14-3-3 proteins interact with a hybrid prenyl-phosphorylation motif to inhibit G proteins.

Signaling through G proteins normally involves conformational switching between GTP- and GDP-bound states. Several Rho GTPases are also regulated by RhoGDI binding and sequestering in the cytosol. Rnd proteins are atypical constitutively GTP-bound Rho proteins, whose regulation remains elusive. Here, we report a high-affinity 14-3-3-binding site at the C terminus of Rnd3 consisting of both the Cys241-farnesyl moiety and a Rho-associated coiled coil containing protein kinase (ROCK)-dependent Ser240 phosphorylation site. 14-3-3 binding to Rnd3 also involves phosphorylation of Ser218 by ROCK and/or Ser210 by protein kinase C (PKC). The crystal structure of a phosphorylated, farnesylated Rnd3 peptide with 14-3-3 reveals a hydrophobic groove in 14-3-3 proteins accommodating the farnesyl moiety. Functionally, 14-3-3 inhibits Rnd3-induced cell rounding by translocating it from the plasma membrane to the cytosol. Rnd1, Rnd2, and geranylgeranylated Rap1A interact similarly with 14-3-3. In contrast to the canonical GTP/GDP switch that regulates most Ras superfamily members, our results reveal an unprecedented mechanism for G protein inhibition by 14-3-3 proteins.

D-Cycloserine destruction by alanine racemase and the limit of irreversible inhibition.

The broad-spectrum antibiotic D-cycloserine (DCS) is a key component of regimens used to treat multi- and extensively drug-resistant tuberculosis. DCS, a structural analog of D-alanine, binds to and inactivates two essential enzymes involved in peptidoglycan biosynthesis, alanine racemase (Alr) and D-Ala:D-Ala ligase. Inactivation of Alr is thought to proceed via a mechanism-based irreversible route, forming an adduct with the pyridoxal 5'-phosphate cofactor, leading to bacterial death. Inconsistent with this hypothesis, Mycobacterium tuberculosis Alr activity can be detected after exposure to clinically relevant DCS concentrations. To address this paradox, we investigated the chemical mechanism of Alr inhibition by DCS. Inhibition of M. tuberculosis Alr and other Alrs is reversible, mechanistically revealed by a previously unidentified DCS-adduct hydrolysis. Dissociation and subsequent rearrangement to a stable substituted oxime explains Alr reactivation in the cellular milieu. This knowledge provides a novel route for discovery of improved Alr inhibitors against M. tuberculosis and other bacteria.

Probing the Catalytic Mechanism and Inhibition of SAMHD1 Using the Differential Properties of Rp- and Sp-dNTPαS Diastereomers.

SAMHD1 is a fundamental regulator of cellular dNTPs that catalyzes their hydrolysis into 2'-deoxynucleoside and triphosphate, restricting the replication of viruses, including HIV-1, in CD4+ myeloid lineage and resting T-cells. SAMHD1 mutations are associated with the autoimmune disease Aicardi-Goutières syndrome (AGS) and certain cancers. More recently, SAMHD1 has been linked to anticancer drug resistance and the suppression of the interferon response to cytosolic nucleic acids after DNA damage. Here, we probe dNTP hydrolysis and inhibition of SAMHD1 using the Rp and Sp diastereomers of dNTPαS nucleotides. Our biochemical and enzymological data show that the α-phosphorothioate substitution in Sp-dNTPαS but not Rp-dNTPαS diastereomers prevents Mg2+ ion coordination at both the allosteric and catalytic sites, rendering SAMHD1 unable to form stable, catalytically active homotetramers or hydrolyze substrate dNTPs at the catalytic site. Furthermore, we find that Sp-dNTPαS diastereomers competitively inhibit dNTP hydrolysis, while Rp-dNTPαS nucleotides stabilize tetramerization and are hydrolyzed with similar kinetic parameters to cognate dNTPs. For the first time, we present a cocrystal structure of SAMHD1 with a substrate, Rp-dGTPαS, in which an Fe-Mg-bridging water species is poised for nucleophilic attack on the Pα. We conclude that it is the incompatibility of Mg2+, a hard Lewis acid, and the α-phosphorothioate thiol, a soft Lewis base, that prevents the Sp-dNTPαS nucleotides coordinating in a catalytically productive conformation. On the basis of these data, we present a model for SAMHD1 stereospecific hydrolysis of Rp-dNTPαS nucleotides and for a mode of competitive inhibition by Sp-dNTPαS nucleotides that competes with formation of the enzyme-substrate complex.

IMP1 KH1 and KH2 domains create a structural platform with unique RNA recognition and re-modelling properties.

IGF2 mRNA-binding protein 1 (IMP1) is a key regulator of messenger RNA (mRNA) metabolism and transport in organismal development and, in cancer, its mis-regulation is an important component of tumour metastasis. IMP1 function relies on the recognition of a diverse set of mRNA targets that is mediated by the combinatorial action of multiple RNA-binding domains. Here, we dissect the structure and RNA-binding properties of two key RNA-binding domains of IMP1, KH1 and KH2, and we build a kinetic model for the recognition of RNA targets. Our data and model explain how the two domains are organized as an intermolecular pseudo-dimer and that the important role they play in mRNA target recognition is underpinned by the high RNA-binding affinity and fast kinetics of this KH1KH2-RNA recognition unit. Importantly, the high-affinity RNA-binding by KH1KH2 is achieved by an inter-domain coupling 50-fold stronger than that existing in a second pseudo-dimer in the protein, KH3KH4. The presence of this strong coupling supports a role of RNA re-modelling in IMP1 recognition of known cancer targets.

Structures of the human Pals1 PDZ domain with and without ligand suggest gated access of Crb to the PDZ peptide-binding groove.

Many components of epithelial polarity protein complexes possess PDZ domains that are required for protein interaction and recruitment to the apical plasma membrane. Apical localization of the Crumbs (Crb) transmembrane protein requires a PDZ-mediated interaction with Pals1 (protein-associated with Lin7, Stardust, MPP5), a member of the p55 family of membrane-associated guanylate kinases (MAGUKs). This study describes the molecular interaction between the Crb carboxy-terminal motif (ERLI), which is required for Drosophila cell polarity, and the Pals1 PDZ domain using crystallography and fluorescence polarization. Only the last four Crb residues contribute to Pals1 PDZ-domain binding affinity, with specificity contributed by conserved charged interactions. Comparison of the Crb-bound Pals1 PDZ structure with an apo Pals1 structure reveals a key Phe side chain that gates access to the PDZ peptide-binding groove. Removal of this side chain enhances the binding affinity by more than fivefold, suggesting that access of Crb to Pals1 may be regulated by intradomain contacts or by protein-protein interaction.

Adenosine-binding motif mimicry and cellular effects of a thieno[2,3-d]pyrimidine-based chemical inhibitor of atypical protein kinase C isoenzymes.

The aPKC [atypical PKC (protein kinase C)] isoforms ι and ζ play crucial roles in the formation and maintenance of cell polarity and represent attractive anti-oncogenic drug targets in Ras-dependent tumours. To date, few isoform-specific chemical biology tools are available to inhibit aPKC catalytic activity. In the present paper, we describe the identification and functional characterization of potent and selective thieno[2,3-d]pyrimidine-based chemical inhibitors of aPKCs. A crystal structure of human PKCι kinase domain bound to a representative compound, CRT0066854, reveals the basis for potent and selective chemical inhibition. Furthermore, CRT0066854 displaces a crucial Asn-Phe-Asp motif that is part of the adenosine-binding pocket and engages an acidic patch used by arginine-rich PKC substrates. We show that CRT0066854 inhibits the LLGL2 (lethal giant larvae 2) phosphorylation in cell lines and exhibits phenotypic effects in a range of cell-based assays. We conclude that this compound can be used as a chemical tool to modulate aPKC activity in vitro and in vivo and may guide the search for further aPKC-selective inhibitors.

Mechanism of substrate hydrolysis by the human nucleotide pool sanitiser DNPH1.

Poly(ADP-ribose) polymerase (PARP) inhibitors are used in the clinic to treat BRCA-deficient breast, ovarian and prostate cancers. As their efficacy is potentiated by loss of the nucleotide salvage factor DNPH1 there is considerable interest in the development of highly specific small molecule DNPH1 inhibitors. Here, we present X-ray crystal structures of dimeric DNPH1 bound to its substrate hydroxymethyl deoxyuridine monophosphate (hmdUMP). Direct interaction with the hydroxymethyl group is important for substrate positioning, while conserved residues surrounding the base facilitate target discrimination. Glycosidic bond cleavage is driven by a conserved catalytic triad and proceeds via a two-step mechanism involving formation and subsequent disruption of a covalent glycosyl-enzyme intermediate. Mutation of a previously uncharacterised yet conserved glutamate traps the intermediate in the active site, demonstrating its role in the hydrolytic step. These observations define the enzyme's catalytic site and mechanism of hydrolysis, and provide important insights for inhibitor discovery.

Structural analysis of human FANCL, the E3 ligase in the Fanconi anemia pathway.

The Fanconi anemia (FA) pathway is essential for the repair of DNA interstrand cross-links. At the heart of this pathway is the monoubiquitination of the FANCI-FANCD2 (ID) complex by the multiprotein "core complex" containing the E3 ubiquitin ligase FANCL. Vertebrate organisms have the eight-protein core complex, whereas invertebrates apparently do not. We report here the structure of the central domain of human FANCL in comparison with the recently solved Drosophila melanogaster FANCL. Our data represent the first structural detail into the catalytic core of the human system and reveal that the central fold of FANCL is conserved between species. However, there are macromolecular differences between the FANCL proteins that may account for the apparent distinctions in core complex requirements between the vertebrate and invertebrate FA pathways. In addition, we characterize the binding of human FANCL with its partners, Ube2t, FANCD2, and FANCI. Mutational analysis reveals which residues are required for substrate binding, and we also show the domain required for E2 binding.

CCP4 Cloud for structure determination and project management in macromolecular crystallography.

Nowadays, progress in the determination of three-dimensional macromolecular structures from diffraction images is achieved partly at the cost of increasing data volumes. This is due to the deployment of modern high-speed, high-resolution detectors, the increased complexity and variety of crystallographic software, the use of extensive databases and high-performance computing. This limits what can be accomplished with personal, offline, computing equipment in terms of both productivity and maintainability. There is also an issue of long-term data maintenance and availability of structure-solution projects as the links between experimental observations and the final results deposited in the PDB. In this article, CCP4 Cloud, a new front-end of the CCP4 software suite, is presented which mitigates these effects by providing an online, cloud-based environment for crystallographic computation. CCP4 Cloud was developed for the efficient delivery of computing power, database services and seamless integration with web resources. It provides a rich graphical user interface that allows project sharing and long-term storage for structure-solution projects, and can be linked to data-producing facilities. The system is distributed with the CCP4 software suite version 7.1 and higher, and an online publicly available instance of CCP4 Cloud is provided by CCP4.

Recognition of an intra-chain tandem 14-3-3 binding site within PKCepsilon.

The phosphoserine/threonine binding protein 14-3-3 stimulates the catalytic activity of protein kinase C-epsilon (PKCepsilon) by engaging two tandem phosphoserine-containing motifs located between the PKCepsilon regulatory and catalytic domains (V3 region). Interaction between 14-3-3 and this region of PKCepsilon is essential for the completion of cytokinesis. Here, we report the crystal structure of 14-3-3zeta bound to a synthetic diphosphorylated PKCepsilon V3 region revealing how a consensus 14-3-3 site and a divergent 14-3-3 site cooperate to bind to 14-3-3 and so activate PKCepsilon. Thermodynamic data show a markedly enhanced binding affinity for two-site phosphopeptides over single-site 14-3-3 binding motifs and identifies Ser 368 as a gatekeeper phosphorylation site in this physiologically relevant 14-3-3 ligand. This dual-site intra-chain recognition has implications for other 14-3-3 targets, which seem to have only a single 14-3-3 motif, as other lower affinity and cryptic 14-3-3 gatekeeper sites might exist.

Mutations in SKI in Shprintzen-Goldberg syndrome lead to attenuated TGF-ß responses through SKI stabilization.

Shprintzen-Goldberg syndrome (SGS) is a multisystemic connective tissue disorder, with considerable clinical overlap with Marfan and Loeys-Dietz syndromes. These syndromes have commonly been associated with enhanced TGF-ß signaling. In SGS patients, heterozygous point mutations have been mapped to the transcriptional co-repressor SKI, which is a negative regulator of TGF-ß signaling that is rapidly degraded upon ligand stimulation. The molecular consequences of these mutations, however, are not understood. Here we use a combination of structural biology, genome editing, and biochemistry to show that SGS mutations in SKI abolish its binding to phosphorylated SMAD2 and SMAD3. This results in stabilization of SKI and consequently attenuation of TGF-ß responses, both in knockin cells expressing an SGS mutation and in fibroblasts from SGS patients. Thus, we reveal that SGS is associated with an attenuation of TGF-ß-induced transcriptional responses, and not enhancement, which has important implications for other Marfan-related syndromes.

A two-site flexible clamp mechanism for RET-GDNF-GFRα1 assembly reveals both conformational adaptation and strict geometric spacing.

RET receptor tyrosine kinase plays vital developmental and neuroprotective roles in metazoans. GDNF family ligands (GFLs) when bound to cognate GFRα co-receptors recognize and activate RET stimulating its cytoplasmic kinase function. The principles for RET ligand-co-receptor recognition are incompletely understood. Here, we report a crystal structure of the cadherin-like module (CLD1-4) from zebrafish RET revealing interdomain flexibility between CLD2 and CLD3. Comparison with a cryo-electron microscopy structure of a ligand-engaged zebrafish RETECD-GDNF-GFRα1a complex indicates conformational changes within a clade-specific CLD3 loop adjacent to the co-receptor. Our observations indicate that RET is a molecular clamp with a flexible calcium-dependent arm that adapts to different GFRα co-receptors, while its rigid arm recognizes a GFL dimer to align both membrane-proximal cysteine-rich domains. We also visualize linear arrays of RETECD-GDNF-GFRα1a suggesting that a conserved contact stabilizes higher-order species. Our study reveals that ligand-co-receptor recognition by RET involves both receptor plasticity and strict spacing of receptor dimers by GFL ligands.

Explosive expansion of betagamma-crystallin genes in the ancestral vertebrate.

In jawed vertebrates, betagamma-crystallins are restricted to the eye lens and thus excellent markers of lens evolution. These betagamma-crystallins are four Greek key motifs/two domain proteins, whereas the urochordate betagamma-crystallin has a single domain. To trace the origin of the vertebrate betagamma-crystallin genes, we searched for homologues in the genomes of a jawless vertebrate (lamprey) and of a cephalochordate (lancelet). The lamprey genome contains orthologs of the gnathostome betaB1-, betaA2- and gammaN-crystallin genes and a single domain gammaN-crystallin-like gene. It contains at least two gamma-crystallin genes, but lacks the gnathostome gammaS-crystallin gene. The genome also encodes a non-lenticular protein containing betagamma-crystallin motifs, AIM1, also found in gnathostomes but not detectable in the uro- or cephalochordate genome. The four cephalochordate betagamma-crystallin genes found encode two-domain proteins. Unlike the vertebrate betagamma-crystallins but like the urochordate betagamma-crystallin, three of the predicted proteins contain calcium-binding sites. In the cephalochordate betagamma-crystallin genes, the introns are located within motif-encoding region, while in the urochordate and in the vertebrate betagamma-crystallin genes the introns are between motif- and/or domain encoding regions. Coincident with the evolution of the vertebrate lens an ancestral urochordate type betagamma-crystallin gene rapidly expanded and diverged in the ancestral vertebrate before the cyclostomes/gnathostomes split. The beta- and gammaN-crystallin genes were maintained in subsequent evolution, and, given the selection pressure imposed by accurate vision, must be essential for lens function. The gamma-crystallin genes show lineage specific expansion and contraction, presumably in adaptation to the demands on vision resulting from (changes in) lifestyle.

Cryo-EM of human Arp2/3 complexes provides structural insights into actin nucleation modulation by ARPC5 isoforms.

The Arp2/3 complex regulates many cellular processes by stimulating formation of branched actin filament networks. Because three of its seven subunits exist as two different isoforms, mammals produce a family of Arp2/3 complexes with different properties that may be suited to different physiological contexts. To shed light on how isoform diversification affects Arp2/3 function, we determined a 4.2 Šresolution cryo-EM structure of the most active human Arp2/3 complex containing ARPC1B and ARPC5L, and compared it with the structure of the least active ARPC1A-ARPC5-containing complex. The architecture of each isoform-specific Arp2/3 complex is the same. Strikingly, however, the N-terminal half of ARPC5L is partially disordered compared to ARPC5, suggesting that this region of ARPC5/ARPC5L is an important determinant of complex activity. Confirming this idea, the nucleation activity of Arp2/3 complexes containing hybrid ARPC5/ARPC5L subunits is higher when the ARPC5L N-terminus is present, thereby providing insight into activity differences between the different Arp2/3 complexes.

Crystal structures of SAMHD1 inhibitor complexes reveal the mechanism of water-mediated dNTP hydrolysis.

SAMHD1 regulates cellular 2'-deoxynucleoside-5'-triphosphate (dNTP) homeostasis by catalysing the hydrolysis of dNTPs into 2'-deoxynucleosides and triphosphate. In CD4+ myeloid lineage and resting T-cells, SAMHD1 blocks HIV-1 and other viral infections by depletion of the dNTP pool to a level that cannot support replication. SAMHD1 mutations are associated with the autoimmune disease Aicardi-Goutières syndrome and hypermutated cancers. Furthermore, SAMHD1 sensitises cancer cells to nucleoside-analogue anti-cancer therapies and is linked with DNA repair and suppression of the interferon response to cytosolic nucleic acids. Nevertheless, despite its requirement in these processes, the fundamental mechanism of SAMHD1-catalysed dNTP hydrolysis remained unknown. Here, we present structural and enzymological data showing that SAMHD1 utilises an active site, bi-metallic iron-magnesium centre that positions a hydroxide nucleophile in-line with the Pα-O5' bond to catalyse phosphoester bond hydrolysis. This precise molecular mechanism for SAMHD1 catalysis, reveals how SAMHD1 down-regulates cellular dNTP and modulates the efficacy of nucleoside-based anti-cancer and anti-viral therapies.

Urochordate betagamma-crystallin and the evolutionary origin of the vertebrate eye lens.

A refracting lens is a key component of our image-forming camera eye; however, its evolutionary origin is unknown because precursor structures appear absent in nonvertebrates. The vertebrate betagamma-crystallin genes encode abundant structural proteins critical for the function of the lens. We show that the urochordate Ciona intestinalis, which split from the vertebrate lineage before the evolution of the lens, has a single gene coding for a single domain monomeric betagamma-crystallin. The crystal structure of Ciona betagamma-crystallin is very similar to that of a vertebrate betagamma-crystallin domain, except for paired, occupied calcium binding sites. The Ciona betagamma-crystallin is only expressed in the palps and in the otolith, the pigmented sister cell of the light-sensing ocellus. The Ciona betagamma-crystallin promoter region targeted expression to the visual system, including lens, in transgenic Xenopus tadpoles. We conclude that the vertebrate betagamma-crystallins evolved from a single domain protein already expressed in the neuroectoderm of the prevertebrate ancestor. The conservation of the regulatory hierarchy controlling betagamma-crystallin expression between organisms with and without a lens shows that the evolutionary origin of the lens was based on co-option of pre-existing regulatory circuits controlling the expression of a key structural gene in a primitive light-sensing system.

Biophysical properties of gammaC-crystallin in human and mouse eye lens: the role of molecular dipoles.

The eye lens is packed with soluble crystallin proteins, providing a lifetime of transparency and light refraction. gamma-Crystallins are major components of the dense, high refractive index central regions of the lens and generally have high solubility, high stability and high levels of cysteine residues. Human gammaC belongs to a group of gamma-crystallins with a pair of cysteine residues at positions 78 and 79. Unlike other gamma-crystallins it has relatively low solubility, whereas mouse gammaC, which has the exposed C79 replaced with arginine, and a novel mouse splice variant, gammaCins, are both highly soluble. Furthermore, human gammaC is extremely stable, while the mouse orthologs are less stable. Evolutionary pressure may have favoured stability over solubility for human gammaC and the reverse for the orthologs in the mouse. Mutation of C79 to R79, in human gammaC, greatly increased solubility, however, neither form produced crystals. Remarkably, when the human gammaD R36S crystallization cataract mutation was mimicked in human gammaC-crystallin, the solubility of gammaC was dramatically increased, although it still did not crystallize. The highly soluble mouse gammaC-crystallin did crystallize. Its X-ray structure was solved and used in homology modelling of human gammaC, and its mutants C79R and R36S. The human gammaD R36S mutant was also modelled from human gammaD coordinates. Molecular dynamics simulation of the six molecules in the solution state showed that the human gammaCs differed from gammaDs in domain pairing, behaviour that correlates with interface sequence changes. When the fluctuations of the calculated molecular dipoles, for the six structures, over time were analysed, characteristic patterns for soluble gammaC and gammaD proteins were observed. Individual sequence changes that increase or decrease solubility correlated well with changes in the magnitude and direction of these dipoles. It is suggested that changes in surface residues have allowed adaptation for the differing needs of human and mouse lenses.

Structure of DNA-CMG-Pol epsilon elucidates the roles of the non-catalytic polymerase modules in the eukaryotic replisome.

Eukaryotic origin firing depends on assembly of the Cdc45-MCM-GINS (CMG) helicase. A key step is the recruitment of GINS that requires the leading-strand polymerase Pol epsilon, composed of Pol2, Dpb2, Dpb3, Dpb4. While a truncation of the catalytic N-terminal Pol2 supports cell division, Dpb2 and C-terminal Pol2 (C-Pol2) are essential for viability. Dpb2 and C-Pol2 are non-catalytic modules, shown or predicted to be related to an exonuclease and DNA polymerase, respectively. Here, we present the cryo-EM structure of the isolated C-Pol2/Dpb2 heterodimer, revealing that C-Pol2 contains a DNA polymerase fold. We also present the structure of CMG/C-Pol2/Dpb2 on a DNA fork, and find that polymerase binding changes both the helicase structure and fork-junction engagement. Inter-subunit contacts that keep the helicase-polymerase complex together explain several cellular phenotypes. At least some of these contacts are preserved during Pol epsilon-dependent CMG assembly on path to origin firing, as observed with DNA replication reconstituted in vitro.

Structural Basis of Eco1-Mediated Cohesin Acetylation.

Sister-chromatid cohesion is established by Eco1-mediated acetylation on two conserved tandem lysines in the cohesin Smc3 subunit. However, the molecular basis of Eco1 substrate recognition and acetylation in cohesion is not fully understood. Here, we discover and rationalize the substrate specificity of Eco1 using mass spectrometry coupled with in-vitro acetylation assays and crystallography. Our structures of the X. laevis Eco2 (xEco2) bound to its primary and secondary Smc3 substrates demonstrate the plasticity of the substrate-binding site, which confers substrate specificity by concerted conformational changes of the central ß hairpin and the C-terminal extension.

Structure of the cohesin loader Scc2.

The functions of cohesin are central to genome integrity, chromosome organization and transcription regulation through its prevention of premature sister-chromatid separation and the formation of DNA loops. The loading of cohesin onto chromatin depends on the Scc2-Scc4 complex; however, little is known about how it stimulates the cohesion-loading activity. Here we determine the large 'hook' structure of Scc2 responsible for catalysing cohesin loading. We identify key Scc2 surfaces that are crucial for cohesin loading in vivo. With the aid of previously determined structures and homology modelling, we derive a pseudo-atomic structure of the full-length Scc2-Scc4 complex. Finally, using recombinantly purified Scc2-Scc4 and cohesin, we performed crosslinking mass spectrometry and interaction assays that suggest Scc2-Scc4 uses its modular structure to make multiple contacts with cohesin.