Quantitative Analysis of Ligand Induced Heterodimerization of Two Distinct Receptors

The induced dimerization of two distinct receptors through a heterobifunctional inducer is prevalent among all levels of cellular signaling processes, yet its complexity poses difficulty for systematic quantitative analysis. This paper first shows how to calculate the amount of any possible complex or monomer of heteroligand and two receptors present at equilibrium. The theory is subsequently applied to the determination of three independent equilibrium parameters involved in the rapamycin induced FKBP and FRB dimerization, in which all parameters were simultaneously estimated using one set of fluorescence resonance energy transfer (FRET) experiments. A MATLAB script is provided for parametric fitting

NMR Studies of the Interaction between Human Programmed Cell Death 5 and Human p53

Human programmed cell death 5 (PDCD5) is a protein playing a significant role in regulating both the apoptotic and paraptotic cell deaths. Resent findings show that PDCD5 is a positive regulator of Tip60 and also has a potential ability to interact with p53. Here we aim to experimentally characterize the nature of the interactions between PDCD5 and the p53 N-terminal domain (NTD) and to depict the binding mode between two proteins. The interprotein binding interfaces were determined by NMR experiments performed with PDCD5 and various fragments of p53 NTD. The binding affinity was investigated using the NMR titration experiments. Analysis revealed that the PDCD5 binding site on p53 is localized within residues 41–56 of p53 TAD2 subdomain while p53 binds preferentially to the positively charged surface region around the C-terminals of helices α3 and α5 and the N-terminal of helix α4 of PDCD5. The binding is mainly mediated through electrostatic interactions. The present data suggested a model for the interaction between PDCD5 and the p53 NTD

The effect of the number of sequence features used on the overall prediction accuracies for the two datasets 25PDB and D8244.

<p>The effect of the number of sequence features used on the overall prediction accuracies for the two datasets 25PDB and D8244.</p

Performance of the blind test using the independent D1185 dataset.

<p>Performance of the blind test using the independent D1185 dataset.</p

Comparison of the jackknife test results between our method and other competing structural class prediction methods using the 25PDB dataset.

<p>Comparison of the jackknife test results between our method and other competing structural class prediction methods using the 25PDB dataset.</p

Structural and Dynamic Insights into the Mechanism of Allosteric Signal Transmission in ERK2-Mediated MKP3 Activation

The mitogen-activated protein kinases (MAPKs) are key components of cellular signal transduction pathways, which are down-regulated by the MAPK phosphatases (MKPs). Catalytic activity of the MKPs is controlled both by their ability to recognize selective MAPKs and by allosteric activation upon binding to MAPK substrates. Here, we use a combination of experimental and computational techniques to elucidate the molecular mechanism for the ERK2-induced MKP3 activation. Mutational and kinetic study shows that the ³³⁴FNFM³³⁷ motif in the MKP3 catalytic domain is essential for MKP3-mediated ERK2 inactivation and is responsible for ERK2-mediated MKP3 activation. The long-term molecular dynamics (MD) simulations further reveal a complete dynamic process in which the catalytic domain of MKP3 gradually changes to a conformation that resembles an active MKP catalytic domain over the time scale of the simulation, providing a direct time-dependent observation of allosteric signal transmission in ERK2-induced MKP3 activation

Structural and Dynamic Insights into the Mechanism of Allosteric Signal Transmission in ERK2-Mediated MKP3 Activation

The mitogen-activated protein kinases (MAPKs) are key components of cellular signal transduction pathways, which are down-regulated by the MAPK phosphatases (MKPs). Catalytic activity of the MKPs is controlled both by their ability to recognize selective MAPKs and by allosteric activation upon binding to MAPK substrates. Here, we use a combination of experimental and computational techniques to elucidate the molecular mechanism for the ERK2-induced MKP3 activation. Mutational and kinetic study shows that the ³³⁴FNFM³³⁷ motif in the MKP3 catalytic domain is essential for MKP3-mediated ERK2 inactivation and is responsible for ERK2-mediated MKP3 activation. The long-term molecular dynamics (MD) simulations further reveal a complete dynamic process in which the catalytic domain of MKP3 gradually changes to a conformation that resembles an active MKP catalytic domain over the time scale of the simulation, providing a direct time-dependent observation of allosteric signal transmission in ERK2-induced MKP3 activation

Structural Basis for the Regulation of Maternal Embryonic Leucine Zipper Kinase

<div><p>MELK (maternal embryonic leucine zipper kinase), which is a member of the AMPK (AMP-activated protein kinase)-related kinase family, plays important roles in diverse cellular processes and has become a promising drug target for certain cancers. However, the regulatory mechanism of MELK remains elusive. Here, we report the crystal structure of a fragment of human MELK that contains the kinase domain and ubiquitin-associated (UBA) domain. The UBA domain tightly binds to the back of the kinase domain, which may contribute to the proper conformation and activity of the kinase domain. Interestingly, the activation segment in the kinase domain displays a unique conformation that contains an intramolecular disulfide bond. The structural and biochemical analyses unravel the molecular mechanisms for the autophosphorylation/activation of MELK and the dependence of its catalytic activity on reducing agents. Thus, our results may provide the basis for designing specific MELK inhibitors for cancer treatment.</p></div

Data_Sheet_1_Causal effect of polyunsaturated fatty acids on bone mineral density and fracture.docx

BackgroundPolyunsaturated fatty acids (PUFAs) are closely related to osteoporosis. To test their causal relationship, we conducted a Mendelian randomization (MR) analysis.MethodsWe analyzed the causal relationship between four PUFAs measures, n-3 PUFAs (n-3), n-6 PUFAs (n-6), the ratio of n-3 PUFAs to total fatty acids (n-3 pct), and the ratio of n-6 PUFAs to n-3 PUFAs (n-6 to n-3), and five measures of osteoporosis, including estimated bone mineral density (eBMD), forearm (FA) BMD, femoral neck (FN) BMD, lumbar spine (LS) BMD, and fracture, using two-sample MR analysis. In order to verify the direct effect between PUFAs and BMD, we chose interleukin-6 (IL-6), tumor necrosis factor-β (TNF-β), and bone morphogenetic proteins 7 (BMP-7), three markers or cytokines strongly related to BMD, as possible confounding factors, and analyzed the possible causal relationships between them and PUFAs or BMD by MR. Inverse variance weighting (IVW), MR-Egger, weighted and weighted median were conducted. MR Pleiotropy RESidual Sum and Outlier (MR-PRESSO) and MR-Egger regression methods were used to evaluate the potential pleiotropy of instrumental variables (IVs) and outliers were identified by MR-PRESSO. Cochran’s Q statistic was used to detect the heterogeneity among IVs. Leave-one-out sensitivity analysis was used to find SNPs that have a significant impact on the results. All results were corrected by the Bonferroni correction.ResultsThe IVW results showed that n-3 PUFAs (OR = 1.030, 95% CI: 1.013, 1.047, P = 0.001) and n-6 PUFAs (OR = 1.053, 95% CI: 1.034, 1.072, P 0.0025). None of IL-6, TNF-β, and BMP-7 had a causal effect on PUFA and BMD simultaneously (all P > 0.05).ConclusionEvidence from this MR study supports the genetically predicted causal effects of n-3, n-6, n-3 pct, and n-6 to n-3 on eBMD. In addition, n-3 not only associate with FA BMD and LS BMD through its own level and n-6 to n-3, but also link to fracture through n-3 pct.</p

Solubility and activity of MELK KD-UBA mutants.

<p>All mutants were overexpressed in <i>E. coli</i> and the soluble proteins were purified to homogeneity as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070031#s2" target="_blank">Materials and Methods</a>. The kinase assay was performed in the presence of 10 µM AMARA, 10 mM DTT and 100 nM MELK in standard kinase assay buffer (mean and s.e.m., n = 3).</p