The nucleosome remodeling and deacetylase (NuRD) complex is essential for metazoan development but has been refractory to biochemical analysis. We present an integrated analysis of the native mammalian NuRD complex, combining quantitative mass spectrometry, cross-linking, protein biochemistry, and electron microscopy to define the architecture of the complex. NuRD is built from a 2:2:4 (MTA, HDAC, and RBBP) deacetylase module and a 1:1:1 (MBD, GATAD2, and Chromodomain-Helicase-DNA-binding [CHD]) remodeling module, and the complex displays considerable structural dynamics. The enigmatic GATAD2 controls the asymmetry of the complex and directly recruits the CHD remodeler. The MTA-MBD interaction acts as a point of functional switching, with the transcriptional regulator PWWP2A competing with MBD for binding to the MTA-HDAC-RBBP subcomplex. Overall, our data address the long-running controversy over NuRD stoichiometry, provide imaging of the mammalian NuRD complex, and establish the biochemical mechanism by which PWWP2A can regulate NuRD composition.

Correction for ‘Twists or turns: stabilising alpha vs. beta turns in tetrapeptides’ by Huy N. Hoang et al., Chem. Sci., 2019, 10, 10595–10600, DOI: 10.1039/C9SC04153B.

Peptides and proteins are not orally bioavailable in mammals, although a few peptides are intestinally absorbed in small amounts. Polypeptides are generally too large and polar to passively diffuse through lipid membranes, while most known active transport mechanisms facilitate cell uptake of only very small peptides. Systematic evaluations of peptides with molecular weights above 500 Da are needed to identify parameters that influence oral bioavailability. Here we describe 125 cyclic peptides Containing four to thirty-seven amino acids that are orally absorbed by mammals. Cyclization minimizes degradation in the gut, blood, and tissues by removing cleavable N- and C-termini and by shielding components from metabolic enzymes. Cyclization also folds peptides into bioactive conformations that determine exposure of polar atoms to solvation by water and lipids and therefore can influence oral bioavailability. Key chemical properties thought to influence oral absorption and bioavailability are analyzed, including molecular weight, octanol water partitioning, hydrogen bond donors/acceptors, rotatable bonds, and polar surface area. The cyclic peptides violated to different degrees all of the limits traditionally considered to be important for oral bioavailability;of drug-like small molecules, although fewer hydrogen bond donors and reduced flexibility generally favored oral absorption.

The N ucleosome R emodeling and D eacetylase (NuRD) complex is a ~1‐MDa 10‐subunit transcriptional co‐regulator that plays essential roles in hematopoietic development, stem cell renewal and DNA repair, and is emerging as a central player in diseases ranging from cancer to thalassemia. Despite the importance of this complex, little is known about its structure and biochemical activity. Hence, knowledge of NuRD structure and function will provide broad insight into mechanisms of eukaryotic gene regulation. To address this deficit, we are using a barrage of biochemical and biophysical approaches. We recently showed that the NuRD complex can exist as a separate stable entity (without CHD4) dubbed the NuDe (Nucleosome Deacetylase) complex — disrupting the architectural paradigm that CHD4 is an integrated core component of the complex (1). In addition, a combination of mass spectrometry and single‐particle electron microscopy has allowed us to piece together the architecture of NuRD through its subcomplexes at (quite) low resolution (2). Last but not least, we are now utilising single‐molecule fluorescence to observe the remodeling activity of individual NuRD complexes in real time. This could reveal the biochemical mechanism by which this complex remodels chromatin during gene transcription and DNA repair. Support or Funding Information Australian Research Council (DE120102857) Australian National Health and Medical Research Council (APP1012161 and 571099)

The nucleosome remodelling and deacetylase (NuRD) complex is essential for the development of complex animals. NuRD has roles in regulating gene expression and repairing damaged DNA. The complex comprises at least six proteins with two or more paralogues of each protein routinely identified when the complex is purified from cell extracts. To understand the structure and function of NuRD, a map of direct subunit interactions is needed. Dozens of published studies have attempted to define direct inter-subunit connectivities. We propose that conclusions reported in many such studies are in fact ambiguous for one of several reasons. First, the expression of many NuRD subunits in bacteria is unlikely to lead to folded, active protein. Second, interaction studies carried out in cells that contain endogenous NuRD complex can lead to false positives through bridging of target proteins by endogenous components. Combining existing information on NuRD structure with a protocol designed to minimize false positives, we report a conservative and robust interaction map for the NuRD complex. We also suggest a 3D model of the complex that brings together the existing data on the complex. The issues and strategies discussed herein are also applicable to the analysis of a wide range of multi-subunit complexes. ENZYMES:Micrococcal nuclease (MNase), EC 3.1.31.1; histone deacetylase (HDAC), EC 3.5.1.98.

Chromatin remodeling enzymes act to dynamically regulate gene accessibility. In many cases, these enzymes function as large multicomponent complexes that in general comprise a central ATP-dependent Snf2 family helicase that is decorated with a variable number of regulatory subunits. The nucleosome remodeling and deacetylase (NuRD) complex, which is essential for normal development in higher organisms, is one such macromolecular machine. The NuRD complex comprises ∼10 subunits, including the histone deacetylases 1 and 2 (HDAC1 and HDAC2), and is defined by the presence of a CHD family remodeling enzyme, most commonly CHD4 (chromodomain helicase DNA-binding protein 4). The existing paradigm holds that CHD4 acts as the central hub upon which the complex is built. We show here that this paradigm does not, in fact, hold and that CHD4 is a peripheral component of the NuRD complex. A complex lacking CHD4 that has HDAC activity can exist as a stable species. The addition of recombinant CHD4 to this nucleosome deacetylase complex reconstitutes a NuRD complex with nucleosome remodeling activity. These data contribute to our understanding of the architecture of the NuRD complex.

AbstractCyclic pentapeptides (e.g. Ac‐(cyclo‐1,5)‐[KAXAD]‐NH2; X=Ala, 1; Arg, 2) in water adopt one α‐helical turn defined by three hydrogen bonds. NMR structure analysis reveals a slight distortion from α‐helicity at the C‐terminal aspartate caused by torsional restraints imposed by the K(i)–D(i+4) lactam bridge. To investigate this effect on helix nucleation, the more water‐soluble 2 was appended to N‐, C‐, or both termini of a palindromic peptide ARAARAARA (≤5 % helicity), resulting in 67, 92, or 100 % relative α‐helicity, as calculated from CD spectra. From the C‐terminus of peptides, 2 can nucleate at least six α‐helical turns. From the N‐terminus, imperfect alignment of the Asp5 backbone amide in 2 reduces helix nucleation, but is corrected by a second unit of 2 separated by 0–9 residues from the first. These cyclic peptides are extremely versatile helix nucleators that can be placed anywhere in 5–25 residue peptides, which correspond to most helix lengths in protein–protein interactions.

Bcl2 Homology (BH) proteins can either trigger or prevent programmed cell death or apoptosis. Deregulation of the BH protein family network leads to evasion of apoptosis, uncontrolled proliferation and is a hallmark of cancer. Inhibition of pro-survival BH proteins is a promising chemotherapeutic strategy for certain cancers. We have examined whether helix-constrained peptides based on the BAD BH3 domain (residues 103-127) can be downsized to much smaller more drug-like peptides. We report the preparation, structural characterisation, in vitro Bcl-xL inhibition and leukemic T-cell killing ability of 45 linear, mono-, bi- and tricyclic helical peptidomimetics between 8- and 19-residues in length. We show that the BAD BH3 can be downsized to 8- 14 residues and still maintain appreciable affinity for Bcl-xL. In addition, the binding efficiency indices (BEI) of the downsized mimetics are significantly higher than the BAD BH3 and similar stapled BH3 mimetics, approaching drug-like molecules. This suggests that bicyclic and monocyclic mimetics based on BH3 domains are much more efficient binding ligands than the longer peptides which they mimic.

Chromatin remodeling enzymes act to dynamically regulate gene accessibility. In many cases, these enzymes function as large multicomponent complexes that, in general, comprise a central ATP-dependent Snf2-family helicase that is decorated with a variable number of regulatory subunits. The Nucleosome Remodeling and Deacetylase complex (NuRD), which is essential for normal development in higher organisms, is one such macromolecular machine. The NuRD complex comprises approximately 10 subunits, including the histone deacetylases 1 and 2 (HDAC1 and HDAC2), and is defined by the presence of a CHD-family remodeling enzyme – most commonly Chromodomain Helicase DNAbinding protein 4 (CHD4). The existing paradigm holds that CHD4 acts as the central hub upon which the complex is built. We show here that this paradigm does not, in fact, hold and that CHD4 is only a peripheral and dynamic component of the NuRD complex. A complex lacking CHD4 that has HDAC activity can exist as a stable species in cells. The addition of recombinant CHD4 to this Nucleosome Deacetylase (NuDe) complex reconstitutes a NuRD complex with nucleosome remodeling activity. These data are an important step towards understanding the architecture of the NuRD complex. Nucleosomes effectively act as a roadblock to all aspects of genome biology. ATPdependent chromatin remodeling enzymes solve this problem by using ATP-derived energy to alter the positions, occupancy and composition of nucleosomes. All remodelers possess a highlyrelated ATPase motor domain from the helicase family and are classified into four subfamilies (INO80, ISWI, SWR1 and CHD) based on sequence similarity (1). Each subfamily is represented in nearly all eukaryotes, suggesting that they catalyse different remodeling events. For example, ISWI proteins reposition (or slide) nucleosomes to create regularly spaced arrays; this periodic organisation is a key characteristic of DNA at the start of genes (2). SWR1 and INO80 enzymes have opposing roles in histone variant dynamics; the former incorporates these histone variants (e.g., H2A.Z) while the latter removes them. These variants set up specific chromatin structures that modulate transcription and replication, although the roles of many variants are http://www.jbc.org/cgi/doi/10.1074/jbc.M115.707018 The latest version is at JBC Papers in Press. Published on May 27, 2016 as Manuscript M115.707018 Copyright 2016 by The American Society for Biochemistry and Molecular Biology, Inc. by gest on M ay 1, 2016 hp://w w w .jb.org/ D ow nladed from CHD4 is a dynamic component of the NuRD complex

Correction for u0027Downsizing the BAD BH3 peptide to small constrained α-helices with improved ligand efficiencyu0027 by Nicholas E. Shepherd et al., Org. Biomol. Chem., 2016, DOI: 10.1039/c6ob02185a.

Canonical single-stranded DNA-binding proteins (SSBs) from the oligosaccharide/oligonucleotide-binding (OB) domain family are present in all known organisms and are critical for DNA replication, recombination and repair. The SSB from the hyperthermophilic crenarchaeote Sulfolobus solfataricus (SsoSSB) has a ‘simple’ domain organization consisting of a single DNA-binding OB fold coupled to a flexible C-terminal tail, in contrast with other SSBs in this family that incorporate up to four OB domains. Despite the large differences in the domain organization within the SSB family, the structure of the OB domain is remarkably similar all cellular life forms. However, there are significant differences in the molecular mechanism of ssDNA binding. We have determined the structure of the SsoSSB OB domain bound to ssDNA by NMR spectroscopy. We reveal that ssDNA recognition is modulated by base-stacking of three key aromatic residues, in contrast with the OB domains of human RPA and the recently discovered human homologue of SsoSSB, hSSB1. We also demonstrate that SsoSSB binds ssDNA with a footprint of five bases and with a defined binding polarity. These data elucidate the structural basis of DNA binding and shed light on the molecular mechanism by which these ‘simple’ SSBs interact with ssDNA.

We have developed an approach for directly isolating an intact multi-protein chromatin remodeling complex from mammalian cell extracts using synthetic peptide affinity reagent 4. FOG1(1–15), a short peptide sequence known to target subunits of the nucleosome remodeling and deacetylase (NuRD) complex, was joined via a 35-atom hydrophilic linker to the StreptagII peptide. Loading this peptide onto Streptactin beads enabled capture of the intact NuRD complex from MEL cell nuclear extract. Gentle biotin elution yielded the desired intact complex free of significant contaminants and in a form that was catalytically competent in a nucleosome remodeling assay. The efficiency of 4 in isolating the NuRD complex was comparable to other reported methods utilising recombinantly produced GST–FOG1(1–45).

Nitroxide labels are useful probes of biomolecule structure that can be detected by NMR and EPR spectroscopy. Although many methods exist for labeling oligonucleotides with nitroxides, most require reagents that are expensive or laborious to prepare. A simpler approach is described herein using commercially available phosphorothioate oligonucleotides and 2-(3-iodoacetamidomethyl)-PROXYL. We describe semi-optimized conditions for labeling DNA and RNA oligonucleotides and methodology for purifying and identifying these reagents by MALDI-MS. The nitroxide label showed some propensity to hydrolyze at high temperature and over prolonged periods at room temperature. Nitroxide-labeled DNA oligonucleotides gave characteristic EPR spectra and caused the disappearance of bound protein signals in 15N HSQC spectra consistent with the paramagnetic relaxation enhancement (PRE) effect.

Nociceptin (orphanin FQ) is a 17-residue neuropeptide hormone with roles in both nociception and analgesia. It is an opioid-like peptide that binds to and activates the G-protein-coupled receptor opioid receptor-like-1 (ORL-1, NOP, orphanin FQ receptor, kappa-type 3 opioid receptor) on central and peripheral nervous tissue, without activating classic delta-, kappa-, or mu-opioid receptors or being inhibited by the classic opioid antagonist naloxone. The three-dimensional structure of ORL-1 was recently published, and the activation mechanism is believed to involve capture by ORL-1 of the high-affinity binding, prohelical C-terminus. This likely anchors the receptor-activating N-terminus of nociception nearby for insertion in the membrane-spanning helices of ORL-1. In search of higher agonist potency, two lysine and two aspartate residues were strategically incorporated into the receptor-binding C-terminus of the nociceptin sequence and two Lys(i)→Asp(i+4) side chain-side chain condensations were used to generate lactam cross-links that constrained nociceptin into a highly stable α-helix in water. A cell-based assay was developed using natively expressed ORL-1 receptors on mouse neuroblastoma cells to measure phosphorylated ERK as a reporter of agonist-induced receptor activation and intracellular signaling. Agonist activity was increased up to 20-fold over native nociceptin using a combination of this helix-inducing strategy and other amino acid modifications. An NMR-derived three-dimensional solution structure is described for a potent ORL-1 agonist derived from nociceptin, along with structure-activity relationships leading to the most potent known α-helical ORL-1 agonist (EC₅₀ 40 pM, pERK, Neuro-2a cells) and antagonist (IC₅₀ 7 nM, pERK, Neuro-2a cells). These α-helix-constrained mimetics of nociceptin(1-17) had enhanced serum stability relative to unconstrained peptide analogues and nociceptin itself, were not cytotoxic, and displayed potent thermal analgesic and antianalgesic properties in rats (ED₅₀ 70 pmol, IC₅₀ 10 nmol, s.c.), suggesting promising uses in vivo for the treatment of pain and other ORL-1-mediated responses.

The realization that gene transcription is much more pervasive than previously thought and that many diverse RNA species exist in simple as well as complex organisms has triggered efforts to develop functionalized RNA-binding proteins (RBPs) that have the ability to probe and manipulate RNA function. Previously, we showed that the RanBP2-type zinc finger (ZF) domain is a good candidate for an addressable single-stranded-RNA (ssRNA) binding domain that can recognize ssRNA in a modular and specific manner. In the present study, we successfully engineered a sequence specificity change onto this ZF scaffold by using a combinatorial approach based on phage display. This work constitutes a foundation from which a set of RanBP2 ZFs might be developed that is able to recognize any given RNA sequence.

HIV-1 Rev is a 116 residue transporter protein that enters the host cell nucleus and uses its 17 amino acid segment (Rev34–50) to bind and capture a specific piece of RNA, the Rev Response Element (RRE), for transport to the cytoplasm. This is critical for HIV replication. In isolation, Rev34–50 shows negligible structure in water, but is alpha helical in a mixture of water and 2,2,2-trifluoroethanol (TFE) or when bound to RRE. Here we report that helix-constrained cyclic pentapeptides, either appended to the N-terminus or incorporated within Rev34–50, are efficient helix nucleators in water. They induce up to 90% alpha helicity for isolated Rev peptides in water and confer high RNA-binding affinity.

Background: The NuRD complex controls gene expression through altering chromatin structure. Results: The MTA1-RbAp48 structure shows how the RbAp46/p48 histone chaperones are recruited to NuRD. Conclusion: The MTA subunits act as scaffolds for NuRD complex assembly. Significance: The MTA/RbAp48 interaction prevents binding of histone H4, which is crucial for understanding the role of the RbAp46/p48 chaperones in the complex. The nucleosome remodeling and deacetylase (NuRD) complex is a widely conserved transcriptional co-regulator that harbors both nucleosome remodeling and histone deacetylase activities. It plays a critical role in the early stages of ES cell differentiation and the reprogramming of somatic to induced pluripotent stem cells. Abnormalities in several NuRD proteins are associated with cancer and aging. We have investigated the architecture of NuRD by determining the structure of a subcomplex comprising RbAp48 and MTA1. Surprisingly, RbAp48 recognizes MTA1 using the same site that it uses to bind histone H4, showing that assembly into NuRD modulates RbAp46/48 interactions with histones. Taken together with other results, our data show that the MTA proteins act as scaffolds for NuRD complex assembly. We further show that the RbAp48-MTA1 interaction is essential for the in vivo integration of RbAp46/48 into the NuRD complex.

Myocardial ischemia and cardioprotection by ischemic preconditioning induce signal networks aimed at survival or cell death if the ischemic period is prolonged. These pathways are mediated by protein post-translational modifications that are hypothesized to cross-talk with and regulate each other. Phosphopeptides and lysine-acetylated peptides were quantified in isolated rat hearts subjected to ischemia or ischemic pre-conditioning, with and without splitomicin inhibition of lysine deacetylation. We show lysine acetylation (acetyl-Lys)-dependent activation of AMP-activated protein kinase, AKT, andPKA kinases during ischemia. Phosphorylation and acetyl-Lys sites mapped onto tertiary structures were proximal in >50% of proteins investigated, yet they were mutually exclusive in 50 ischemic pre-conditioning-and/or ischemia-associated peptides containing the KXXS basophilic protein kinase consensus motif. Modifications in this motif were modeled in the C terminus of muscle-type creatine kinase. Acetyl-Lys increased proximal dephosphorylation by 10-fold. Structural analysis of modified muscle-type creatine kinase peptide variants by two-dimensional NMR revealed stabilization via a lysine-phosphate salt bridge, which was disrupted by acetyl-Lys resulting in backbone flexibility and increased phosphatase accessibility.