The Sir4 H-BRCT domain interacts with phospho-proteins to sequester and repress yeast heterochromatin.

In Saccharomyces cerevisiae, the silent information regulator (SIR) proteins Sir2/3/4 form a complex that suppresses transcription in subtelomeric regions and at the homothallic mating-type (HM) loci. Here, we identify a non-canonical BRCA1 C-terminal domain (H-BRCT) in Sir4, which is responsible for tethering telomeres to the nuclear periphery. We show that Sir4 H-BRCT and the closely related Dbf4 H-BRCT serve as selective phospho-epitope recognition domains that bind to a variety of phosphorylated target peptides. We present detailed structural information about the binding mode of established Sir4 interactors (Esc1, Ty5, Ubp10) and identify several novel interactors of Sir4 H-BRCT, including the E3 ubiquitin ligase Tom1. Based on these findings, we propose a phospho-peptide consensus motif for interaction with Sir4 H-BRCT and Dbf4 H-BRCT. Ablation of the Sir4 H-BRCT phospho-peptide interaction disrupts SIR-mediated repression and perinuclear localization. In conclusion, the Sir4 H-BRCT domain serves as a hub for recruitment of phosphorylated target proteins to heterochromatin to properly regulate silencing and nuclear order.

Influenza virus uses transportin 1 for vRNP debundling during cell entry.

Influenza A virus is a pathogen of great medical impact. To develop novel antiviral strategies, it is essential to understand the molecular aspects of virus-host cell interactions in detail. During entry, the viral ribonucleoproteins (vRNPs) that carry the RNA genome must be released from the incoming particle before they can enter the nucleus for replication. The uncoating process is facilitated by histone deacetylase 6 (ref.1). However, the precise mechanism of shell opening and vRNP debundling is unknown. Here, we show that transportin 1, a member of the importin-ß family proteins, binds to a PY-NLS2 sequence motif close to the amino terminus of matrix protein (M1) exposed during acid priming of the viral core. It promotes the removal of M1 and induces disassembly of vRNP bundles. Next, the vRNPs interact with importin-α/ß and enter the nucleus. Thus, influenza A virus uses dual importin-ßs for distinct steps in host cell entry.

Evolutionary plasticity of the NHL domain underlies distinct solutions to RNA recognition.

RNA-binding proteins regulate all aspects of RNA metabolism. Their association with RNA is mediated by RNA-binding domains, of which many remain uncharacterized. A recently reported example is the NHL domain, found in prominent regulators of cellular plasticity like the C. elegans LIN-41. Here we employ an integrative approach to dissect the RNA specificity of LIN-41. Using computational analysis, structural biology, and in vivo studies in worms and human cells, we find that a positively charged pocket, specific to the NHL domain of LIN-41 and its homologs (collectively LIN41), recognizes a stem-loop RNA element, whose shape determines the binding specificity. Surprisingly, the mechanism of RNA recognition by LIN41 is drastically different from that of its more distant relative, the fly Brat. Our phylogenetic analysis suggests that this reflects a rapid evolution of the domain, presenting an interesting example of a conserved protein fold that acquired completely different solutions to RNA recognition.

Structural Basis of Mec1-Ddc2-RPA Assembly and Activation on Single-Stranded DNA at Sites of Damage.

Mec1-Ddc2 (ATR-ATRIP) is a key DNA-damage-sensing kinase that is recruited through the single-stranded (ss) DNA-binding replication protein A (RPA) to initiate the DNA damage checkpoint response. Activation of ATR-ATRIP in the absence of DNA damage is lethal. Therefore, it is important that damage-specific recruitment precedes kinase activation, which is achieved at least in part by Mec1-Ddc2 homodimerization. Here, we report a structural, biochemical, and functional characterization of the yeast Mec1-Ddc2-RPA assembly. High-resolution co-crystal structures of Ddc2-Rfa1 and Ddc2-Rfa1-t11 (K45E mutant) N termini and of the Ddc2 coiled-coil domain (CCD) provide insight into Mec1-Ddc2 homodimerization and damage-site targeting. Based on our structural and functional findings, we present a Mec1-Ddc2-RPA-ssDNA composite structural model. By way of validation, we show that RPA-dependent recruitment of Mec1-Ddc2 is crucial for maintaining its homodimeric state at ssDNA and that Ddc2's recruitment domain and CCD are important for Mec1-dependent survival of UV-light-induced DNA damage.

Structural insights into HDAC6 tubulin deacetylation and its selective inhibition.

We report crystal structures of zebrafish histone deacetylase 6 (HDAC6) catalytic domains in tandem or as single domains in complex with the (R) and (S) enantiomers of trichostatin A (TSA) or with the HDAC6-specific inhibitor nexturastat A. The tandem domains formed, together with the inter-domain linker, an ellipsoid-shaped complex with pseudo-twofold symmetry. We identified important active site differences between both catalytic domains and revealed the binding mode of HDAC6 selective inhibitors. HDAC inhibition assays with (R)- and (S)-TSA showed that (R)-TSA was a broad-range inhibitor, whereas (S)-TSA had moderate selectivity for HDAC6. We identified a uniquely positioned α-helix and a flexible tryptophan residue in the loop joining α-helices H20 to H21 as critical for deacetylation of the physiologic substrate tubulin. Using single-molecule measurements and biochemical assays we demonstrated that HDAC6 catalytic domain 2 deacetylated α-tubulin lysine 40 in the lumen of microtubules, but that its preferred substrate was unpolymerized tubulin.

The TRIM-NHL protein LIN-41 controls the onset of developmental plasticity in Caenorhabditis elegans.

The mechanisms controlling cell fate determination and reprogramming are fundamental for development. A profound reprogramming, allowing the production of pluripotent cells in early embryos, takes place during the oocyte-to-embryo transition. To understand how the oocyte reprogramming potential is controlled, we sought Caenorhabditis elegans mutants in which embryonic transcription is initiated precociously in germ cells. This screen identified LIN-41, a TRIM-NHL protein and a component of the somatic heterochronic pathway, as a temporal regulator of pluripotency in the germline. We found that LIN-41 is expressed in the cytoplasm of developing oocytes, which, in lin-41 mutants, acquire pluripotent characteristics of embryonic cells and form teratomas. To understand LIN-41 function in the germline, we conducted structure-function studies. In contrast to other TRIM-NHL proteins, we found that LIN-41 is unlikely to function as an E3 ubiquitin ligase. Similar to other TRIM-NHL proteins, the somatic function of LIN-41 is thought to involve mRNA regulation. Surprisingly, we found that mutations predicted to disrupt the association of LIN-41 with mRNA, which otherwise compromise LIN-41 function in the heterochronic pathway in the soma, have only minor effects in the germline. Similarly, LIN-41-mediated repression of a key somatic mRNA target is dispensable for the germline function. Thus, LIN-41 appears to function in the germline and the soma via different molecular mechanisms. These studies provide the first insight into the mechanism inhibiting the onset of embryonic differentiation in developing oocytes, which is required to ensure a successful transition between generations.

Memo is a copper-dependent redox protein with an essential role in migration and metastasis.

Memo is an evolutionarily conserved protein with a critical role in cell motility. We found that Memo was required for migration and invasion of breast cancer cells in vitro and spontaneous lung metastasis from breast cancer cell xenografts in vivo. Biochemical assays revealed that Memo is a copper-dependent redox enzyme that promoted a more oxidized intracellular milieu and stimulated the production of reactive oxygen species (ROS) in cellular structures involved in migration. Memo was also required for the sustained production of the ROS O2- by NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase 1 (NOX1) in breast cancer cells. Memo abundance was increased in >40% of the primary breast tumors tested, was correlated with clinical parameters of aggressive disease, and was an independent prognostic factor of early distant metastasis.

Dimerization of Sir3 via its C-terminal winged helix domain is essential for yeast heterochromatin formation.

Gene silencing in budding yeast relies on the binding of the Silent Information Regulator (Sir) complex to chromatin, which is mediated by extensive interactions between the Sir proteins and nucleosomes. Sir3, a divergent member of the AAA+ ATPase-like family, contacts both the histone H4 tail and the nucleosome core. Here, we present the structure and function of the conserved C-terminal domain of Sir3, comprising 138 amino acids. This module adopts a variant winged helix-turn-helix (wH) architecture that exists as a stable homodimer in solution. Mutagenesis shows that the self-association mediated by this domain is essential for holo-Sir3 dimerization. Its loss impairs Sir3 loading onto nucleosomes in vitro and eliminates silencing at telomeres and HM loci in vivo. Replacing the Sir3 wH domain with an unrelated bacterial dimerization motif restores both HM and telomeric repression in sir3Δ cells. In contrast, related wH domains of archaeal and human members of the Orc1/Sir3 family are monomeric and have DNA binding activity. We speculate that a dimerization function for the wH evolved with Sir3's ability to facilitate heterochromatin formation.

Acyl coenzyme A thioesterase Them5/Acot15 is involved in cardiolipin remodeling and fatty liver development.

Acyl coenzyme A (acyl-CoA) thioesterases hydrolyze thioester bonds in acyl-CoA metabolites. The majority of mammalian thioesterases are α/ß-hydrolases and have been studied extensively. A second class of Hotdog-fold enzymes has been less well described. Here, we present a structural and functional analysis of a new mammalian mitochondrial thioesterase, Them5. Them5 and its paralog, Them4, adopt the classical Hotdog-fold structure and form homodimers in crystals. In vitro, Them5 shows strong thioesterase activity with long-chain acyl-CoAs. Loss of Them5 specifically alters the remodeling process of the mitochondrial phospholipid cardiolipin. Them5(-/-) mice show deregulation of lipid metabolism and the development of fatty liver, exacerbated by a high-fat diet. Consequently, mitochondrial morphology is affected, and functions such as respiration and ß-oxidation are impaired. The novel mitochondrial acyl-CoA thioesterase Them5 has a critical and specific role in the cardiolipin remodeling process, connecting it to the development of fatty liver and related conditions.

Structure of human POFUT2: insights into thrombospondin type 1 repeat fold and O-fucosylation.

Protein O-fucosylation is a post-translational modification found on serine/threonine residues of thrombospondin type 1 repeats (TSR). The fucose transfer is catalysed by the protein O-fucosyltransferase 2 (POFUT2) and >40 human proteins contain the TSR consensus sequence for POFUT2-dependent fucosylation. To better understand O-fucosylation on TSR, we carried out a structural and functional analysis of human POFUT2 and its TSR substrate. Crystal structures of POFUT2 reveal a variation of the classical GT-B fold and identify sugar donor and TSR acceptor binding sites. Structural findings are correlated with steady-state kinetic measurements of wild-type and mutant POFUT2 and TSR and give insight into the catalytic mechanism and substrate specificity. By using an artificial mini-TSR substrate, we show that specificity is not primarily encoded in the TSR protein sequence but rather in the unusual 3D structure of a small part of the TSR. Our findings uncover that recognition of distinct conserved 3D fold motifs can be used as a mechanism to achieve substrate specificity by enzymes modifying completely folded proteins of very wide sequence diversity and biological function.

Peters Plus syndrome is a new congenital disorder of glycosylation and involves defective Omicron-glycosylation of thrombospondin type 1 repeats.

Peters Plus syndrome is an autosomal recessive disorder characterized by anterior eye chamber defects, disproportionate short stature, developmental delay, and cleft lip and/or palate. It is caused by splice site mutations in what was thought to be a beta1,3-galactosyltransferase-like gene (B3GALTL). Recently, we and others found this gene to encode a beta1,3-glucosyltransferase involved in the synthesis of the disaccharide Glc-beta1,3-Fuc-Omicron-that occurs on thrombospondin type 1 repeats of many biologically important proteins. No functional tests have been performed to date on the presumed glycosylation defect in Peters Plus syndrome. We have established a sensitive immunopurification-mass spectrometry method, using multiple reaction monitoring, to analyze Omicron-fucosyl glycans. It was used to compare the reporter protein properdin from Peters Plus patients with that from control heterozygous relatives. In properdin from patients, we could not detect the Glc-beta1,3-Fuc-Omicron-disaccharide, and we only found Fuc-Omicron-at all four Omicron-fucosylation sites. In contrast, properdin from heterozygous relatives and a healthy volunteer carried the Glc-beta1,3-Fuc-Omicron-disaccharide. These data firmly establish Peters Plus syndrome as a new congenital disorder of glycosylation.

Identification and characterization of abeta1,3-glucosyltransferase that synthesizes the Glc-beta1,3-Fuc disaccharide on thrombospondin type 1 repeats.

Thrombospondin type 1 repeats (TSRs) are biologically important domains of extracellular proteins. They are modified with a unique Glcbeta1,3Fucalpha1-O-linked disaccharide on either serine or threonine residues. Here we identify the putative glycosyltransferase, B3GTL, as the beta1,3-glucosyltransferase involved in the biosynthesis of this disaccharide. This enzyme is conserved from Caenorhabditis elegans to man and shares 28% sequence identity with Fringe, the beta1,3-N-acetylglucosaminyltransferase that modifies O-linked fucosyl residues in proteins containing epidermal growth factor-like domains, such as Notch. beta1,3-Glucosyltransferase glucosylates properly folded TSR-fucose but not fucosylated epidermal growth factor-like domain or the non-fucosylated modules. Specifically, the glucose is added in a beta1,3-linkage to the fucose in TSR. The activity profiles of beta1,3-glucosyltransferase and protein O-fucosyltransferase 2, the enzyme that carries out the first step in TSR O-fucosylation, superimpose in endoplasmic reticulum subfractions obtained by density gradient centrifugation. Both enzymes are soluble proteins that efficiently modify properly folded TSR modules. The identification of the beta1,3-glucosyltransferase gene allows us to manipulate the formation of the rare Glcbeta1,3Fucalpha1 structure to investigate its biological function.