Structural mechanism of HP1⍺-dependent transcriptional repression and chromatin compaction.

Heterochromatin protein 1 (HP1) plays a central role in establishing and maintaining constitutive heterochromatin. However, the mechanisms underlying HP1-nucleosome interactions and their contributions to heterochromatin functions remain elusive. Here, we present the cryoelectron microscopy (cryo-EM) structure of an HP1α dimer bound to an H2A.

Structural mechanism of HP1α-dependent transcriptional repression and chromatin compaction.

Heterochromatin protein 1 (HP1) plays a central role in establishing and maintaining constitutive heterochromatin. However, the mechanisms underlying HP1-nucleosome interactions and their contributions to heterochromatin functions remain elusive. In this study, we employed a multidisciplinary approach to unravel the interactions between human HP1α and nucleosomes.

DNA methylation and hydroxymethylation analysis using a high throughput and low bias direct injection mass spectrometry platform.

DNA modifications are small covalent chemical groups that modify nucleotides to regulate DNA readout. Anomalous abundance and genome-wide localization of these modifications can negatively tune gene expression and propagate into unbalanced epigenetics regulation, which is known to be associated with multiple conditions such as cancer, diabetes and aging. We present a direct injection mass spectrometry (DI-MS) platform that offers fast, accurate and precise quantitation of global levels of DNA cytidine methylation (mC) and hydroxymethylation (hmC) in less than one minute per sample.

Best Practices for Aggregate Quantitation of Antibody Therapeutics by Sedimentation Velocity Analytical Ultracentrifugation.

Analytical ultracentrifugation (AUC) is a critical analytical tool supporting the development and manufacture of protein therapeutics. AUC is routinely used as an assay orthogonal to size exclusion chromatography for aggregate quantitation. This article distills the experimental and analysis procedures used by the authors for sedimentation velocity AUC into a series of best-practices considerations.

INI1/SMARCB1 Rpt1 domain mimics TAR RNA in binding to integrase to facilitate HIV-1 replication.

INI1/SMARCB1 binds to HIV-1 integrase (IN) through its Rpt1 domain and exhibits multifaceted role in HIV-1 replication. Determining the NMR structure of INI1-Rpt1 and modeling its interaction with the IN-C-terminal domain (IN-CTD) reveal that INI1-Rpt1/IN-CTD interface residues overlap with those required for IN/RNA interaction. Mutational analyses validate our model and indicate that the same IN residues are involved in both INI1 and RNA binding.

Conformational and lipid bilayer-perturbing properties of Marburg virus GP2 segments containing the fusion loop and membrane-proximal external region/transmembrane domain.

Fusion of host and viral membranes is a crucial step during infection by enveloped viruses. In the structurally-defined "class I″ viral glycoproteins, the formation of a highly stable α-helical bundle by the ectodomain of the fusion subunit (e.g.

Ca(II) and Zn(II) Cooperate To Modulate the Structure and Self-Assembly of S100A12.

S100A12 is a member of the Ca binding S100 family of proteins that functions within the human innate immune system. Zinc sequestration by S100A12 confers antimicrobial activity when the protein is secreted by neutrophils. Here, we demonstrate that Ca binding to S100A12's EF-hand motifs and Zn binding to its dimeric interface cooperate to induce reversible self-assembly of the protein.

Chemical Generation of Hydroxyl Radical for Oxidative 'Footprinting'.

Background: For almost four decades, hydroxyl radical chemically generated by Fenton chemistry has been a mainstay for the oxidative 'footprinting' of macromolecules.

Objective: In this article, we start by reviewing the application of chemical generation of hydroxyl radical to the development of oxidative footprinting of DNA and RNA and the subsequent application of the method to oxidative footprinting of proteins. We next discuss a novel strategy for generating hydroxyl radicals by Fenton chemistry that immobilizes catalytic iron on a solid surface (Pyrite Shrink Wrap laminate) for the application of nucleic acid and protein footprinting.

Dynamic intramolecular regulation of the histone chaperone nucleoplasmin controls histone binding and release.

Nucleoplasmin (Npm) is a highly conserved histone chaperone responsible for the maternal storage and zygotic release of histones H2A/H2B. Npm contains a pentameric N-terminal core domain and an intrinsically disordered C-terminal tail domain. Though intrinsically disordered regions are common among histone chaperones, their roles in histone binding and chaperoning remain unclear.

Structures of the L27 Domain of Disc Large Homologue 1 Protein Illustrate a Self-Assembly Module.

Disc large 1 (Dlg1) proteins, members of the MAGUK protein family, are linked to cell polarity via their participation in multiprotein assemblies. At their N-termini, Dlg1 proteins contain a L27 domain. Typically, the L27 domains participate in the formation of obligate hetero-oligomers with the L27 domains from their cognate partners.

An Opportunity for Industry-Academia Partnership: Training the Next Generation of Industrial Researchers in Characterizing Higher Order Protein Structure.

Training researchers for positions in the United States biopharmaceutical industry has long been driven by academia. This commentary explores how the changing landscape of academic training will impact the industrial workforce, particularly with regard to the development of protein therapeutics in the area of biophysical and higher order structural characterization. We discuss how to balance future training and employment opportunities, how academic-industrial partnerships can help young scientists acquire the skills needed by their future employer, and how an appropriately trained workforce can facilitate the translation of new technology from academic to industrial laboratories.

Bright monomeric near-infrared fluorescent proteins as tags and biosensors for multiscale imaging.

Monomeric near-infrared (NIR) fluorescent proteins (FPs) are in high demand as protein tags and components of biosensors for deep-tissue imaging and multicolour microscopy. We report three bright and spectrally distinct monomeric NIR FPs, termed miRFPs, engineered from bacterial phytochrome, which can be used as easily as GFP-like FPs. miRFPs are 2-5-fold brighter in mammalian cells than other monomeric NIR FPs and perform well in protein fusions, allowing multicolour structured illumination microscopy.

MeCP2 Binding Cooperativity Inhibits DNA Modification-Specific Recognition.

Methyl-CpG binding protein 2 (MeCP2) is a multifunctional protein that guides neuronal development through its binding to DNA, recognition of sites of methyl-CpG (mCpG) DNA modification, and interaction with other regulatory proteins. Our study explores the relationship between mCpG and hydroxymethyl-CpG (hmCpG) recognition mediated by its mCpG binding domain (MBD) and binding cooperativity mediated by its C-terminal polypeptide. Previous study of the isolated MBD of MeCP2 documented an unusual mechanism by which ion uptake is required for discrimination of mCpG and hmCpG from CpG.

Assembly and Molecular Architecture of the Phosphoinositide 3-Kinase p85α Homodimer.

Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases that are activated by growth factor and G-protein-coupled receptors and propagate intracellular signals for growth, survival, proliferation, and metabolism. p85α, a modular protein consisting of five domains, binds and inhibits the enzymatic activity of class IA PI3K catalytic subunits. Here, we describe the structural states of the p85α dimer, based on data from in vivo and in vitro solution characterization.

Different inhibition of Gβγ-stimulated class IB phosphoinositide 3-kinase (PI3K) variants by a monoclonal antibody. Specific function of p101 as a Gβγ-dependent regulator of PI3Kγ enzymatic activity.

Class IB phosphoinositide 3-kinases γ (PI3Kγ) are second-messenger-generating enzymes downstream of signalling cascades triggered by G-protein-coupled receptors (GPCRs). PI3Kγ variants have one catalytic p110γ subunit that can form two different heterodimers by binding to one of a pair of non-catalytic subunits, p87 or p101. Growing experimental data argue for a different regulation of p87-p110γ and p101-p110γ allowing integration into distinct signalling pathways.

Structural and Functional Studies on the Marburg Virus GP2 Fusion Loop.

Marburg virus (MARV) and the ebolaviruses belong to the family Filoviridae (the members of which are filoviruses) that cause severe hemorrhagic fever. Infection requires fusion of the host and viral membranes, a process that occurs in the host cell endosomal compartment and is facilitated by the envelope glycoprotein fusion subunit, GP2. The N-terminal fusion loop (FL) of GP2 is a hydrophobic disulfide-bonded loop that is postulated to insert and disrupt the host endosomal membrane during fusion.

Protein footprinting by pyrite shrink-wrap laminate.

The structure of macromolecules and their complexes dictate their biological function. In "footprinting", the solvent accessibility of the residues that constitute proteins, DNA and RNA can be determined from their reactivity to an exogenous reagent such as the hydroxyl radical (·OH). While ·OH generation for protein footprinting is achieved by radiolysis, photolysis and electrochemistry, we present a simpler solution.

Conditional trimerization and lytic activity of HIV-1 gp41 variants containing the membrane-associated segments.

Fusion of host and viral membranes is a critical step during infection by membrane-bound viruses. The HIV-1 glycoproteins gp120 (surface subunit) and gp41 (fusion subunit) represent the prototypic system for studying this process; in the prevailing model, the gp41 ectodomain forms a trimeric six-helix bundle that constitutes a critical intermediate and provides the energetic driving force for overcoming barriers associated with membrane fusion. However, most structural studies of gp41 variants have been performed either on ectodomain constructs lacking one or more of the membrane-associated segments (the fusion peptide, FP, the membrane-proximal external region, MPER, and the transmembrane domain, TM) or on variants consisting of these isolated segments alone without the ectodomain.

Unusual characteristics of the DNA binding domain of epigenetic regulatory protein MeCP2 determine its binding specificity.

The protein MeCP2 mediates epigenetic regulation by binding methyl-CpG (mCpG) sites on chromatin. MeCP2 consists of six domains of which one, the methyl binding domain (MBD), binds mCpG sites in duplex DNA. We show that solution conditions with physiological or greater salt concentrations or the presence of nonspecific competitor DNA is necessary for the MBD to discriminate mCpG from CpG with high specificity.

Mapping the kinetic barriers of a Large RNA molecule's folding landscape.

The folding of linear polymers into discrete three-dimensional structures is often required for biological function. The formation of long-lived intermediates is a hallmark of the folding of large RNA molecules due to the ruggedness of their energy landscapes. The precise thermodynamic nature of the barriers (whether enthalpic or entropic) that leads to intermediate formation is still poorly characterized in large structured RNA molecules.

An RNA aptamer possessing a novel monovalent cation-mediated fold inhibits lysozyme catalysis by inhibiting the binding of long natural substrates.

RNA aptamers are being developed as inhibitors of macromolecular and cellular function, diagnostic tools, and potential therapeutics. Our understanding of the physical nature of this emerging class of nucleic acid-protein complexes is limited; few atomic resolution structures have been reported for aptamers bound to their protein target. Guided by chemical mapping, we systematically minimized an RNA aptamer (Lys1) selected against hen egg white lysozyme.