Wang-Landau molecular dynamics technique to search for low-energy conformational space of proteins

Multicanonical molecular dynamics (MD) is a powerful technique for sampling conformations on rugged potential surfaces such as protein. However, it is notoriously difficult to estimate the multicanonical temperature effectively. Wang and Landau developed a convenient method for estimating the density of states based on a multicanonical Monte Carlo method. In their method, the density of states is calculated autonomously during a simulation. In this paper we develop a set of techniques to effectively apply the Wang-Landau method to MD simulations. In the multicanonical MD, the estimation of the derivative of the density of states is critical. In order to estimate it accurately, we devise two original improvements. First, the correction for the density of states is made smooth by using the Gaussian distribution obtained by a short canonical simulation. Second, an approximation is applied to the derivative, which is based on the Gaussian distribution and the multiple weighted histogram technique. A test of this method was performed with small polypeptides, Met-enkephalin and Trp-cage, and it is demonstrated that Wang-Landau MD is consistent with replica exchange MD but can sample much larger conformational space.Comment: 8 pages, 7 figures, accepted for publication in Physical Review

A Link among DNA Replication, Recombination, and Gene Expression Revealed by Genetic and Genomic Analysis of TEBICHI Gene of Arabidopsis thaliana

Spatio-temporal regulation of gene expression during development depends on many factors. Mutations in Arabidopsis thaliana TEBICHI (TEB) gene encoding putative helicase and DNA polymerase domains-containing protein result in defects in meristem maintenance and correct organ formation, as well as constitutive DNA damage response and a defect in cell cycle progression; but the molecular link between these phenotypes of teb mutants is unknown. Here, we show that mutations in the DNA replication checkpoint pathway gene, ATR, but not in ATM gene, enhance developmental phenotypes of teb mutants, although atr suppresses cell cycle defect of teb mutants. Developmental phenotypes of teb mutants are also enhanced by mutations in RAD51D and XRCC2 gene, which are involved in homologous recombination. teb and teb atr double mutants exhibit defects in adaxial-abaxial polarity of leaves, which is caused in part by the upregulation of ETTIN (ETT)/AUXIN RESPONSIVE FACTOR 3 (ARF3) and ARF4 genes. The Helitron transposon in the upstream of ETT/ARF3 gene is likely to be involved in the upregulation of ETT/ARF3 in teb. Microarray analysis indicated that teb and teb atr causes preferential upregulation of genes nearby the Helitron transposons. Furthermore, interestingly, duplicated genes, especially tandemly arrayed homologous genes, are highly upregulated in teb or teb atr. We conclude that TEB is required for normal progression of DNA replication and for correct expression of genes during development. Interplay between these two functions and possible mechanism leading to altered expression of specific genes will be discussed

ß2-Glycoprotein I/HLA class II complexes are novel autoantigens in antiphospholipid syndrome.

Antiphospholipid syndrome (APS) is an autoimmune disorder characterized by thrombosis and/or pregnancy complications. β2-glycoprotein I (β2GPI) complexed with phospholipid is recognized as a major target for autoantibodies in APS; however, less than half the patients with clinical manifestations of APS possess autoantibodies against the complexes. Therefore, the range of autoantigens involved in APS remains unclear. Recently, we found that human leukocyte antigen (HLA) class II molecules transport misfolded cellular proteins to the cell surface via association with their peptide-binding grooves. Furthermore, immunoglobulin G heavy chain/HLA class II complexes were specific targets for autoantibodies in rheumatoid arthritis. Here, we demonstrate that intact β2GPI, not peptide, forms a complex with HLA class II molecules. Strikingly, 100 (83.3%) of the 120 APS patients analyzed, including those whose antiphospholipid antibody titers were within normal range, possessed autoantibodies that recognize β2GPI/HLA class II complexes in the absence of phospholipids. In situ association between β2GPI and HLA class II was observed in placental tissues of APS patients but not in healthy controls. Furthermore, autoantibodies against β2GPI/HLA class II complexes mediated complement-dependent cytotoxicity against cells expressing the complexes. These data suggest that β2GPI/HLA class II complexes are a target in APS that might be involved in the pathogenesis

Disruption of CRAF-mediated MEK activation is required for effective MEK inhibition in KRAS mutant tumors

MEK inhibitors are clinically active in BRAF V600E melanomas, but only marginally so in KRAS-mutant tumors. While MEK inhibitor treatment resulted in sustained inhibition of ERK phopshorylation in BRAF V600E tumors, it was associated with a rebound in ERK phosphorylation after prolonged exposure in KRAS mutant tumors. To understand this phenomenon, we performed an RNAi screen in a KRAS-mutant model and found that CRAF knockdown enhanced the antiproliferative effects of MEK inhibition both in vivo and in vitro. In agreement with previous findings, MEK inhibitor treatment in KRAS mutant cells resulted in CRAF reactivation and induction of MEK phosphorylation. However, MEK activated by CRAF was less susceptible to traditional MEK inhibitors, such as PD0325901, compared to when activated by BRAF V600E. Consistent with this observation, the rebound in ERK phosphorylation following MEK inhibitor treatment in KRAS mutant tumors was dependent on intact CRAF expression. Furthermore, traditional MEK inhibitors induced RAF-MEK complexes in KRAS mutant models, and disrupting these complexes enhanced the inhibition of CRAF-dependent ERK signaling. In an effort to identify more effective compounds we found that newer MEK inhibitors, such as trametinib and CH5126766, do not only target the catalytic activity of MEK, but in addition, they also impair its reactivation by CRAF. This occurs through two distinct mechanisms: trametinib disrupts RAF-MEK complex formation, as evidenced by co-immunoprecipitation of endogenous proteins and surface plasmon resonance assays with purified enzymes. Instead, CH512676 induces the interaction of MEK with RAF, yet it prevents the phosphorylation of MEK in this complex. To elucidate the mechanism responsible for the latter property, we determined the ternary structure of CH512676-bound MEK1. CH5126766 interacts with Asn 221 and Ser 222 residues in MEK1 to displace the activation segment and disrupt RAF-mediated phosphorylation. These data provide a blueprint for developing better MEK inhibitors that effectively inhibit ERK signaling in KRAS mutant tumors

Two B3 domain transcriptional repressors prevent sugar-inducible expression of seed maturation genes in Arabidopsis seedlings

During development of plant seeds, embryos import nutrients and store massive amounts of reserves. Seed reserves are rapidly degraded and mobilized to support seedling development after germination. HIGH-LEVEL EXPRESSION OF SUGAR-INDUCIBLE GENE 2 (HSI2) of Arabidopsis thaliana is a B3 DNA-binding domain protein that represses the transcription of sugar-inducible reporter gene. Although disruption of HSI2 or HSI2-Like 1 (HSL1) did not affect growth, seeds with disruption of both HSI2 and HSL1 (KK mutant) developed abortive seedlings that stopped growing 7–9 days after imbibition. KK seedlings developed swollen hypocotyls that accumulated seed storage proteins and oil on medium containing sucrose or other metabolizable sugars, and calluses developed from KK seedlings also accumulated seed storage reserves. The expression of seed maturation genes, which include LEAFY COTYLEDON-type master regulators, in KK seedlings depended on the concentration of sucrose, suggesting that sugar controls the expression of seed maturation genes. Our results suggest that HSI2 and HSL1 repress the sugar-inducible expression of the seed maturation program in seedlings and play an essential role in regulating the transition from seed maturation to seedling growth