binding to nucleosomes. Here, we performed dynamic atomic force microscopy and NMR analyses to clarify how the densely phosphorylated AID masks the DNA binding interface of the high-mobility-group domain (HMG). Dynamic atomic force microscopy of the nearly intact FACT revealed that a small globule temporally appears but quickly vanishes within each mobile tail-like image, corresponding to the HMG-containing IDR. The lifespan of the globule increases upon phosphorylation. NMR analysis indicated that phosphorylation induces no ordered structure but increases the number of binding sites in AID to HMG with an adjacent basic segment, thereby retaining the robust electrostatic intramolecular interaction within FACT even in the presence of DNA. These data lead to the conclusion that the inhibitory effect of nucleosome binding is ascribed to the increase in the probability of encounter between HMG and the phosphorylated IDR.

In the continuing study directed toward the development of peroxisome proliferator-activated receptor gamma (hPPARγ) agonist, we attempted to improve the water solubility of our previously developed hPPARγ-selective agonist 3, which is insufficiently soluble for practical use, by employing two strategies: introducing substituents to reduce its molecular planarity and decreasing its hydrophobicity via replacement of the adamantyl group with a heteroaromatic ring. The first approach proved ineffective, but the second was productive. Here, we report the design and synthesis of a series of α-benzyl phenylpropanoic acid-type hPPARγ partial agonists with improved aqueous solubility. Among them, we selected (R)-7j, which activates hPPARγ to the extent of about 65% of the maximum observed with a full agonist, for further evaluation. The ligand-binding mode and the reason for the partial-agonistic activity are discussed based on X-ray-determined structure of the complex of hPPARγ ligand-binding domain (LBD) and (R)-7j with previously reported ligand-LDB structures. Preliminal apoptotic effect of (R)-7j against human scirrhous gastric cancer cell line OCUM-2MD3 is also described.

In this issue of Structure, Nakae et al. (2016) report the structure of the archaeal EndoMS endonuclease, which cleaves DNA containing mismatched base pairs. Their data demonstrate a unique dual base flipping mechanism, providing intriguing insights into the molecular evolution of protein machineries involved in DNA mismatch repair.

The N and C termini of CENP-T undergo tension-dependent separation, suggesting that CENP-T elongation is responsible for changes in the shape of the inner kinetochore., The kinetochore forms a dynamic interface with microtubules from the mitotic spindle. Live-cell light microscopy–based observations on the dynamic structural changes within the kinetochore suggest that molecular rearrangements within the kinetochore occur upon microtubule interaction. However, the source of these rearrangements is still unclear. In this paper, we analyze vertebrate kinetochore ultrastructure by immunoelectron microscopy (EM) in the presence or absence of tension from spindle microtubules. We found that the inner kinetochore region defined by CENP-A, CENP-C, CENP-R, and the C-terminal domain of CENP-T is deformed in the presence of tension, whereas the outer kinetochore region defined by Ndc80, Mis12, and CENP-E is not stretched even under tension. Importantly, based on EM, fluorescence microscopy, and in vitro analyses, we demonstrated that the N and C termini of CENP-T undergo a tension-dependent separation, suggesting that CENP-T elongation is at least partly responsible for changes in the shape of the inner kinetochore.

Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-mediated transcription factor with roles in glucose, lipid, and lipoprotein homeostasis, and PPARγ ligands are expected have therapeutic potential in these as well as other areas. We report here the design, synthesis, crystallographic analysis, and computational studies of α-benzylphenylpropanoic acid PPARγ agonists. Interestingly, these compounds show a reversal of the stereochemistry-transactivation activity relationship observed with other phenylpropanoic acid ligands.

The nuclear receptor, peroxisome proliferator-activated receptor γ (PPARγ), recognizes various synthetic and endogenous ligands by the ligand-binding domain. Fatty-acid metabolites reportedly activate PPARγ through conformational changes of the Ω loop. Here, we report that serotonin metabolites act as endogenous agonists for PPARγ to regulate macrophage function and adipogenesis by directly binding to helix H12. A cyclooxygenase inhibitor, indomethacin, is a mimetic agonist of these metabolites. Crystallographic analyses revealed that an indole acetate functions as a common moiety for the recognition by the sub-pocket near helix H12. Intriguingly, a serotonin metabolite and a fatty-acid metabolite each bind to distinct sub-pockets, and the PPARγ antagonist, T0070907, blocked the fatty-acid agonism, but not that of the serotonin metabolites. Mutational analyses on receptor-mediated transcription and coactivator binding revealed that each metabolite individually uses coregulator and/or heterodimer interfaces in a ligand-type-specific manner. Furthermore, the inhibition of the serotonin metabolism reduced the expression of the endogenous PPARγ-target gene. Collectively, these results suggest a novel agonism, in which PPARγ functions as a multiple sensor in response to distinct metabolites.

The important roles of a nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) are widely accepted in various biological processes as well as metabolic diseases. Despite the worldwide quest for pharmaceutical manipulation of PPARgamma activity through the ligand-binding domain, very little information about the activation mechanism of the N-terminal activation function-1 (AF-1) domain. Here, we demonstrate the molecular and structural basis of the phosphorylation-dependent regulation of PPARgamma activity by a peptidyl-prolyl isomerase, Pin1. Pin1 interacts with the phosphorylated AF-1 domain, thereby inhibiting the polyubiquitination of PPARgamma. The interaction and inhibition are dependent upon the WW domain of Pin1 but are independent of peptidyl-prolyl cis/trans-isomerase activity. Gene knockdown experiments revealed that Pin1 inhibits the PPARgamma-dependent gene expression in THP-1 macrophage-like cells. Thus, our results suggest that Pin1 regulates macrophage function through the direct binding to the phosphorylated AF-1 domain of PPARgamma.

FACT is a heterodimer of SPT16 and SSRP1, which each contain several conserved regions in the primary structure. The interaction of FACT with nucleosomes induces chromatin remodeling through the combinatorial action of its distinct functional protein regions. However, there is little mechanistic insight into how these regions cooperatively contribute to FACT functions, particularly regarding the recognition of nucleosomal DNA. Here, we report the identification of novel phosphorylation sites of Drosophila melanogaster FACT (dFACT) expressed in Sf9 cells. These sites are densely concentrated in the acidic intrinsically disordered (ID) region of the SSRP1 subunit and control nucleosomal DNA binding by dFACT. This region and the adjacent segment of the HMG domain form weak electrostatic intramolecular interactions, which is reinforced by the phosphorylation, thereby blocking DNA binding competitively. Importantly, this control mechanism appears to support rapid chromatin transactions during early embryogenesis through the dephosphorylation of some sites in the maternally transmitted dSSRP1.

The structures of the ligand-binding domains (LBDs) of human peroxisome proliferator-activated receptors (PPARα, PPARγ and PPARδ) in complexes with a pan agonist, an α/δ dual agonist and a PPARδ-specific agonist were determined. The results explain how each ligand is recognized by the PPAR LBDs at an atomic level., Peroxisome proliferator-activated receptors (PPARs) belong to the nuclear hormone receptor family, which is defined as transcriptional factors that are activated by the binding of ligands to their ligand-binding domains (LBDs). Although the three PPAR subtypes display different tissue distribution patterns and distinct pharmacological profiles, they all are essentially related to fatty-acid and glucose metabolism. Since the PPARs share similar three-dimensional structures within the LBDs, synthetic ligands which simultaneously activate two or all of the PPARs could be potent candidates in terms of drugs for the treatment of abnormal metabolic homeostasis. The structures of several PPAR LBDs were determined in complex with synthetic ligands, derivatives of 3-(4-alkoxy­phenyl)propanoic acid, which exhibit unique agonistic activities. The PPARα and PPARγ LBDs were complexed with the same pan agonist, TIPP-703, which activates all three PPARs and their crystal structures were determined. The two LBD–ligand complex structures revealed how the pan agonist is adapted to the similar, but significantly different, ligand-binding pockets of the PPARs. The structures of the PPARδ LBD in complex with an α/δ-selective ligand, TIPP-401, and with a related δ-­specific ligand, TIPP-204, were also determined. The comparison between the two PPARδ complexes revealed how each ligand exhibits either a ‘dual selective’ or ‘single specific’ binding mode.

A series of 3-(4-alkoxypheny)propanoic acid derivatives was prepared as candidate peroxisome proliferator-activated receptor (PPAR) delta-selective agonists, based on our previously discovered potent human PPARalpha/delta dual agonist TIPP-401 as a lead compound. Structure-activity relationship studies clearly indicated the importance of the chain length of the alkoxy group at the 4-position, and the n-butoxy compound exhibited the most potent PPARdelta transactivation activity and highest PPARdelta selectivity. The (S)-enantiomer of a representative compound (TIPP-204) exhibited extremely potent PPARdelta transactivation activity, comparable to that of the known PPARdelta-selective agonist GW-501516. To understand why TIPP-204 shows high selectivity for hPPARdelta among hPPAR subtypes, and why TIPP-401, a structurally related compound, is a hPPARalpha/delta dual agonist, computational docking of TIPP-401 to the ligand binding domains of hPPARalpha and hPPARdelta and X-ray structure analysis of TIPP-204-hPPARdelta ligand binding domain were carried out. The results allowed identification of certain amino acids as putative determinants of the hPPARdelta selectivity of TIPP-204. To confirm the significance of these amino acids, GAL4-fusion proteins of mutated hPPARdeltas and hPPARalphas were prepared, and the transactivation activity of TIPP-204 toward the mutants was evaluated. The amino acid(s) that predominantly influence the potency and selectivity of TIPP-204 are different from that of the well-known PPARdelta-selective agonist GW-501516, which belongs to a different chemical class. The significance of these amino acids was confirmed by the examination of the complex structure between TIPP-204 and hPPARdelta. The results revealed several interactions relevant to the hPPARdelta-selectivity of the two ligands and will be useful for logical hPPARdelta ligand design.

The 3D structure of the ternary complex, consisting of DNA ligase, the proliferating cell nuclear antigen (PCNA) clamp, and DNA, was investigated by single-particle analysis. This report presents the structural view, where the crescent-shaped DNA ligase with 3 distinct domains surrounds the central DNA duplex, encircled by the closed PCNA ring, thus forming a double-layer structure with dual contacts between the 2 proteins. The relative orientations of the DNA ligase domains, which remarkably differ from those of the known crystal structures, suggest that a large domain rearrangement occurs upon ternary complex formation. A second contact was found between the PCNA ring and the middle adenylation domain of the DNA ligase. Notably, the map revealed a substantial DNA tilt from the PCNA ring axis. This structure allows us to propose a switching mechanism for the replication factors operating on the PCNA ring.

Metabotropic glutamate receptors (mGluRs) are involved in the regulation of many physiological and pathological processes in the central nervous system. The extracellular domain (ECD) of mGluR subtype 3 (mGluR3) was produced using the baculovirus expression system and purified from the culture medium. However, the recombinant protein showed heterogeneity in molecular weight on SDS-PAGE analysis. It was found that the unglycosylation of Asn414 significantly reduced the heterogeneity. Consequently, three site-specifically unglycosylated mutant proteins of mGluR3 ECD, replacing Asn414 only or replacing Asn414 in combination with other glycosylation sites, were successfully crystallized in the presence of L-glutamate. Among them, crystals of the N414/439Q mutant diffracted X-rays to 2.35 A resolution using synchrotron radiation. The crystal belonged to the monoclinic space group P2(1), with unit-cell parameters a = 84.0, b = 97.5, c = 108.1 A, beta = 93.0 degrees . Assuming the presence of two protomers per crystallographic asymmetric unit, the Matthews coefficient V(M) was calculated to be 3.5 A(3) Da(-1) and the solvent content was 65%.

We developed a potent peroxisome proliferator-activated receptor pan agonist (a candidate drug for treatment of altered metabolic homeostasis) by introducing fluorine atoms at appropriate position(s) of the known phenylpropionic acid-type pan agonist TIPP-703.

The RuvAB complex promotes migration of Holliday junction at the late stage of homologous recombination. The RuvA tetramer specifically recognizes Holliday junction to form two types of complexes. A single tetramer is bound to the open configuration of the junction DNA in complex I, while the octameric RuvA core structure sandwiches the same junction in complex II. The hexameric RuvB rings, symmetrically bound to both sides of RuvA on Holliday junction, pump out DNA duplexes, depending upon ATP hydrolysis. We investigated functional differences between the wild-type RuvA from Thermus thermophilus and mutants impaired the ability of complex II formation. These mutant RuvA, exclusively forming complex I, reduced activities of branch migration and ATP hydrolysis, suggesting that the octameric RuvA is essential for efficient branch migration. Together with our recent electron microscopic analysis, this finding provides important insights into functional roles of complex II in the coordinated branch migration mechanism.

Lipid-mediated regulatory mechanism of the C-terminal ligand binding to PDZ domains is not fully understood, despite their roles in subcellular organization. Here, we provide structural insights into the phosphatidylinositol 4,5-bisphosphate (PIP 2 ) recognition mode of a PDZ domain, as revealed from the crystal structure of the phosphate-bound PDZ domain. Two adjacent phosphate ions bind to the basic residues close to the amino terminus of the α2 helix in the Tamalin PDZ domain, reflecting an interaction mode of the two phosphate groups of PIP 2 . Based on the observed location of the two phosphate molecules within the PDZ domain, we built the docking model of PIP 2 with the PDZ domain of the well-known PIP 2 -binding protein, syntenin-1. This model suggests that the hydrophobic diacylglycerol group of PIP 2 could contact the ligand-binding groove of the PDZ domain. These structural features well explain biological phenomena, which were previously reported for the PIP 2 -mediated PDZ ligand-binding regulation.

Nucleosomes act as a barrier in nuclear processes, such as DNA transcription, replication, and repair. Facilitates chromatin transcription (FACT) plays essential roles in nucleosome dynamics during these processes. Two different mechanisms so far have been proposed for nucleosome reorganization by FACT. However, these interpretations have left some unsolved problems in terms of FACTmediated eviction and exchange of histones within nucleosome. Most recently, we have proposed the integrated reorganization mechanism, in which FACT retains nucleosome in transiently loosened structures. This accessible structure allows us to explain most reasonably about how FACT directs further disruption of nucleosome and histone replacement in concert with other factors.

When the response mechanism is believed to be not missing at random (NMAR), a valid analysis requires stronger assumptions on the response mechanism than standard statistical methods would otherwise require. Semiparametric estimators have been developed under the parametric model assumptions on the response mechanism. In this paper, a new statistical test is proposed to guarantee model identifiability without using instrumental variable assumption. Furthermore, we develop optimal semiparametric estimation for parameters such as the population mean. Specifically, we propose two semiparametric optimal estimators that do not require any model assumptions other than the response mechanism. Asymptotic properties of the proposed estimators are discussed. An extensive simulation study is presented to compare with some existing methods. We present an application of our method using Korean labor and income panel survey data.

Glutamate is the major excitatory neurotransmitter and its metabotropic glutamate receptor (mGluR) plays an important role in the central nervous system. The ligand-binding domain (LBD) of mGluR subtype 7 (mGluR7) was produced using the baculovirus expression system and purified from the culture medium. The purified protein was characterized by gel-filtration chromatography, SDS-PAGE and a ligand-binding assay. Crystals of mGluR7 LBD were grown at 293 K by the hanging-drop vapour-diffusion method. The crystals diffracted X-rays to 3.30 A resolution using synchrotron radiation and belong to the trigonal space group P3(1)21, with unit-cell parameters a = b = 92.4, c = 114.3 A. Assuming the presence of one protomer per crystallographic asymmetric unit, the Matthews coefficient V(M) was calculated to be 2.5 A3 Da(-1) and the solvent content was 51%.

Metabotropic glutamate receptors (mGluRs) function as neuronal G-protein-coupled receptors and this requires efficient membrane targeting through associations with cytoplasmic proteins. However, the molecular mechanism regulating mGluR cell-surface trafficking remains unknown. We report here that mGluR trafficking is controlled by the autoregulatory assembly of a scaffold protein Tamalin. In the absence of mGluR, Tamalin self-assembles into autoinhibited conformations, through its PDZ domain and C-terminal intrinsic ligand motif. X-ray crystallographic analyses visualized integral parts of the oligomeric self-assemblies of Tamalin, which require not only the novel hydrophobic dimerization interface but also canonical and noncanonical PDZ/ligand autoinhibitory interactions. The mGluR cytoplasmic region can competitively bind to Tamalin at a higher concentration, disrupting weak inhibitory interactions. The atomic view of mGluR association suggests that this rearrangement is dominated by electrostatic attraction and repulsion. We also observed in mammalian cells that the association liberates the intrinsic ligand toward a motor protein receptor, thereby facilitating mGluR cell-surface trafficking. Our study suggests a novel regulatory mechanism of the PDZ domain, by which Tamalin switches between the trafficking-inhibited and -active forms depending on mGluR association.

Facilitates chromatin transcription (FACT) plays essential roles in chromatin remodeling during DNA transcription, replication, and repair. Our structural and biochemical studies of human FACT–histone interactions present precise views of nucleosome reorganization, conducted by the FACT-SPT16 (suppressor of Ty 16) Mid domain and its adjacent acidic AID segment. AID accesses the H2B N-terminal basic region exposed by partial unwrapping of the nucleosomal DNA, thereby triggering the invasion of FACT into the nucleosome. The crystal structure of the Mid domain complexed with an H3–H4 tetramer exhibits two separate contact sites; the Mid domain forms a novel intermolecular β structure with H4. At the other site, the Mid–H2A steric collision on the H2A-docking surface of the H3–H4 tetramer within the nucleosome induces H2A–H2B displacement. This integrated mechanism results in disrupting the H3 αN helix, which is essential for retaining the nucleosomal DNA ends, and hence facilitates DNA stripping from histone.

Summary An ATP-dependent protease, FtsH, digests misassembled membrane proteins in order to maintain membrane integrity and digests short-lived soluble proteins in order to control their cellular regulation. This enzyme has an N-terminal transmembrane segment and a C-terminal cytosolic region consisting of an AAA + ATPase domain and a protease domain. Here we present two crystal structures: the protease domain and the whole cytosolic region. The cytosolic region fully retains an ATP-dependent protease activity and adopts a three-fold-symmetric hexameric structure. The protease domains displayed a six-fold symmetry, while the AAA + domains, each containing ADP, alternate two orientations relative to the protease domain, making "open" and "closed" interdomain contacts. Apparently, ATPase is active only in the closed form, and protease operates in the open form. The protease catalytic sites are accessible only through a tunnel following from the AAA + domain of the adjacent subunit, raising a possibility of translocation of polypeptide substrate to the protease sites through this tunnel.

Replicative DNA polymerase interacts with processivity factors, the beta-subunit of DNA polymerase III or proliferating cell nuclear antigen (PCNA), in order to function with a long template DNA. The archaeal replicative DNA polymerase from Pyrococcus furiosus interacts with PCNA via its PCNA-interacting protein (PIP) motif at the C-terminus. The PCNA homotrimeric ring contains one PIP interacting site on each monomer and since the ring can accommodate up to three molecules simultaneously, formation of a stable stoichiometric complex of PCNA with its interacting protein has been difficult to control in vitro. A stable complex of the DNA polymerase with PCNA, using a PCNA monomer mutant, has been purified and crystallized. The best ordered crystal diffracted to 3.0 A resolution using synchrotron radiation. The crystals belong to space group P2(1)2(1)2, with unit-cell parameters a = 225.3, b = 123.3, c = 91.3 A.

Summary XPF/Rad1/Mus81/Hef proteins recognize and cleave branched DNA structures. XPF and Rad1 proteins cleave the 5′ side of nucleotide excision repair bubble, while Mus81 and Hef cleave similar sites of the nicked Holliday junction, fork, or flap structure. These proteins all function as dimers and consist of catalytic and helix-hairpin-helix DNA binding (HhH) domains. We have determined the crystal structure of the HhH domain of Pyrococcus furiosus Hef nuclease (HefHhH), which revealed the distinct mode of protein dimerization. Our structural and biochemical analyses also showed that each of the catalytic and HhH domains binds to distinct regions within the fork-structured DNA: each HhH domain from two separate subunits asymmetrically binds to the arm region, while the catalytic domain binds near the junction center. Upon binding to DNA, Hef nuclease disrupts base pairs near the cleavage site. It is most likely that this bipartite binding mode is conserved in the XPF/Rad1/Mus81 nuclease family.

Gene expression in eukaryotes depends upon positioning, mobility and packaging of nucleosomes; thus, we need the detailed information of the human nucleosome core particle (NCP) structure, which could clarify chromatin properties. Here, we report the 2.5 A crystal structure of a human NCP. The overall structure is similar to those of other NCPs reported previously. However, the DNA path of human NCP is remarkably different from that taken within other NCPs with an identical DNA sequence. A comparison of the structural parameters between human and Xenopus laevis DNA reveals that the DNA path of human NCP consecutively shifts by 1 bp in the regions of superhelix axis location -5.0 to -2.0 and 5.0 to 7.0. This alteration of the human DNA path is caused predominantly by tight DNA-DNA contacts within the crystal. It is also likely that the conformational change in the human H2B tail induces the local alteration of the DNA path. In human NCP, the region with the altered DNA path lacks Mn2+ ions and the B-factors of the DNA phosphate groups are substantially high. Therefore, in contrast to the histone octamer, the nucleosomal DNA is sufficiently flexible and mobile and can undergo drastic conformational changes, depending upon the environment.