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About a quarter of total human cancers carry mutations in Ras isoforms. Accumulating evidence suggests that small GTPases, RalA and RalB, and their activators, Ral guanine nucleotide exchange factors (RalGEFs), play an essential role in oncogenic Ras-induced signalling. We studied the interaction between human K-Ras4B and the Ras association (RA) domain of Rgl2 (Rgl2RA), one of the RA-containing RalGEFs. We show that the G12V oncogenic K-Ras4B mutation increases the affinity with Rgl2RA. The crystal structure of the K-Ras4BG12V: Rgl2RAcomplex shows a 2:2 heterotetramer where the Switch I and Switch II regions of each K-RasG12Vinteract with both Rgl2RAmolecules. This structural arrangement is highly similar to the H-RasE31K:RALGDSRAcrystal structure and is distinct from the well-characterised Ras:Raf complex. Importantly, the G12V mutation was found at the dimer interface of K-Ras4BG12Vwith its partner. Our study reveals a potentially distinct mode of Ras:effector complex formation by RalGEFs, and offers a possible mechanistic explanation for how the oncogenic K-Ras4BG12Vhyperactivates the RalA/B pathway.Summary BlurbThe work reports that the K-RAS4B.G12V oncogenic mutation increases the affinity with Rgl2, an activator of RalA/B and provides a structural explanation by analysing the K-Ras4B(G12V):Rgl2 complex crystal structure where the G12V mutation resides at the interface of the complex.

Histone deacetylases 1 and 2 (HDAC1/2) serve as the catalytic subunit of six distinct families of nuclear complexes. These complexes repress gene transcription through removing acetyl groups from lysine residues in histone tails. In addition to the deacetylase subunit, these complexes typically contain transcription factor and/or chromatin binding activities. The MIER:HDAC complex has hitherto been poorly characterized. Here we show that MIER1 unexpectedly co-purifies with an H2A:H2B histone dimer. We show that MIER1 is also able to bind a complete histone octamer. Intriguingly, we found that a larger MIER1:HDAC1:BAHD1:C1QBP complex additionally co-purifies with an intact nucleosome on which H3K27 is either di- or tri-methylated. Together this suggests that the MIER1 complex acts downstream of PRC2 to expand regions of repressed chromatin and to deposit histone octamer onto nucleosome-depleted regions of DNA.

Class I histone deacetylase complexes play essential roles in many nuclear processes. Whilst they contain a common catalytic subunit, they have diverse modes of action determined by associated factors in the distinct complexes. The deacetylase module from the NuRD complex contains three protein domains that control the recruitment of chromatin to the deacetylase enzyme, HDAC1/2. Using biochemical approaches and cryo-electron microscopy, we have determined how three chromatin-binding domains (MTA1-BAH, MBD2/3 and RBBP4/7) are assembled in relation to the core complex so as to facilitate interaction of the complex with the genome. We observe a striking arrangement of the BAH domains suggesting a potential mechanism for binding to di-nucleosomes. We also find that the WD40 domains from RBBP4 are linked to the core with surprising flexibility that is likely important for chromatin engagement. A single MBD2 protein binds asymmetrically to the dimerisation interface of the complex. This symmetry mismatch explains the stoichiometry of the complex. Finally, our structures suggest how the holo-NuRD might assemble on a di-nucleosome substrate.

Summary The transcriptional corepressor complex CoREST is one of seven histone deacetylase complexes that regulate the genome through controlling chromatin acetylation. The CoREST complex is unique in containing both histone demethylase and deacetylase enzymes, LSD1 and HDAC1, held together by the RCOR1 scaffold protein. To date, it has been assumed that the enzymes function independently within the complex. Now, we report the assembly of the ternary complex. Using both structural and functional studies, we show that the activity of the two enzymes is closely coupled and that the complex can exist in at least two distinct states with different kinetics. Electron microscopy of the complex reveals a bi-lobed structure with LSD1 and HDAC1 enzymes at opposite ends of the complex. The structure of CoREST in complex with a nucleosome reveals a mode of chromatin engagement that contrasts with previous models., Graphical Abstract, Highlights • The activities of LSD1 and HDAC1 are closely coupled in the CoREST complex • Both LSD1 and HDAC1 exist in two different kinetic states • CoREST has a bi-lobed, flexible structure with the two enzymes located at opposite ends • CoREST interacts with methylated nucleosomes via LSD1, but not HDAC1 or RCOR1, Using a real-time NMR assay, Song et al. characterize crosstalk between LSD1 and HDAC1 in the CoREST complex. Activation or inhibition of one enzyme strongly affects activity of the other. Electron microscopy studies of the complex reveal a bi-lobed structure with implications for the mode of interaction with nucleosomes.

We have identified a proteolysis targeting chimera (PROTAC) of class I HDACs 1, 2 and 3. The most active degrader consists of a benzamide HDAC inhibitor, an alkyl linker, and the von Hippel-Lindau E3 ligand. Our PROTAC increased histone acetylation levels and compromised colon cancer HCT116 cell viability, establishing a degradation strategy as an alternative to class I HDAC inhibition.

The file attached to this record is the author's final peer reviewed version. The Publisher's final version can be found by following the DOI link. The circadian clock is an endogenous time-keeping system that is ubiquitous in animals and plants as well as some bacteria. In mammals, the clock regulates the sleep-wake cycle via 2 basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) domain proteins-CLOCK and BMAL1. There is emerging evidence to suggest that heme affects circadian control, through binding of heme to various circadian proteins, but the mechanisms of regulation are largely unknown. In this work we examine the interaction of heme with human CLOCK (hCLOCK). We present a crystal structure for the PAS-A domain of hCLOCK, and we examine heme binding to the PAS-A and PAS-B domains. UV-visible and electron paramagnetic resonance spectroscopies are consistent with a bis-histidine ligated heme species in solution in the oxidized (ferric) PAS-A protein, and by mutagenesis we identify His144 as a ligand to the heme. There is evidence for flexibility in the heme pocket, which may give rise to an additional Cys axial ligand at 20K (His/Cys coordination). Using DNA binding assays, we demonstrate that heme disrupts binding of CLOCK to its E-box DNA target. Evidence is presented for a conformationally mobile protein framework, which is linked to changes in heme ligation and which has the capacity to affect binding to the E-box. Within the hCLOCK structural framework, this would provide a mechanism for heme-dependent transcriptional regulation.

[Image: see text] Syntheses of Fmoc amino acids having zinc-binding groups were prepared and incorporated into substrate inhibitor H3K27 peptides using Fmoc/(t)Bu solid-phase peptide synthesis (SPPS). Peptide 11, prepared using Fmoc-Asu(NHO(t)Bu)-OH, is a potent inhibitor (IC(50) = 390 nM) of the core NuRD corepressor complex (HDAC1–MTA1–RBBP4). The Fmoc amino acids have the potential to facilitate the rapid preparation of substrate peptidomimetic inhibitor (SPI) libraries in the search for selective HDAC inhibitors.

The Aster proteins (encoded by the Gramd1a-c genes) contain a ligand-binding fold structurally similar to a START domain and mediate nonvesicular plasma membrane (PM) to endoplasmic reticulum (ER) cholesterol transport. In an effort to develop small molecule modulators of Asters, we identified 20α-hydroxycholesterol (HC) and U18666A as lead compounds. Unfortunately, both 20α-HC and U18666A target other sterol homeostatic proteins, limiting their utility. 20α-HC inhibits sterol regulatory element-binding protein 2 (SREBP2) processing, and U18666A is an inhibitor of the vesicular trafficking protein Niemann-Pick C1 (NPC1). To develop potent and selective Aster inhibitors, we synthesized a series of compounds by modifying 20α-HC and U18666A. Among these, AI (Aster inhibitor)-1l, which has a longer side chain than 20α-HC, selectively bound to Aster-C. The crystal structure of Aster-C in complex with AI-1l suggests that sequence and flexibility differences in the loop that gates the binding cavity may account for the ligand specificity for Aster C. We further identified the U18666A analog AI-3d as a potent inhibitor of all three Aster proteins. AI-3d blocks the ability of Asters to bind and transfer cholesterol in vitro and in cells. Importantly, AI-3d also inhibits the movement of low-density lipoprotein (LDL) cholesterol to the ER, although AI-3d does not block NPC1. This finding positions the nonvesicular Aster pathway downstream of NPC1-dependent vesicular transport in the movement of LDL cholesterol to the ER. Selective Aster inhibitors represent useful chemical tools to distinguish vesicular and nonvesicular sterol transport mechanisms in mammalian cells.

MiDAC is one of seven distinct, large multi-protein complexes that recruit class I histone deacetylases to the genome to regulate gene expression. Despite implications of involvement in cell cycle regulation and in several cancers, surprisingly little is known about the function or structure of MiDAC. Here we show that MiDAC is important for chromosome alignment during mitosis in cancer cell lines. Mice lacking the MiDAC proteins, DNTTIP1 or MIDEAS, die with identical phenotypes during late embryogenesis due to perturbations in gene expression that result in heart malformation and haematopoietic failure. This suggests that MiDAC has an essential and unique function that cannot be compensated by other HDAC complexes. Consistent with this, the cryoEM structure of MiDAC reveals a unique and distinctive mode of assembly. Four copies of HDAC1 are positioned at the periphery with outward-facing active sites suggesting that the complex may target multiple nucleosomes implying a processive deacetylase function., The MiDAC complex recruits class I histone deacetylases to chromatin but little is known about its precise structure and function. Here, the authors explore the role of MiDAC in the cell cycle and during mouse embryogenesis, and present cryoEM structures that provide insight into MiDAC’s mode of assembly.

A 23-year-old man and his grandmother with hyperthyroxinemia and hypercortisolemia were heterozygous for an ALB mutation (p. Arg218Pro), known to cause familial dysalbuminemic hyperthyroxinemia (FDH). However, serum-free cortisol levels in these individuals were normal and total cortisol concentrations fell markedly after depletion of albumin from their serum. We conclude that binding of steroid as well as iodothyronines to mutant albumin causes raised circulating cortisol as well as thyroid hormones in euthyroid euadrenal individuals with R218P FDH, with potential for misdiagnosis, unnecessary investigation, and inappropriate treatment.

Histone acetylation regulates chromatin structure and gene expression and is removed by histone deacetylases (HDACs). HDACs are commonly found in various protein complexes to confer distinct cellular functions, but how the multi-subunit complexes influence deacetylase activities and site-selectivities in chromatin is poorly understood. Previously we reported the results of studies on the HDAC1 containing CoREST complex and acetylated nucleosome substrates which revealed a notable preference for deacetylation of histone H3 acetyl-Lys9 vs. acetyl-Lys14 (Wu et al, 2018). Here we analyze the enzymatic properties of five class I HDAC complexes: CoREST, NuRD, Sin3B, MiDAC and SMRT with site-specific acetylated nucleosome substrates. Our results demonstrate that these HDAC complexes show a wide variety of deacetylase rates in a site-selective manner. A Gly13 in the histone H3 tail is responsible for a sharp reduction in deacetylase activity of the CoREST complex for H3K14ac. These studies provide a framework for connecting enzymatic and biological functions of specific HDAC complexes.

BCL6 is a transcriptional repressor. Two domains of the protein, the N-terminal BTB-POZ domain and the RD2 domain are responsible for recruitment of co-repressor molecules and histone deacetylases. The BTB-POZ domain is found in a large and diverse range of proteins that play important roles in development, homeostasis and neoplasia. Crystal structures of several BTB-POZ domains, including BCL6 have been determined. The BTB-POZ domain of BCL6 not only mediates dimerisation but is also responsible for recruitment of co-repressors such as SMRT, NCOR and BCOR. Interestingly both SMRT and BCOR bind to the same site within the BCL6 BTB-POZ domain despite having very different primary sequences. Since both peptides and small molecules have been shown to bind to the co-repressor binding site it would suggest that the BTB_POZ domain is a suitable target for drug discovery. Here we report near complete backbone 15N, 13C and 1H assignments for the BTB-POZ domain of BCL6 to assist in the analysis of binding modes for small molecules.

Following the first isolation of nuclear receptor (NR) genes, genetic disorders caused by NR gene mutations were initially discovered by a candidate gene approach based on their known roles in endocrine pathways and physiologic processes. Subsequently, the identification of disorders has been informed by phenotypes associated with gene disruption in animal models or by genetic linkage studies. More recently, whole exome sequencing has associated pathogenic genetic variants with unexpected, often multisystem, human phenotypes. To date, defects in 20 of 48 human NR genes have been associated with human disorders, with different mutations mediating phenotypes of varying severity or several distinct conditions being associated with different changes in the same gene. Studies of individuals with deleterious genetic variants can elucidate novel roles of human NRs, validating them as targets for drug development or providing new insights into structure-function relationships. Importantly, human genetic discoveries enable definitive disease diagnosis and can provide opportunities to therapeutically manage affected individuals. Here we review germline changes in human NR genes associated with "monogenic" conditions, including a discussion of the structural basis of mutations that cause distinctive changes in NR function and the molecular mechanisms mediating pathogenesis.

Histone deacetylase (HDAC) inhibitors are proven anticancer therapeutics and have potential in the treatment of many other diseases including HIV infection, Alzheimer's disease, and Friedreich's ataxia. A problem with the currently available HDAC inhibitors is that they have limited specificity and target multiple deacetylases. Designing isoform-selective inhibitors has proven challenging due to similarities in the structure and chemistry of HDAC active sites. However, the fact that HDACs 1, 2, and 3 are recruited to several large multi-subunit complexes, each with particular biological functions, raises the possibility of specifically inhibiting individual complexes. This may be assisted by recent structural and functional information about the assembly of these complexes. Here, we review the available structural information and discuss potential targeting strategies.

SummaryDrugs that recapitulate aspects of the exercise adaptive response have the potential to provide better treatment for diseases associated with physical inactivity. We previously observed reduced skeletal muscle class IIa HDAC (histone deacetylase) transcriptional repressive activity during exercise. Here, we find that exercise-like adaptations are induced by skeletal muscle expression of class IIa HDAC mutants that cannot form a corepressor complex. Adaptations include increased metabolic gene expression, mitochondrial capacity, and lipid oxidation. An existing HDAC inhibitor, Scriptaid, had similar phenotypic effects through disruption of the class IIa HDAC corepressor complex. Acute Scriptaid administration to mice increased the expression of metabolic genes, which required an intact class IIa HDAC corepressor complex. Chronic Scriptaid administration increased exercise capacity, whole-body energy expenditure and lipid oxidation, and reduced fasting blood lipids and glucose. Therefore, compounds that disrupt class IIa HDAC function could be used to enhance metabolic health in chronic diseases driven by physical inactivity.

Summary The death-inducing signaling complex (DISC) initiates death receptor-induced apoptosis. DISC assembly and activation are controlled by c-FLIP isoforms, which function as pro-apoptotic (c-FLIPL only) or anti-apoptotic (c-FLIPL/c-FLIPS) regulators of procaspase-8 activation. Current models assume that c-FLIP directly competes with procaspase-8 for recruitment to FADD. Using a functional reconstituted DISC, structure-guided mutagenesis, and quantitative LC-MS/MS, we show that c-FLIPL/S binding to the DISC is instead a co-operative procaspase-8-dependent process. FADD initially recruits procaspase-8, which in turn recruits and heterodimerizes with c-FLIPL/S via a hierarchical binding mechanism. Procaspase-8 activation is regulated by the ratio of unbound c-FLIPL/S to procaspase-8, which determines composition of the procaspase-8:c-FLIPL/S heterodimer. Thus, procaspase-8:c-FLIPL exhibits localized enzymatic activity and is preferentially an activator, promoting DED-mediated procaspase-8 oligomer assembly, whereas procaspase-8:c-FLIPS lacks activity and potently blocks procaspase-8 activation. This co-operative hierarchical binding model explains the dual role of c-FLIPL and crucially defines how c-FLIP isoforms differentially control cell fate., Graphical Abstract, Highlights • c-FLIP isoforms (L/S) do not directly compete with caspase-8 for binding to FADD • c-FLIP binds to the DISC via a co-operative hierarchical caspase-8-dependent process • Co-operative and hierarchical binding crucially explains the dual function of c-FLIPL • Our unified model defines how c-FLIP isoforms differentially direct cell fate, It is unclear how c-FLIP isoforms can differentially regulate caspase-8 activation to direct cell fate. Hughes et al. show that c-FLIPL/S recruitment to FADD is indirect and requires caspase-8. This co-operative hierarchical binding process explains the conundrum of the dual role of c-FLIPL and defines how c-FLIPL/S differentially control cell fate.

Prokaryotic expression systems have been widely used to express proteins for structural studies. Such expression systems have the advantage of being economical, straightforward and fast. However, for many eukaryotic proteins and particularly protein complexes, bacterial expression systems do not produce significant yields of soluble protein. This may result from failure to efficiently transcribe/translate the required protein or may result from the formation of insoluble aggregates known as inclusion bodies. Mammalian expression systems can often produce natively folded proteins, sometimes with native post-translational modifications. However, such expression systems are underutilized due to the perception that they are costly, technically challenging and result in limited protein yields. In fact, HEK 293F cells are straightforward to grow, transfect with high efficiency and often produce significant yields of recombinant proteins. In this unit, we describe a method to express and purify milligram quantities of a human protein complex from HEK 293F cells grown in suspension transiently transfected with the appropriate plasmids. © 2017 by John Wiley & Sons, Inc.

Fission yeast MOZART1 homologue Mzt1 associates with γ-tubulin and is found at MTOCs. It plays an essential role in microtubule regulation, as well as in septum formation and cytokinesis. Recombinant Mzt1 interacts directly with GCP3Alp6. This interaction may assist MTOC activity of the γ-tubulin complex., In humans, MOZART1 plays an essential role in mitotic spindle formation as a component of the γ-tubulin ring complex. We report that the fission yeast homologue of MOZART1, Mzt1/Tam4, is located at microtubule-organizing centers (MTOCs) and coimmunoprecipitates with γ-tubulin Gtb1 from cell extracts. We show that mzt1/tam4 is an essential gene in fission yeast, encoding a 64–amino acid peptide, depletion of which leads to aberrant microtubule structure, including malformed mitotic spindles and impaired interphase microtubule array. Mzt1/Tam4 depletion also causes cytokinesis defects, suggesting a role of the γ-tubulin complex in the regulation of cytokinesis. Yeast two-hybrid analysis shows that Mzt1/Tam4 forms a complex with Alp6, a fission yeast homologue of γ-tubulin complex protein 3 (GCP3). Biophysical methods demonstrate that there is a direct interaction between recombinant Mzt1/Tam4 and the N-terminal region of GCP3Alp6. Together our results suggest that Mzt1/Tam4 contributes to the MTOC function through regulation of GCP3Alp6.

Summary BAK activation represents a key step during apoptosis, but how it converts into a mitochondria-permeabilizing pore remains unclear. By further delineating the structural rearrangements involved, we reveal that BAK activation progresses through a series of independent steps: BH3-domain exposure, N-terminal change, oligomerization, and membrane insertion. Employing a “BCL-XL-addiction” model, we show that neutralization of BCL-XL by the BH3 mimetic ABT-737 resulted in death only when cells were reconstituted with BCL-XL:BAK, but not BCL-2/ BCL-XL:BIM complexes. Although this resembles the indirect model, release of BAK from BCL-XL did not result in spontaneous adoption of the pore conformation. Commitment to apoptosis required association of the direct activator BIM with oligomeric BAK promoting its conversion to a membrane-inserted pore. The sequential nature of this cascade provides multiple opportunities for other BCL-2 proteins to interfere with or promote BAK activation and unites aspects of the indirect and direct activation models., Graphical Abstract, Highlights • A BH3 mimetic displaces BAK from BCL-XL consistent with the indirect activation model • Released BAK undergoes a series of distinct independent conformational changes • Oligomerization is not the final step in the activation cascade • BIM promotes pre-pore-to-pore conversion supporting the “direct” activation model, BAK activation is a key step in apoptosis, but how it converts into a mitochondrial permeabilizing pore remains unclear. By analyzing the structural rearrangements involved, Cohen and colleagues now define how BAK activation occurs in a stepwise manner. Whereas the authors find some aspects consistent with the indirect activation model (where antiapoptotic BCL2 proteins sequester effectors), they provide data arguing that association of the direct activator BIM with oligomeric BAK is required for commitment to apoptosis.

Transient transfection of human embryonic kidney cells (HEK 293) enables the rapid and affordable labscale production of recombinant proteins. In this chapter protocols for the expression and purification of both secreted and intracellular proteins using transient expression in HEK 293 cells are described.

The NuRD complex is a multi-protein transcriptional corepressor that couples histone deacetylase and ATP-dependent chromatin remodelling activities. The complex regulates the higher-order structure of chromatin, and has important roles in the regulation of gene expression, DNA damage repair and cell differentiation. HDACs 1 and 2 are recruited by the MTA1 corepressor to form the catalytic core of the complex. The histone chaperone protein RBBP4, has previously been shown to bind to the carboxy-terminal tail of MTA1. We show that MTA1 recruits a second copy of RBBP4. The crystal structure reveals an extensive interface between MTA1 and RBBP4. An EM structure, supported by SAXS and crosslinking, reveals the architecture of the dimeric HDAC1:MTA1:RBBP4 assembly which forms the core of the NuRD complex. We find evidence that in this complex RBBP4 mediates interaction with histone H3 tails, but not histone H4, suggesting a mechanism for recruitment of the NuRD complex to chromatin. DOI: http://dx.doi.org/10.7554/eLife.13941.001, eLife digest The correct regulation of our genes is essential for life. Genes are actively switched on or off through the action of assemblies of proteins that act together as molecular machines. Some of these machines alter the way that DNA is packaged inside cells. Packaged DNA – called chromatin – consists of DNA wrapped around proteins called histones, which together form structures called nucleosomes. Changing how tightly nucleosomes are packed together can alter whether a gene is active: tighter packing makes it harder to access the genes in that stretch of DNA and therefore inactivates them. In humans, an assembly of proteins called the NuRD complex makes chromatin more compact by removing acetyl groups from nucleosomes. This complex is important for early development and for the stability and repair of our genes. Three proteins make up its core: HDAC1, which removes the acetyl group from the nucleosome; MTA1, which acts as a scaffold to hold the complex together; and RBBP4, which enables the complex to interact with nucleosomes. Understanding how protein complexes are assembled tells us a lot about how they work. Millard et al. have therefore used a number of structural techniques to investigate the three-dimensional architecture of the three core proteins in the NuRD complex. The resulting structures have revealed how the HDAC1, MTA1 and RBBP4 proteins interact to influence how the complex is recruited to nucleosomes. The next step will be to assemble all the remaining proteins of the NuRD complex to understand its architecture as a whole. DOI: http://dx.doi.org/10.7554/eLife.13941.002

Summary Formation of the death-inducing signaling complex (DISC) is a critical step in death receptor-mediated apoptosis, yet the mechanisms underlying assembly of this key multiprotein complex remain unclear. Using quantitative mass spectrometry, we have delineated the stoichiometry of the native TRAIL DISC. While current models suggest that core DISC components are present at a ratio of 1:1, our data indicate that FADD is substoichiometric relative to TRAIL-Rs or DED-only proteins; strikingly, there is up to 9-fold more caspase-8 than FADD in the DISC. Using structural modeling, we propose an alternative DISC model in which procaspase-8 molecules interact sequentially, via their DED domains, to form a caspase-activating chain. Mutating key interacting residues in procaspase-8 DED2 abrogates DED chain formation in cells and disrupts TRAIL/CD95 DISC-mediated procaspase-8 activation in a functional DISC reconstitution model. This provides direct experimental evidence for a DISC model in which DED chain assembly drives caspase-8 dimerization/activation, thereby triggering cell death., Graphical Abstract Highlights ► The TRAIL DISC is a soluble >700 kDa complex of TRAIL-Rs, FADD, and DED-only proteins ► LC-MS/MS defines FADD as substoichiometric relative to TRAIL-Rs/DED-only proteins ► Structural modeling reveals that FADD recruits multiple DED-only proteins to the DISC ► We uncover a crucial role for caspase-8 DED chain assembly in triggering cell death

Highlights ► Co-repressors SMRT/NCoR act as “hub proteins”. ► SMRT/NCoR integrate transcription factor and histone modifying enzyme signaling. ► Structures of co-repressor complexes. ► Structural studies provide mechanistic, and potentially therapeutic, insights., Co-repressor proteins, such as SMRT and NCoR, mediate the repressive activity of unliganded nuclear receptors and other transcription factors. They appear to act as intrinsically disordered “hub proteins” that integrate the activities of a range of transcription factors with a number of histone modifying enzymes. Although these co-repressor proteins are challenging targets for structural studies due to their largely unstructured character, a number of structures have recently been determined of co-repressor interaction regions in complex with their interacting partners. These have yielded considerable insight into the mechanism of assembly of these complexes, the structural basis for the specificity of the interactions and also open opportunities for targeting these interactions therapeutically.

The E3 ubiquitin ligase IDOL (inducible degrader of the LDL receptor) regulates LDL receptor (LDLR)-dependent cholesterol uptake, but its mechanism of action, including the molecular basis for its stringent specificity, is poorly understood. Here we show that IDOL uses a singular strategy among E3 ligases for target recognition. The IDOL FERM domain binds directly to a recognition sequence in the cytoplasmic tails of lipoprotein receptors. This physical interaction is independent of IDOL's really interesting new gene (RING) domain E3 ligase activity and its capacity for autoubiquitination. Furthermore, IDOL controls its own stability through autoubiquitination of a unique FERM subdomain fold not present in other FERM proteins. Key residues defining the IDOL–LDLR interaction and IDOL autoubiquitination are functionally conserved in their insect homologs. Finally, we demonstrate that target recognition by IDOL involves a tripartite interaction between the FERM domain, membrane phospholipids, and the lipoprotein receptor tail. Our data identify the IDOL–LDLR interaction as an evolutionarily conserved mechanism for the regulation of lipid uptake and suggest that this interaction could potentially be exploited for the pharmacologic modulation of lipid metabolism.

Nuclear receptors are arguably the best understood transcriptional regulators. We know a great deal about the mechanisms through which they activate transcription in response to ligand binding and about the mechanisms through which they repress transcription in the absence of ligand. However, endocrine regulation often requires that ligand-bound receptors repress transcription of a subset of genes. An understanding of the mechanism for ligand-induced repression and how this differs from activation has proven elusive. A number of recent studies have directly or indirectly addressed this problem. Yet it seems the more evidence that accumulates, the more complex the mystery becomes.

SummaryPeroxisome proliferator-activated receptor γ (PPARγ) is a lipid-activated transcription factor regulating lipid metabolism and inflammatory response in macrophages and dendritic cells (DCs). These immune cells exposed to distinct inflammatory milieu show cell type specification as a result of altered gene expression. We demonstrate here a mechanism how inflammatory molecules modulate PPARγ signaling in distinct subsets of cells. Proinflammatory molecules inhibited whereas interleukin-4 (IL-4) stimulated PPARγ activity in macrophages and DCs. Furthermore, IL-4 signaling augmented PPARγ activity through an interaction between PPARγ and signal transducer and activators of transcription 6 (STAT6) on promoters of PPARγ target genes, including FABP4. Thus, STAT6 acts as a facilitating factor for PPARγ by promoting DNA binding and consequently increasing the number of regulated genes and the magnitude of responses. This interaction, underpinning cell type-specific responses, represents a unique way of controlling nuclear receptor signaling by inflammatory molecules in immune cells.

Summary Objective Androgen insensitivity syndrome (AIS) is associated with mutations throughout the androgen receptor (AR) gene. Different mutations at the same codon have been identified in individuals with various phenotypes suggesting the nature of the codon substituted may influence the degree of AIS. We investigated if phenotype could be predicted by comparing the functionality of AR mutations with those at the same codon of known phenotype. Patients We identified patients from the Cambridge Disorders of Sex Development Database with the AR substitutions: Phe754Ser with microphallus without hypospadias and Asp690Val with complete AIS. Mutations Phe754Leu, Phe754Val and Asp690deletion (Asp690del) have previously been reported to be associated with different degrees of AIS. Design We characterized the functional properties of Phe754Ser, Phe754Leu, Phe754Val, Asp690Val and Asp690del receptor mutants in vitro and used the crystal structure of the AR ligand binding domain to model the mutations. Results The receptor mutants Phe754Ser, Phe754Leu and Phe754Val bound androgen with decreasing affinity, while Asp690Val showed reduced affinity compared to Asp690del. A similar pattern of reduced activation was seen on androgen responsive elements. We suggest how the mutations could affect AR structure, resulting in the observed phenotypes. Conclusions The relative functional properties of Phe754 and Asp690 mutant AR receptors correlate broadly with their specific phenotypes. Therefore, comparing the molecular consequences of novel mutations with others at the same codon may be a useful aid to AIS patient management, particularly for sex of rearing decisions when prediction of functionality is important.

The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) has important roles in adipogenesis and immune response as well as roles in both lipid and carbohydrate metabolism. Although synthetic agonists for PPARgamma are widely used as insulin sensitizers, the identity of the natural ligand(s) for PPARgamma is still not clear. Suggested natural ligands include 15-deoxy-delta12,14-prostaglandin J2 and oxidized fatty acids such as 9-HODE and 13-HODE. Crystal structures of PPARgamma have revealed the mode of recognition for synthetic compounds. Here we report structures of PPARgamma bound to oxidized fatty acids that are likely to be natural ligands for this receptor. These structures reveal that the receptor can (i) simultaneously bind two fatty acids and (ii) couple covalently with conjugated oxo fatty acids. Thermal stability and gene expression analyses suggest that such covalent ligands are particularly effective activators of PPARgamma and thus may serve as potent and biologically relevant ligands.

Histone deacetylase 3 removes acetyl groups from lysine residues, thereby modifying protein function. It is found in both the nucleus and the cytoplasm. We have discovered that it can indirectly deacetylate tubulin, a cytoplasmic protein that forms microtubules, thus modifying the microtubule., Histone deacetylase 3 (HDAC3), a member of the Class I subfamily of HDACs, is found in both the nucleus and the cytoplasm. Its roles in the nucleus have been well characterized, but its cytoplasmic roles are still not elucidated fully. We found that blocking HDAC3 activity using MI192, a compound specific for HDAC3, modulated tubulin acetylation in the human prostate cancer cell line PC3. A brief 1 h treatment of PC3 cells with MI192 significantly increased levels of tubulin acetylation and ablated the dynamic behaviour of microtubules in live cells. siRNA-mediated knockdown (KD) of HDAC3 in PC3 cells, significantly increased levels of tubulin acetylation, and overexpression reduced it. However, the active HDAC3–silencing mediator of retinoic and thyroid receptors (SMRT)–deacetylase-activating domain (DAD) complex did not directly deacetylate tubulin in vitro. These data suggest that HDAC3 indirectly modulates tubulin acetylation.

Nuclear receptor corepressors are key components of large multi-protein complexes that regulate gene expression. These complexes are recruited to specific genomic loci through the interaction with unliganded, or antagonist-bound, nuclear receptors as well as other repressive transcription factors. The activity of these complexes is mediated in large part through stably associated enzymes that act on the chromatin in the vicinity of the recruitment sites. The best-studied corepressors associated with nuclear receptors are the homologous proteins SMRT and NCoR. These are large proteins that contain extensive intrinsically disordered regions. These regions contain many short conserved sequence motifs that mediate the interactions with transcription factors as well as other proteins. A structured core region within the corepressors mediates assembly of SMRT and NCoR with two scaffold proteins GPS2 and TBL1 as well as the histone deacetylase enzyme HDAC3. Importantly, HDAC3 is activated through assembly into the complex and in the context of the complex its activity is regulated by inositol phosphates.

Loss-of-function mutations in the ligand-binding domain of human peroxisome proliferator-activated receptor gamma (PPARgamma) are associated with a novel syndrome characterized by partial lipodystrophy and severe insulin resistance. Here we have further characterized the properties of natural dominant-negative PPARgamma mutants (P467L, V290M) and evaluated the efficacy of putative natural ligands and synthetic thiazolidinedione (TZD) or tyrosine-based (TA) receptor agonists in rescuing mutant receptor function. A range of natural ligands failed to activate the PPARgamma mutants and their transcriptional responses to TZDs (e.g. pioglitazone, rosiglitazone) were markedly attenuated, whereas TAs (e.g. farglitazar) corrected defects in ligand binding and coactivator recruitment by the PPARgamma mutants, restoring transcriptional function comparable with wild-type receptor. Transcriptional silencing via recruitment of corepressor contributes to dominant-negative inhibition of wild type by the P467L and V290M mutants and the introduction of an artificial mutation (L318A) disrupting corepressor interaction abrogated their dominant-negative activity. More complete ligand-dependent corepressor release and reversal of dominant-negative inhibition was achieved with TA than TZD agonists. Modeling suggests a structural basis for these observations: both mutations destabilize helix 12 to favor receptor-corepressor interaction; conversely, farglitazar makes more extensive contacts than rosiglitazone within the ligand-binding pocket, to stabilize helix 12, facilitating corepressor release and transcriptional activation. Farglitazar was a more potent inducer of PPARgamma target gene (aP2) expression in peripheral blood mononuclear cells with the P467L mutation. Having shown that rosiglitazone is of variable and limited efficacy in these subjects, we suggest that TAs may represent a more rational therapeutic approach.

Spen proteins regulate the expression of key transcriptional effectors in diverse signaling pathways. They are large proteins characterized by N-terminal RNA-binding motifs and a highly conserved C-terminal SPOC domain. The specific biological role of the SPOC domain (Spenparalog andorthologC-terminal domain), and hence, the common function of Spen proteins, has been unclear to date. The Spen protein, SHARP (SMRT/HDAC1-associatedrepressorprotein), was identified as a component of transcriptional repression complexes in both nuclear receptor and Notch/RBP-Jκ signaling pathways. We have determined the 1.8 Å crystal structure of the SPOC domain from SHARP. This structure shows that essentially all of the conserved surface residues map to a positively charged patch. Structure-based mutational analysis indicates that this conserved region is responsible for the interaction between SHARP and the universal transcriptional corepressor SMRT/NCoR (silencingmediator forretinoid andthyroid receptors/nuclear receptorcorepressor. We demonstrate that this interaction involves a highly conserved acidic motif at the C terminus of SMRT/NCoR. These findings suggest that the conserved function of the SPOC domain is to mediate interaction with SMRT/NCoR corepressors, and that Spen proteins play an essential role in the repression complex.

Nuclear receptors are transcription factors that activate gene expression in response to ligands. The C-terminal helix (helix 12) of the ligand-binding domain plays a critical role in the activation mechanism. When bound to activating ligands, helix 12 adopts a conformation that promotes the binding of co-activator proteins. Helix 12 also adopts this 'active' position in several ligand-free structures, raising questions as to the exact role of helix 12. We proposed that the dynamic properties of helix 12 may be critical for the activation mechanism and, to test this, have used fluorescence anisotropy techniques to directly monitor the mobility of helix 12 in PPARgamma. Our results suggest that helix 12 is significantly more mobile than the main body of the protein. Upon ligand binding, helix 12 shows reduced mobility, accounting for its role as a molecular switch. We also show that natural mutations in human PPARgamma, associated with severe insulin resistance and diabetes mellitus, exhibit perturbations in the dynamic behavior of helix 12. Our findings provide the first direct observations of the mobility of helix 12 and suggest that the dynamic properties of this helix are key to the regulation of transcriptional activity.

The expression and purification of large amounts of recombinant protein complexes is an essential requirement for structural biology studies. For over two decades, prokaryotic expression systems such as E. coli have dominated the scientific literature over costly and less efficient eukaryotic cell lines. Despite the clear advantage in terms of yields and costs of expressing recombinant proteins in bacteria, the absence of specific co-factors, chaperones and post-translational modifications may cause loss of function, mis-folding and can disrupt protein-protein interactions of certain eukaryotic multi-subunit complexes, surface receptors and secreted proteins. The use of mammalian cell expression systems can address these drawbacks since they provide a eukaryotic expression environment. However, low protein yields and high costs of such methods have until recently limited their use for structural biology. Here we describe a simple and accessible method for expressing and purifying milligram quantities of protein by performing transient transfections of suspension grown HEK (Human Embryonic Kidney) 293 F cells.

BCL6 is a transcriptional repressor that is over-expressed due to chromosomal translocations, or other abnormalities, in ~40% of diffuse large B-cell lymphoma. BCL6 interacts with co-repressor, SMRT, and this is essential for its role in lymphomas. Peptide or small molecule inhibitors, which prevent the association of SMRT with BCL6, inhibit transcriptional repression and cause apoptosis of lymphoma cells in vitro and in vivo. In order to discover compounds, which have the potential to be developed into BCL6 inhibitors, we screened a natural product library. The ansamycin antibiotic, rifamycin SV, inhibited BCL6 transcriptional repression and NMR spectroscopy confirmed a direct interaction between rifamycin SV and BCL6. To further determine the characteristics of compounds binding to BCL6-POZ we analyzed four other members of this family and showed that rifabutin, bound most strongly. An X-ray crystal structure of the rifabutin-BCL6 complex revealed that rifabutin occupies a partly non-polar pocket making interactions with tyrosine58, asparagine21 and arginine24 of the BCL6-POZ domain. Importantly these residues are also important for the interaction of BLC6 with SMRT. This work demonstrates a unique approach to developing a structure activity relationship for a compound that will form the basis of a therapeutically useful BCL6 inhibitor.

Objective: Decipher mechanisms that underlie cervical remodeling (CR) during pregnancy. Design and population: Examine the proteomic profile of mice cervices in early and late pregnancy and verify data generated using Western Blot and real time PCR analysis. Main outcome measures: Identify signature proteins. Results: In total, seventy three (73) signature proteins were identified and were found up regulated in pregnancy, namely proteins associated with: a) collagen presence and organization (4), notably biglycan and lumican, b) immune scavenging functions and c) cytoskeletal activity. These proteomic trends were verified through Western blot and real time PCR analysis. Conclusion: These signature proteins and their expression profile collectively suggest a complex biological process involving extracellular matrix (ECM)- and immune`-associated factors. Based on these data, we propose that biglycan may provide the link between natural birth and inflammation-mediated pre-term birth.

Co-repressor proteins mediate transcriptional repression by nuclear receptors in the absence of ligand. The identification of a co-repressor-receptor interaction motif, and the finding that compressors and co-activators compete for the same site on the receptor, suggests a simple mechanism for the switch from repression to activation upon ligand binding. Defects in this mechanism result in dominant-negative receptors that repress transcription. Such receptors have been implicated in several clinically important diseases, including thyroid hormone resistance and diabetes mellitus.

The syndrome of resistance to thyroid hormone is associated with diverse mutations in the ligand-binding domain of the thyroid hormone β receptor, localizing to three clusters around the hormone binding cavity. Here, we report three novel resistance to thyroid hormone mutations (S314C, S314F, and S314Y), due to different nucleotide substitutions in the same codon, occurring in six separate families. Functional characterization of these mutant receptors showed marked differences in their properties. S314F and S314Y receptor mutants exhibited significant transcriptional impairment in keeping with negligible ligand binding and were potent dominant negative inhibitors of wild-type receptor action. In contrast, the S314C mutant bound ligand with reduced affinity, such that its functional impairment and dominant negative activity manifest at low concentrations of thyroid hormone, but are more reversible at higher T3 concentrations. The degree of functional impairment of mutant receptors in vitro may correlate with the magnitude of thyroid dysfunction in vivo. Modelling these mutations using the crystal structure of thyroid hormone receptor β shows why ligand binding is perturbed and why the phenylalanine/tyrosine mutations are more deleterious than cysteine.

Members of the family of fungal Zn2Cys6 DNA-binding proteins discriminate between their DNA targets using neither their DNA-binding module, nor their dimerization domain, but primarily through the relatively flexible linker between them.

The biological activities of the retinoids are mediated by two nuclear hormone receptors: the retinoic acid receptor (RAR) and the retinoid-X receptor (RXR). RXR (and its insect homologue ultraspiracle) is a common heterodimeric partner for many other nuclear receptors, including the insect ecdysone receptor. As part of a continuing analysis of nuclear receptor function, we noticed that, whereas RXR can be readily expressed in Escherichia coli to produce soluble protein, many of its heterodimeric partners cannot. For example, overexpression of RAR results mostly in inclusion bodies with the residual soluble component unable to interact with RXR or ligand efficiently. Similar results are seen with other RXR/ultraspiracle partners. To overcome these problems, we designed a novel double cistronic vector to coexpress RXR and its partner ligand-binding domains in the same bacterial cell. This resulted in a dramatic increase in production of soluble and apparently stable heterodimer. Hormone-binding studies using the purified RXR–RAR heterodimer reveal increased ligand-binding capacity of both components of 5- to 10-fold, resulting in virtually complete functionality. Based on these studies we find that bacterially expressed receptors can exist in one of three distinct states: insoluble, soluble but unable to bind ligand, or soluble with full ligand-binding capacity. These results suggest that coexpression may represent a general strategy for biophysical and structural analysis of receptor complexes.

Nuclear receptors are transcription factors that regulate gene expression through the ligand-controlled recruitment of a diverse group of proteins known as coregulators. Most nuclear receptor coregulators function in large multi-protein complexes that modify chromatin and thereby regulate the transcription of target genes. Structural and functional studies are beginning to reveal how these complexes are assembled bringing together multiple functionalities that mediate: recruitment to specific genomic loci through interaction with transcription factors; recruitment of enzymatic activities that either modify or remodel chromatin and targeting the complexes to their chromatin substrate. These activities are regulated by post-translational modifications, alternative splicing and small signalling molecules. This review focuses on our current understanding of coregulator complexes and aims to highlight the common principles that are beginning to emerge.

Understanding how proteins recognize DNA in a sequence-specific manner is central to our understanding of the regulation of transcription and other cellular processes. In this article we review the principles of DNA recognition that have emerged from the large number of high-resolution crystal structures determined over the last 10 years. The DNA-binding domains of transcription factors exhibit surprisingly diverse protein architectures, yet all achieve a precise complementarity of shape facilitating specific chemical recognition of their particular DNA targets. Although general rules for recognition can be derived, the complex nature of the recognition mechanism precludes a simple recognition code. In particular, it has become evident that the structure and flexibility of DNA and contacts mediated by water molecules contribute to the recognition process. Nevertheless, based on known structures it has proven possible to design proteins with novel recognition specificities. Despite this considerable practical success, the thermodynamic and kinetic properties of protein/DNA recognition remain poorly understood.

The term zinc finger was first used to describe a 30-residue, repeated sequence motif found in an unusually abundant Xenopus transcription factor. It was proposed that each motif is folded around a central zinc ion to form an independent minidomain and that adjacent zinc fingers are combined as modules to make up a DNA-binding domain with the modules "gripping" the DNA (hence the term finger). We now know that these proposals were correct and that these DNA-binding motifs are found in many eukaryotic DNA-binding proteins. More recently, crystal structures of three different complexes between zinc finger domains and their target DNA binding sites have revealed a remarkably simple mode of interaction with DNA. The simplicity of the zinc finger structure, and of its interaction with DNA, is a very striking feature of this protein domain. After the discovery of the zinc finger motif, patterns of potential zinc ligands have been found in several other proteins, some of which also bind to DNA. Structural studies of these domains have revealed how zinc can stabilize quite diverse protein architectures. In total, 10 such small zinc-binding domains have been studied structurally. These form a diverse collection, but each in turn has been termed a zinc finger motif-although clearly what they have in common is only their zinc-binding property, which stabilizes an apparently autonomously folded unit.

Background: Structural studies of protein–DNA complexes have tended to give the impression that DNA recognition requires a unique molecular interface. However, many proteins recognize DNA targets that differ from what is thought to be their ideal target sequence. The steroid hormone receptors illustrate this problem in recognition rather well, since consensus DNA targets are rare. Results Here we describe the structure, at 2.6 a resolution, of a complex between a dimer of the DNA-binding domain from the human oestrogen receptor (ERDBD) and a non-consensus DNA target site in which there is a single base substitution in one half of the palindromic binding site. This substitution results in a 10-fold increase in the dissociation constant of the ERDBD–DNA complex. Comparison of this structure with a structure containing a consensus DNA-binding site determined previously, shows that recognition of the non-consensus sequence is achieved by the rearrangement of a lysine side chain so as to make an alternative base contact. Conclusion This study suggests that proteins adapt to recognize different DNA sequences by rearranging side chains at the protein–DNA interface so as to form alternative patterns of intermolecular contacts.