Affinity-directed substrate/H+-antiport by a MATE transporter.

Multidrug and toxin extrusion (MATE) family transporters excrete toxic compounds coupled to Na+/H+ influx. Although structures of MATE transporters are available, the mechanism by which substrate export is coupled to ion influx remains unknown. To address this issue, we conducted a structural analysis of Pyrococcus furiosus MATE (PfMATE) using solution nuclear magnetic resonance (NMR). The NMR analysis, along with thorough substitutions of all non-exposed acidic residues, confirmed that PfMATE is under an equilibrium between inward-facing (IF) and outward-facing (OF) conformations, dictated by the Glu163 protonation. Importantly, we found that only the IF conformation exhibits a mid-µM affinity for substrate recognition. In contrast, the OF conformation exhibited only weak mM substrate affinity, suitable for releasing substrate to the extracellular side. These results indicate that PfMATE is an affinity-directed H+ antiporter where substrates selectively bind to the protonated IF conformation in the equilibrium, and subsequent proton release mechanistically ensures H+-coupled substrate excretion by the transporter.

The GTP responsiveness of PI5P4Kß evolved from a compromised trade-off between activity and specificity.

Unlike most kinases, phosphatidylinositol 5-phosphate 4-kinase ß (PI5P4Kß) utilizes GTP as a physiological phosphate donor and regulates cell growth under stress (i.e., GTP-dependent stress resilience). However, the genesis and evolution of its GTP responsiveness remain unknown. Here, we reveal that PI5P4Kß has acquired GTP preference by generating a short dual-nucleotide-recognizing motif called the guanine efficient association (GEA) motif. Comparison of nucleobase recognition with 660 kinases and 128 G proteins has uncovered that most kinases and PI5P4Kß use their main-chain atoms for adenine recognition, while the side-chain atoms are required for guanine recognition. Mutational analysis of the GEA motif revealed that the acquisition of GTP reactivity is accompanied by an extended activity toward inosine triphosphate (ITP) and xanthosine triphosphate (XTP). Along with the evolutionary analysis data that point to strong negative selection of the GEA motif, these results suggest that the GTP responsiveness of PI5P4Kß has evolved from a compromised trade-off between activity and specificity, underpinning the development of the GTP-dependent stress resilience.

Targeting the cryptic sites: NMR-based strategy to improve protein druggability by controlling the conformational equilibrium.

Cryptic ligand binding sites, which are not evident in the unligated structures, are beneficial in tackling with difficult but attractive drug targets, such as protein-protein interactions (PPIs). However, cryptic sites have thus far not been rationally pursued in the early stages of drug development. Here, we demonstrated by nuclear magnetic resonance that the cryptic site in Bcl-xL exists in a conformational equilibrium between the open and closed conformations under the unligated condition. While the fraction of the open conformation in the unligated wild-type Bcl-xL is estimated to be low, F143W mutation that is distal from the ligand binding site can substantially elevate the population. The F143W mutant showed a higher hit rate in a phage-display peptide screening, and the hit peptide bound to the cryptic site of the wild-type Bcl-xL. Therefore, by controlling the conformational equilibrium in the cryptic site, the opportunity to identify a PPI inhibitor could be improved.

Semiquantitative assessment of the relative apical sparing pattern of longitudinal strain for cardiac amyloidosis identification.

BACKGROUNDS: The relative apical sparing pattern (RASP) of left ventricular (LV) longitudinal strain (LS) is frequently associated with cardiac amyloidosis (CA). However, the visual assessment of RASP is inconsistent, and the quantitative assessment of RASP is time-consuming. This study aimed to compare assessments of RASP for the identification of CA with conventional assessments and investigate their reproducibility and relevance on the assessments. METHODS: Forty patients with biopsy-proven CA were compared with 80 hypertrophied patients matched for mean LV wall thickness. We compared the discriminative abilities of three assessments of RASP to identify CA (visual, quantitative, and semiquantitative). Nine patterns of semiquantitative RASP were investigated; finally, it was defined as "reduction of LS" (≥ -10%) in ≥5 (of 6) basal segments, relative to "preserved LS" (< -15%) in at least one apical segment. RESULTS: The concordance between the two observers for visual RASP was modest (κ = 0.65). On the other hand, the consistency for semiquantitative RASP was perfect (κ = 1.00). The discriminative ability of semiquantitative RASP (area under the curve [AUC]  = 0.74) was significantly better than that of visual RASP (AUC = 0.65) and equivalent to that of binary quantitative RASP. CONCLUSION: Semiquantitative RASP assessment is reproducible and accurately discriminates CA. This simple assessment may help readily refine the risk stratification of patients with diffuse LV hypertrophy.

Prognostic assessment of relative apical sparing pattern of longitudinal strain for severe aortic valve stenosis.

BACKGROUNDS: The relative apical sparing pattern (RASP) of left ventricular (LV) longitudinal strain (LS) is frequently associated with cardiac amyloidosis. Elderly patients with aortic valve stenosis (AS) complicated by transthyretin amyloid cardiomyopathy have poor prognosis. Furthermore, deteriorated basal LS in AS patients has been reported to be associated with adverse outcome. We investigated the association between RASP and outcomes in patients with severe AS. METHODS: We retrospectively studied 156 consecutive patients with severe AS and preserved LV ejection fraction. RASP was assessed by both of semi-quantitative (sRASP) and quantitative (qRASP) methods. sRASP was defined as a deterioration of LS (≥-10%) in ≥ 5 (of 6) basal segments, relative to preserved LS (<-15%) in at least 1 apical segment. qRASP was calculated using the following formula: average apical LS/(average basal LS + average mid-ventricle LS); qRASP ≥ 1 was defined as positive. Patients were followed up to determine outcomes, which included sudden cardiac death or unexpected admission due to heart failure, over a median of 1.9 years. RESULTS: sRASP and qRASP were assessed in all patients, but 24 and 42 patients fulfilled the criteria for sRASP and qRASP, respectively. Both assessments were significantly associated with outcomes (n = 44; 28%). Furthermore, sRASP was significantly associated with outcome after adjusting for EuroSCORE, NYHA ≥ II, or global longitudinal strain. A model based on these covariates for predicting outcomes significantly improved by adding sRASP. CONCLUSION: RASP is observed in some patients with severe AS and provides additive prognostic information over conventional parameters.

Mutation-induced change in chignolin stability from π-turn to α-turn.

Chignolin, which consists of 10 amino acids, adopts two stable states in simulations at room temperature at 1 atm: the native and misfolded states. The sequence of chignolin is optimized to form a stable π-turn and thus the native state has a π-turn from Asp3 to Thr8. On the other hand, the misfolded state adopts an α-turn from Asp3 to Gly7. We previously investigated the differences in the stability mechanism of the two states using computational techniques. Our previous detailed energy analysis implied that the native state was stabilized by hydrogen bonding between the side chain atoms of Thr6 and Thr8, and Thr8 was not involved in stabilization of the misfolded state. Thus, we predicted that mutation of Thr8 to a neutral amino acid could stabilize the misfolded structure over the native structure. In the present work, we performed 4 µs molecular dynamics simulations for 19 mutants of the 8th residue. Among them, the T8I, T8F, T8P, T8N, and T8Y mutants, in which the 8th residue was changed to a neutral residue, formed only the misfolded structure at room temperature. Even at high temperature, for the T8P mutant, the native structure was not observed, as the T8P mutant cannot form the native structure because of steric hindrance caused by the distinctive cyclic structure of proline. Interestingly, the T8P mutant at high temperature has trans and cis conformations in the Gly7-Pro8 sequence, with the trans conformation corresponding to the misfolded state. NMR analysis of the T8P mutant supported our results.

Conformational equilibrium defines the variable induction of the multidrug-binding transcriptional repressor QacR.

QacR, a multidrug-binding transcriptional repressor in pathogenic bacteria Staphylococcus aureus, modulates the transcriptional level of the multidrug transporter gene, qacA, in response to engaging a set of diverse ligands. However, the structural basis that defines the variable induction level remains unknown. Here, we reveal that the conformational equilibrium between the repressive and inducive conformations in QacR defines the induction level of the transporter gene. In addition, the unligated QacR is already partly populated in the inducive conformation, allowing the basal expression of the transporter. We also showed that, in the known constitutively active QacR mutants, the equilibrium is shifted more toward the inducive conformation, even in the unligated state. These results highlight the unexpected structural mechanism, connecting the promiscuous multidrug binding to the variable transcriptional regulation of QacR, which provide clues to dysfunctioning of the multidrug resistance systems.

Dynamic equilibrium on DNA defines transcriptional regulation of a multidrug binding transcriptional repressor, LmrR.

LmrR is a multidrug binding transcriptional repressor that controls the expression of a major multidrug transporter, LmrCD, in Lactococcus lactis. Promiscuous compound ligations reduce the affinity of LmrR for the lmrCD operator by several fold to release the transcriptional repression; however, the affinity reduction is orders of magnitude smaller than that of typical transcriptional repressors. Here, we found that the transcriptional regulation of LmrR is achieved through an equilibrium between the operator-bound and non-specific DNA-adsorption states in vivo. The effective dissociation constant of LmrR for the lmrCD operator under the equilibrium is close to the endogenous concentration of LmrR, which allows a substantial reduction of LmrR occupancy upon compound ligations. Therefore, LmrR represents a dynamic type of transcriptional regulation of prokaryotic multidrug resistance systems, where the small affinity reduction induced by compounds is coupled to the functional relocalization of the repressor on the genomic DNA via nonspecific DNA adsorption.

Nitrogen-detected TROSY yields comparable sensitivity to proton-detected TROSY for non-deuterated, large proteins under physiological salt conditions.

Direct detection of the TROSY component of proton-attached (15)N nuclei ((15)N-detected TROSY) yields high quality spectra with high field magnets, by taking advantage of the slow (15)N transverse relaxation. The slow transverse relaxation and narrow line width of the (15)N-detected TROSY resonances are expected to compensate for the inherently low (15)N sensitivity. However, the sensitivity of (15)N-detected TROSY in a previous report was one-order of magnitude lower than in the conventional (1)H-detected version. This could be due to the fact that the previous experiments were performed at low salt (0-50 mM), which is advantageous for (1)H-detected experiments. Here, we show that the sensitivity gap between (15)N and (1)H becomes marginal for a non-deuterated, large protein (τ c = 35 ns) at a physiological salt concentration (200 mM). This effect is due to the high salt tolerance of the (15)N-detected TROSY. Together with the previously reported benefits of the (15)N-detected TROSY, our results provide further support for the significance of this experiment for structural studies of macromolecules when using high field magnets near and above 1 GHz.

The radish genome and comprehensive gene expression profile of tuberous root formation and development.

Understanding the processes that regulate plant sink formation and development at the molecular level will contribute to the areas of crop breeding, food production and plant evolutionary studies. We report the annotation and analysis of the draft genome sequence of the radish Raphanus sativus var. hortensis (long and thick root radish) and transcriptome analysis during root development. Based on the hybrid assembly approach of next-generation sequencing, a total of 383 Mb (N50 scaffold: 138.17 kb) of sequences of the radish genome was constructed containing 54,357 genes. Syntenic and phylogenetic analyses indicated that divergence between Raphanus and Brassica coincide with the time of whole genome triplication (WGT), suggesting that WGT triggered diversification of Brassiceae crop plants. Further transcriptome analysis showed that the gene functions and pathways related to carbohydrate metabolism were prominently activated in thickening roots, particularly in cell proliferating tissues. Notably, the expression levels of sucrose synthase 1 (SUS1) were correlated with root thickening rates. We also identified the genes involved in pungency synthesis and their transcription factors.

Microevolution of Virulence-Related Genes in Helicobacter pylori Familial Infection.

Helicobacter pylori, a bacterial pathogen that can infect human stomach causing gastritis, ulcers and cancer, is known to have a high degree of genome/epigenome diversity as the result of mutation and recombination. The bacteria often infect in childhood and persist for the life of the host. One of the reasons of the rapid evolution of H. pylori is that it changes its genome drastically for adaptation to a new host. To investigate microevolution and adaptation of the H. pylori genome, we undertook whole genome sequencing of the same or very similar sequence type in multi-locus sequence typing (MLST) with seven genes in members of the same family consisting of parents and children in Japan. Detection of nucleotide substitutions revealed likely transmission pathways involving children. Nonsynonymous (amino acid changing) mutations were found in virulence-related genes (cag genes, vacA, hcpDX, tnfα, ggt, htrA and the collagenase gene), outer membrane protein (OMP) genes and other cell surface-related protein genes, signal transduction genes and restriction-modification genes. We reconstructed various pathways by which H. pylori can adapt to a new human host, and our results raised the possibility that the mutational changes in virulence-related genes have a role in adaptation to a child host. Changes in restriction-modification genes might remodel the methylome and transcriptome to help adaptation. This study has provided insights into H. pylori transmission and virulence and has implications for basic research as well as clinical practice.

Strigolactone regulates anthocyanin accumulation, acid phosphatases production and plant growth under low phosphate condition in Arabidopsis.

Phosphate is an essential macronutrient in plant growth and development; however, the concentration of inorganic phosphate (Pi) in soil is often suboptimal for crop performance. Accordingly, plants have developed physiological strategies to adapt to low Pi availability. Here, we report that typical Pi starvation responses in Arabidopsis are partially dependent on the strigolactone (SL) signaling pathway. SL treatment induced root hair elongation, anthocyanin accumulation, activation of acid phosphatase, and reduced plant weight, which are characteristic responses to phosphate starvation. Furthermore, the expression profile of SL-response genes correlated with the expression of genes induced by Pi starvation. These results suggest a potential overlap between SL signaling and Pi starvation signaling pathways in plants.

Dynamic multidrug recognition by multidrug transcriptional repressor LmrR.

LmrR is a multidrug transcriptional repressor that controls the expression of a major multidrug transporter, LmrCD, in Lactococcus lactis. However, the molecular mechanism by which LmrR binds to structurally unrelated compounds and is released from the promoter region remains largely unknown. Here, we structurally and dynamically characterized LmrR in the apo, compound-bound and promoter-bound states. The compound-binding site of LmrR exhibits ps-µs dynamics in the apo state, and compound ligation shifts the preexisting conformational equilibrium to varying extents to achieve multidrug recognition. Meanwhile, the compound binding induces redistribution of ps-ns dynamics to the allosteric sites, which entropically favors the high-affinity recognition. Furthermore, the reciprocal compound/promoter binding by LmrR is achieved by the incompatible conformational ensembles between the compound- and promoter-bound states. Collectively, the data show how LmrR can dynamically exert its functions through promiscuous multi-target interactions, in a manner that cannot be understood by a static structural view.

Sense transgene-induced post-transcriptional gene silencing in tobacco compromises the splicing of endogenous counterpart genes.

Sense transgene-induced post-transcriptional gene silencing (S-PTGS) is thought to be a type of RNA silencing in which ARGONAUTE1 directs the small interfering RNA (siRNA)-mediated cleavage of a target mRNA in the cytoplasm. Here, we report that the altered splicing of endogenous counterpart genes is a main cause for the reduction of their mature mRNA levels. After the S-PTGS of a tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) gene, 3'-truncated, polyadenylated endo-NtFAD3 transcripts and 5'-truncated, intron-containing endo-NtFAD3 transcripts were detected in the total RNA fraction. Although transcription proceeded until the last exon of the endogenous NtFAD3 gene, intron-containing NtFAD3 transcripts accumulated in the nucleus of the S-PTGS plants. Several intron-containing NtFAD3 transcripts harboring most of the exon sequences were generated when an endogenous silencing suppressor gene, rgs-CaM, was overexpressed in the S-PTGS plants. These intron-containing NtFAD3 splice variants were generated in the presence of NtFAD3 siRNAs that are homologous to the nucleotide sequences of these splice variants. The results of this study indicate that the inhibition of endo-NtFAD3 gene expression is primarily directed via the alteration of splicing and not by cytoplasmic slicer activity. Our results suggest that the transgene and intron-containing endogenous counterpart genes are differentially suppressed in S-PTGS plants.

Abundant sequence divergence in the native Japanese cattle Mishima-Ushi (Bos taurus) detected using whole-genome sequencing.

The native Japanese cattle Mishima-Ushi, a designated national natural treasure, are bred on a remote island, which has resulted in the conservation of their genealogy. We examined the genetic characteristics of 8 Mishima-Ushi individuals by using single nucleotide polymorphisms (SNPs), insertions, and deletions obtained by whole-genome sequencing. Mapping analysis with various criteria showed that predicted heterozygous SNPs were more prevalent than predicted homozygous SNPs in the exonic region, especially non-synonymous SNPs. From the identified 6.54 million polymorphisms, we found 400 non-synonymous SNPs in 313 genes specific to each of the 8 Mishima-Ushi individuals. Additionally, 3,170,833 polymorphisms were found between the 8 Mishima-Ushi individuals. Phylogenetic analysis confirmed that the Mishima-Ushi population diverged from another strain of Japanese cattle. This study provides a framework for further genetic studies of Mishima-Ushi and research on the function of SNP-containing genes as well as understanding the genetic relationship between the domestic and native Japanese cattle breeds.

High-resolution DNA methylome analysis of primordial germ cells identifies gender-specific reprogramming in mice.

Dynamic epigenetic reprogramming occurs during mammalian germ cell development, although the targets of this process, including DNA demethylation and de novo methylation, remain poorly understood. We performed genome-wide DNA methylation analysis in male and female mouse primordial germ cells at embryonic days 10.5, 13.5, and 16.5 by whole-genome shotgun bisulfite sequencing. Our high-resolution DNA methylome maps demonstrated gender-specific differences in CpG methylation at genome-wide and gene-specific levels during fetal germline progression. There was extensive intra- and intergenic hypomethylation with erasure of methylation marks at imprinted, X-linked, or germline-specific genes during gonadal sex determination and partial methylation at particular retrotransposons. Following global demethylation and sex determination, CpG sites switched to de novo methylation in males, but the X-linked genes appeared resistant to the wave of de novo methylation. Significant differential methylation at a subset of imprinted loci was identified in both genders, and non-CpG methylation occurred only in male gonocytes. Our data establish the basis for future studies on the role of epigenetic modifications in germline development and other biological processes.

Contribution of intragenic DNA methylation in mouse gametic DNA methylomes to establish oocyte-specific heritable marks.

Genome-wide dynamic changes in DNA methylation are indispensable for germline development and genomic imprinting in mammals. Here, we report single-base resolution DNA methylome and transcriptome maps of mouse germ cells, generated using whole-genome shotgun bisulfite sequencing and cDNA sequencing (mRNA-seq). Oocyte genomes showed a significant positive correlation between mRNA transcript levels and methylation of the transcribed region. Sperm genomes had nearly complete coverage of methylation, except in the CpG-rich regions, and showed a significant negative correlation between gene expression and promoter methylation. Thus, these methylome maps revealed that oocytes and sperms are widely different in the extent and distribution of DNA methylation. Furthermore, a comparison of oocyte and sperm methylomes identified more than 1,600 CpG islands differentially methylated in oocytes and sperm (germline differentially methylated regions, gDMRs), in addition to the known imprinting control regions (ICRs). About half of these differentially methylated DNA sequences appear to be at least partially resistant to the global DNA demethylation that occurs during preimplantation development. In the absence of Dnmt3L, neither methylation of most oocyte-methylated gDMRs nor intragenic methylation was observed. There was also genome-wide hypomethylation, and partial methylation at particular retrotransposons, while maintaining global gene expression, in oocytes. Along with the identification of the many Dnmt3L-dependent gDMRs at intragenic regions, the present results suggest that oocyte methylation can be divided into 2 types: Dnmt3L-dependent methylation, which is required for maternal methylation imprinting, and Dnmt3L-independent methylation, which might be essential for endogenous retroviral DNA silencing. The present data provide entirely new perspectives on the evaluation of epigenetic markers in germline cells.

Docking simulation of polyamines on a kissing-loop RNA dimer.

Polyamines, especially branched polyamines such as tetrakis(3-aminopropyl)ammonium (Taa), stabilize the tertiary structure of RNA molecules. In this study, we examined the polyamine binding site of the HIV-1 dimerization initiation site (DIS) in the kissing-loop dimer by the docking simulation. It was found that Taa binds predominantly to the kissing loop interaction site of DIS.