FFAT motif phosphorylation controls formation and lipid transfer function of inter-organelle contacts.

Organelles are physically connected in membrane contact sites. The endoplasmic reticulum possesses three major receptors, VAP-A, VAP-B, and MOSPD2, which interact with proteins at the surface of other organelles to build contacts. VAP-A, VAP-B, and MOSPD2 contain an MSP domain, which binds a motif named FFAT (two phenylalanines in an acidic tract). In this study, we identified a non-conventional FFAT motif where a conserved acidic residue is replaced by a serine/threonine. We show that phosphorylation of this serine/threonine is critical for non-conventional FFAT motifs (named Phospho-FFAT) to be recognized by the MSP domain. Moreover, structural analyses of the MSP domain alone or in complex with conventional and Phospho-FFAT peptides revealed new mechanisms of interaction. Based on these new insights, we produced a novel prediction algorithm, which expands the repertoire of candidate proteins with a Phospho-FFAT that are able to create membrane contact sites. Using a prototypical tethering complex made by STARD3 and VAP, we showed that phosphorylation is instrumental for the formation of ER-endosome contacts, and their sterol transfer function. This study reveals that phosphorylation acts as a general switch for inter-organelle contacts.

A Direct Assay for Measuring the Activity and Inhibition of Coactivator-Associated Arginine Methyltransferase 1.

Coactivator-associated arginine methyltransferase 1 (CARM1) is a member of the family of protein arginine methyltransferases. CARM1 catalyzes methyl group transfer from the cofactor S-adenosyl-l-methionine (AdoMet) to both histone and nonhistone protein substrates. CARM1 is involved in a range of cellular processes, mainly involving RNA transcription and gene regulation. As the aberrant expression of CARM1 has been linked to tumorigenesis, the enzyme is a potential therapeutic target, leading to the development of inhibitors and tool compounds engaging with CARM1. To evaluate the effects of these compounds on the activity of CARM1, sensitive and specific analytical methods are needed. While different methods are currently available to assess the activity of methyltransferases, these assays mainly focus on either the measurement of the cofactor product S-adenosyl-l-homocysteine (AdoHcy) or employ radioactive or expensive reagents, each with their own advantages and limitations. To complement the tools currently available for the analysis of CARM1 activity, we here describe the development of a convenient assay employing peptide substrates derived from poly(A)-binding protein 1 (PABP1). This operationally straightforward liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approach allows for the direct detection of substrate methylation with minimal workup. The method was validated, and its value in characterizing CARM1 activity and inhibition was demonstrated through a comparative analysis involving a set of established small molecules and peptide-based CARM1 inhibitors.

Structural Studies Provide New Insights into the Role of Lysine Acetylation on Substrate Recognition by CARM1 and Inform the Design of Potent Peptidomimetic Inhibitors.

The dynamic interplay of post-translational modifications (PTMs) in chromatin provides a communication system for the regulation of gene expression. An increasing number of studies have highlighted the role that such crosstalk between PTMs plays in chromatin recognition. In this study, (bio)chemical and structural approaches were applied to specifically probe the impact of acetylation of Lys18 in the histone H3 tail peptide on peptide recognition by the protein methyltransferase coactivator-associated arginine methyltransferase 1 (CARM1). Peptidomimetics that recapitulate the transition state of protein arginine N-methyltransferases, were designed based on the H3 peptide wherein the target Arg17 was flanked by either a free or an acetylated lysine. Structural studies with these peptidomimetics and the catalytic domain of CARM1 provide new insights into the binding of the H3 peptide within the enzyme active site. While the co-crystal structures reveal that lysine acetylation results in minor conformational differences for both CARM1 and the H3 peptide, acetylation of Lys18 does lead to additional interactions (Van der Waals and hydrogen bonding) and likely reduces the cost of desolvation upon binding, resulting in increased affinity. Informed by these findings a series of smaller peptidomimetics were also prepared and found to maintain potent and selective CARM1 inhibition. These findings provide new insights both into the mechanism of crosstalk between arginine methylation and lysine acetylation as well as towards the development of peptidomimetic CARM1 inhibitors.

Aminobenzosuberone derivatives as PfA-M1 inhibitors: Molecular recognition and antiplasmodial evaluation.

Aminobenzosuberone-based PfA-M1 inhibitors were explored as novel antimalarial agents against two different Plasmodium falciparum strains. The 4-phenyl derivative 7c exhibited the most encouraging growth inhibitory activity with IC50 values of 6.5-11.2 µM. X-ray crystal structures and early assessment of DMPK/ADME-Tox parameters allowed us to initiate structure-based drug design approach and understand the liabilities (such as potential metabolic and aqueous solubility issues) as well as identify the opportunities for improvement of this aminobenzosuberone series. It also suggested that compound 7c should be regarded as an attractive chemical tool to investigate the different biological roles of this multifunctional PfA-M1 protein.

Transition state mimics are valuable mechanistic probes for structural studies with the arginine methyltransferase CARM1.

Coactivator associated arginine methyltransferase 1 (CARM1) is a member of the protein arginine methyltransferase (PRMT) family and methylates a range of proteins in eukaryotic cells. Overexpression of CARM1 is implicated in a number of cancers, and it is therefore seen as a potential therapeutic target. Peptide sequences derived from the well-defined CARM1 substrate poly(A)-binding protein 1 (PABP1) were covalently linked to an adenosine moiety as in the AdoMet cofactor to generate transition state mimics. These constructs were found to be potent CARM1 inhibitors and also formed stable complexes with the enzyme. High-resolution crystal structures of CARM1 in complex with these compounds confirm a mode of binding that is indeed reflective of the transition state at the CARM1 active site. Given the transient nature of PRMT-substrate complexes, such transition state mimics represent valuable chemical tools for structural studies aimed at deciphering the regulation of arginine methylation mediated by the family of arginine methyltransferases.

SECIS-binding protein 2 interacts with the SMN complex and the methylosome for selenoprotein mRNP assembly and translation.

Selenoprotein synthesis requires the co-translational recoding of a UGASec codon. This process involves an RNA structural element, called Selenocysteine Insertion Sequence (SECIS) and the SECIS binding protein 2 (SBP2). Several selenoprotein mRNAs undergo unusual cap hypermethylation by the trimethylguanosine synthase 1 (Tgs1), which is recruited by the ubiquitous Survival of MotoNeurons (SMN) protein. SMN, the protein involved in spinal muscular atrophy, is part of a chaperone complex that collaborates with the methylosome for RNP assembly. Here, we analyze the role of individual SMN and methylosome components in selenoprotein mRNP assembly and translation. We show that SBP2 interacts directly with four proteins of the SMN complex and the methylosome core proteins. Nevertheless, SBP2 is not a methylation substrate of the methylosome. We found that both SMN and methylosome complexes are required for efficient translation of the selenoprotein GPx1 in vivo. We establish that the steady-state level of several selenoprotein mRNAs, major regulators of oxidative stress damage in neurons, is specifically reduced in the spinal cord of SMN-deficient mice and that cap hypermethylation of GPx1 mRNA is affected. Altogether we identified a new function of the SMN complex and the methylosome in selenoprotein mRNP assembly and expression.

Insight into the remarkable affinity and selectivity of the aminobenzosuberone scaffold for the M1 aminopeptidases family based on structure analysis.

Aminopeptidases are ubiquitous hydrolases that cleave the N-terminal residues of proteins and oligopeptides. They are broadly distributed throughout all kingdoms of life and have been implicated in a wide variety of physiological processes, including viral infection, parasite metabolism, protein processing, regulation of peptide hormones, and cancer cell proliferation. Members of the M1 family, also termed gluzincins, are defined by two highly conserved motifs in the catalytic domain: a zinc-binding motif, HEXXH-(X18)-E; and an exopeptidase motif, GXMEN. We report the high-resolution X-ray structures of E. coli aminopeptidase N (PepN) in complex with three aminobenzosuberone scaffolds that display various Ki values (50, 0.33, and 0.034 µM) and provide a compelling view of the outstanding selectivity of these chemical entities for the M1 aminopeptidases. This series of inhibitors interacts as transition state mimics with highly conserved residues of the catalytic machinery and substrate recognition sites. Structural comparisons and model-building studies allowed a deep interpretation of the SAR observed for bacterial, as well as mammalian enzymes. Proteins 2017; 85:1413-1421. © 2017 Wiley Periodicals, Inc.

Functional insights from high resolution structures of mouse protein arginine methyltransferase 6.

PRMT6 is a protein arginine methyltransferase involved in transcriptional regulation, human immunodeficiency virus pathogenesis, DNA base excision repair, and cell cycle progression. Like other PRMTs, PRMT6 is overexpressed in several cancer types and is therefore considered as a potential anti-cancer drug target. In the present study, we described six crystal structures of PRMT6 from Mus musculus, solved and refined at 1.34 Å for the highest resolution structure. The crystal structures revealed that the folding of the helix αX is required to stabilize a productive active site before methylation of the bound peptide can occur. In the absence of cofactor, metal cations can be found in the catalytic pocket at the expected position of the guanidinium moiety of the target arginine substrate. Using mass spectrometry under native conditions, we show that PRMT6 dimer binds two cofactor and a single H4 peptide molecules. Finally, we characterized a new site of in vitro automethylation of mouse PRMT6 at position 7.

Structural basis for the inhibition of histone deacetylase 8 (HDAC8), a key epigenetic player in the blood fluke Schistosoma mansoni.

The treatment of schistosomiasis, a disease caused by blood flukes parasites of the Schistosoma genus, depends on the intensive use of a single drug, praziquantel, which increases the likelihood of the development of drug-resistant parasite strains and renders the search for new drugs a strategic priority. Currently, inhibitors of human epigenetic enzymes are actively investigated as novel anti-cancer drugs and have the potential to be used as new anti-parasitic agents. Here, we report that Schistosoma mansoni histone deacetylase 8 (smHDAC8), the most expressed class I HDAC isotype in this organism, is a functional acetyl-L-lysine deacetylase that plays an important role in parasite infectivity. The crystal structure of smHDAC8 shows that this enzyme adopts a canonical α/ß HDAC fold, with specific solvent exposed loops corresponding to insertions in the schistosome HDAC8 sequence. Importantly, structures of smHDAC8 in complex with generic HDAC inhibitors revealed specific structural changes in the smHDAC8 active site that cannot be accommodated by human HDACs. Using a structure-based approach, we identified several small-molecule inhibitors that build on these specificities. These molecules exhibit an inhibitory effect on smHDAC8 but show reduced affinity for human HDACs. Crucially, we show that a newly identified smHDAC8 inhibitor has the capacity to induce apoptosis and mortality in schistosomes. Taken together, our biological and structural findings define the framework for the rational design of small-molecule inhibitors specifically interfering with schistosome epigenetic mechanisms, and further support an anti-parasitic epigenome targeting strategy to treat neglected diseases caused by eukaryotic pathogens.

Structural insight into arginine methylation by the mouse protein arginine methyltransferase 7: a zinc finger freezes the mimic of the dimeric state into a single active site.

Protein arginine methyltransferase 7 (PRMT7) is a type III arginine methyltransferase which has been implicated in several biological processes such as transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation and metastasis. PRMT7 is a unique but less characterized member of the family of PRMTs. The crystal structure of full-length PRMT7 from Mus musculus refined at 1.7 Šresolution is described. The PRMT7 structure is composed of two catalytic modules in tandem forming a pseudo-dimer and contains only one AdoHcy molecule bound to the N-terminal module. The high-resolution crystal structure presented here revealed several structural features showing that the second active site is frozen in an inactive state by a conserved zinc finger located at the junction between the two PRMT modules and by the collapse of two degenerated AdoMet-binding loops.

The structure of an Iws1/Spt6 complex reveals an interaction domain conserved in TFIIS, Elongin A and Med26.

Binding of elongation factor Spt6 to Iws1 provides an effective means for coupling eukaryotic mRNA synthesis, chromatin remodelling and mRNA export. We show that an N-terminal region of Spt6 (Spt6N) is responsible for interaction with Iws1. The crystallographic structures of Encephalitozoon cuniculi Iws1 and the Iws1/Spt6N complex reveal two conserved binding subdomains in Iws1. The first subdomain (one HEAT repeat; HEAT subdomain) is a putative phosphoprotein-binding site most likely involved in an Spt6-independent function of Iws1. The second subdomain (two ARM repeats; ARM subdomain) specifically recognizes a bipartite N-terminal region of Spt6. Mutations that alter this region of Spt6 cause severe phenotypes in vivo. Importantly, the ARM subdomain of Iws1 is conserved in several transcription factors, including TFIIS, Elongin A and Med26. We show that the homologous region in yeast TFIIS enables this factor to interact with SAGA and the Mediator subunits Spt8 and Med13, suggesting the molecular basis for TFIIS recruitment at promoters. Taken together, our results provide new structural information about the Iws1/Spt6 complex and reveal a novel interaction domain used for the formation of transcription networks.

Structural basis for group A trichothiodystrophy.

Patients with the rare neurodevelopmental repair syndrome known as group A trichothiodystrophy (TTD-A) carry mutations in the gene encoding the p8 subunit of the transcription and DNA repair factor TFIIH. Here we describe the crystal structure of a minimal complex between Tfb5, the yeast ortholog of p8, and the C-terminal domain of Tfb2, the yeast p52 subunit of TFIIH. The structure revealed that these two polypeptides adopt the same fold, forming a compact pseudosymmetric heterodimer via a beta-strand addition and coiled coils interactions between terminal alpha-helices. Furthermore, Tfb5 protects a hydrophobic surface in Tfb2 from solvent, providing a rationale for the influence of p8 in the stabilization of p52 and explaining why mutations that weaken p8-p52 interactions lead to a reduced intracellular TFIIH concentration and a defect in nucleotide-excision repair, a common feature of TTD cells.

Turning Nonselective Inhibitors of Type I Protein Arginine Methyltransferases into Potent and Selective Inhibitors of Protein Arginine Methyltransferase 4 through a Deconstruction-Reconstruction and Fragment-Growing Approach.

Protein arginine methyltransferases (PRMTs) are important therapeutic targets, playing a crucial role in the regulation of many cellular processes and being linked to many diseases. Yet, there is still much to be understood regarding their functions and the biological pathways in which they are involved, as well as on the structural requirements that could drive the development of selective modulators of PRMT activity. Here we report a deconstruction-reconstruction approach that, starting from a series of type I PRMT inhibitors previously identified by us, allowed for the identification of potent and selective inhibitors of PRMT4, which regardless of the low cell permeability show an evident reduction of arginine methylation levels in MCF7 cells and a marked reduction of proliferation. We also report crystal structures with various PRMTs supporting the observed specificity and selectivity.

Structural insights into transcription complexes.

Control of transcription allows the regulation of cell activity in response to external stimuli and research in the field has greatly benefited from efforts in structural biology. In this review, based on specific examples from the European SPINE2-COMPLEXES initiative, we illustrate the impact of structural proteomics on our understanding of the molecular basis of gene expression. While most atomic structures were obtained by X-ray crystallography, the impact of solution NMR and cryo-electron microscopy is far from being negligible. Here, we summarize some highlights and illustrate the importance of specific technologies on the structural biology of protein-protein or protein/DNA transcription complexes: structure/function analysis of components the eukaryotic basal and activated transcription machinery with focus on the TFIID and TFIIH multi-subunit complexes as well as transcription regulators such as members of the nuclear hormone receptor families. We also discuss molecular aspects of promoter recognition and epigenetic control of gene expression.

Noncanonical tandem SH2 enables interaction of elongation factor Spt6 with RNA polymerase II.

Src homology 2 (SH2) domains are mostly found in multicellular organisms where they recognize phosphotyrosine-containing signaling proteins. Spt6, a conserved transcription factor and putative histone chaperone, contains a C-terminal SH2 domain conserved from yeast to human. In mammals, this SH2 domain recognizes phosphoserines rather than phosphotyrosines and is essential for the recruitment of Spt6 by elongating RNA polymerase II (RNAPII), enabling Spt6 to participate in the coupling of transcription elongation, chromatin modulation, and mRNA export. We have determined the structure of the entire Spt6 C-terminal region from Antonospora locustae, revealing the presence of two highly conserved tandem SH2 domains rather than a single SH2 domain. Although the first SH2 domain has a canonical organization, the second SH2 domain is highly noncanonical and appears to be unique in the SH2 family. However, both SH2 domains have phosphate-binding determinants. Our biochemical and genetic data demonstrate that the complete tandem, but not the individual SH2 domains, are necessary and sufficient for the interaction of Spt6 with RNAPII and are important for Spt6 function in vivo. Furthermore, our data suggest that binding of RNAPII to the Spt6 tandem SH2 is more extensive than the mere recognition of a doubly phosphorylated C-terminal domain peptide by the tandem SH2. Taken together, our results show that Spt6 interaction with RNAPII via a novel arrangement of canonical and noncanonical SH2 domains is crucial for Spt6 function in vivo.

Functional insights from structures of coactivator-associated arginine methyltransferase 1 domains.

Coactivator-associated arginine methyltransferase 1 (CARM1), a protein arginine methyltransferase recruited by several transcription factors, methylates a large variety of proteins and plays a critical role in gene expression. We report, in this paper, four crystal structures of isolated modules of CARM1. The 1.7 A crystal structure of the N-terminal domain of CARM1 reveals an unexpected PH domain, a scaffold frequently found to regulate protein-protein interactions in a large variety of biological processes. Three crystal structures of the CARM1 catalytic module, two free and one cofactor-bound forms (refined at 2.55 A, 2.4 A and 2.2 A, respectively) reveal large structural modifications including disorder to order transition, helix to strand transition and active site modifications. The N-terminal and the C-terminal end of CARM1 catalytic module contain molecular switches that may inspire how CARM1 regulates its biological activities by protein-protein interactions.

Acyl derivatives of p-aminosulfonamides and dapsone as new inhibitors of the arginine methyltransferase hPRMT1.

Arginine methylation is an epigenetic modification that receives increasing interest as it plays an important role in several diseases. This is especially true for hormone-dependent cancer, seeing that histone methylation by arginine methyltransferase I (PRMT1) is involved in the activation of sexual hormone receptors. Therefore, PRMT inhibitors are potential drugs and interesting tools for cell biology. A dapsone derivative called allantodapsone previously identified by our group served as a lead structure for inhibitor synthesis. Acylated derivatives of p-aminobenzenesulfonamides and the antilepra drug dapsone were identified as new inhibitors of PRMT1 by in vitro testing. The bis-chloroacetyl amide of dapsone selectively inhibited human PRMT1 in the low micromolar region and was selective for PRMT1 as compared to the arginine methyltransferase CARM1 and the lysine methyltransferase Set7/9. It showed anticancer activity on MCF7a and LNCaP cells and blocked androgen dependent transcription specifically in a reporter gene system. Likewise, a transcriptional block was also demonstrated in LNCaP cells using quantitative RT-PCR on the mRNA of androgen dependent genes.

Structure, Activity and Function of the PRMT2 Protein Arginine Methyltransferase.

PRMT2 belongs to the protein arginine methyltransferase (PRMT) family, which catalyzes the arginine methylation of target proteins. As a type I enzyme, PRMT2 produces asymmetric dimethyl arginine and has been shown to have weak methyltransferase activity on histone substrates in vitro, suggesting that its authentic substrates have not yet been found. PRMT2 contains the canonical PRMT methylation core and a unique Src homology 3 domain. Studies have demonstrated its clear implication in many different cellular processes. PRMT2 acts as a coactivator of several nuclear hormone receptors and is known to interact with a multitude of splicing-related proteins. Furthermore, PRMT2 is aberrantly expressed in several cancer types, including breast cancer and glioblastoma. These reports highlight the crucial role played by PRMT2 and the need for a better characterization of its activity and cellular functions.

Structure determination of the minimal complex between Tfb5 and Tfb2, two subunits of the yeast transcription/DNA-repair factor TFIIH: a retrospective study.

Tfb5 interacts with the Tfb2 subunit of the general transcription factor TFIIH to ensure efficient nucleotide-excision repair in eukaryotes. The crystal structure of the complex between Tfb5 and the C-terminal region of Tfb2 (Tfb2C) from Saccharomyces cerevisiae has recently been reported. Here, the structure-determination process is described as a case study. Although crystals were obtained readily, it was not possible to determine experimental phases from a first crystal form (Tfb2(412-513)-Tfb5(2-72)) that diffracted to 2.6 A resolution. Shortening of the Tfb2C from its N-terminus was decisive and modified the crystal packing, leading to a second crystal form (Tfb2(435-513)-Tfb5(2-72)). These crystals diffracted to 1.7 A resolution with excellent mosaicity and allowed structure determination by conventional approaches using heavy atoms. The refined structure from the second crystal form was used to solve the structure of the first crystal form by molecular replacement. Comparison of the two structures revealed that the N-terminal region of Tfb2C and (to a lesser extent) the C-terminal region of Tfb5 contributed to the crystal packing. A detailed analysis illustrates how variation in domain boundaries influences crystal packing and quality.

Crystallization and preliminary crystallographic analysis of eukaryotic transcription and mRNA export factor Iws1 from Encephalitozoon cuniculi.

Transcription elongation by eukaryotic RNA polymerase II requires the coupling of mRNA synthesis and mRNA processing and export. The essential protein Iws1 is at the interface of these processes through its interaction with histone chaperone and elongation factor Spt6 as well as with complexes involved in mRNA processing and export. Upon crystallization of the evolutionarily conserved domain of Iws1 from Encephalitozoon cuniculi, four different crystal forms were obtained. Three of the crystal forms belonged to space group P2(1) and one belonged to space group P222(1). Preliminary X-ray crystallographic analysis of one of the crystal forms allowed the collection of data to 2.5 A resolution.