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1.
Nat Commun ; 14(1): 6809, 2023 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-37884503

RESUMO

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.


Assuntos
Nucleotídeos , Humanos , Modelos Moleculares , Hidrólise , Domínio Catalítico , Catálise
2.
Acta Crystallogr D Struct Biol ; 78(Pt 9): 1079-1089, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36048148

RESUMO

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.


Assuntos
Computação em Nuvem , Software , Cristalografia por Raios X , Substâncias Macromoleculares/química
3.
Biochemistry ; 60(21): 1682-1698, 2021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-33988981

RESUMO

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.


Assuntos
Desoxirribonucleotídeos/química , Proteína 1 com Domínio SAM e Domínio HD/antagonistas & inibidores , Proteína 1 com Domínio SAM e Domínio HD/química , Regulação Alostérica , Catálise , Domínio Catalítico , Cristalografia por Raios X/métodos , Nucleotídeos de Desoxiguanina/química , Desoxirribonucleotídeos/metabolismo , Humanos , Hidrólise , Cinética , Modelos Moleculares , Proteínas Monoméricas de Ligação ao GTP/química , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Replicação Viral/fisiologia
4.
Structure ; 29(7): 694-708.e7, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-33484636

RESUMO

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.


Assuntos
Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Caderinas/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Modelos Moleculares , Complexos Multiproteicos/química , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Proto-Oncogênicas c-ret/química , Proteínas de Peixe-Zebra/química
5.
Elife ; 102021 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-33416497

RESUMO

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.


Assuntos
Aracnodactilia/genética , Craniossinostoses/genética , Proteínas de Ligação a DNA/genética , Síndrome de Marfan/genética , Mutação , Proteínas Proto-Oncogênicas/genética , Fator de Crescimento Transformador beta/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Humanos , Masculino , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo
6.
Biol Open ; 9(7)2020 07 31.
Artigo em Inglês | MEDLINE | ID: mdl-32661131

RESUMO

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.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/ultraestrutura , Actinas/metabolismo , Microscopia Crioeletrônica , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Complexo 2-3 de Proteínas Relacionadas à Actina/química , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/química , Humanos , Modelos Moleculares , Mutação , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
7.
Nat Commun ; 11(1): 3165, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576829

RESUMO

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.


Assuntos
Nucleosídeo-Trifosfatase/química , Proteína 1 com Domínio SAM e Domínio HD/química , Água/química , Doenças Autoimunes do Sistema Nervoso/metabolismo , Domínio Catalítico , Cristalografia por Raios X , HIV-1/genética , HIV-1/fisiologia , Humanos , Hidrólise , Interferons , Modelos Moleculares , Mutação , Malformações do Sistema Nervoso/metabolismo , Polifosfatos , Conformação Proteica , Proteína 1 com Domínio SAM e Domínio HD/genética , Replicação Viral/fisiologia
8.
Nat Chem Biol ; 16(6): 686-694, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32203411

RESUMO

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.


Assuntos
Alanina Racemase/metabolismo , Antibióticos Antituberculose/química , Ciclosserina/química , Proteínas Recombinantes/metabolismo , Alanina/química , Alanina/metabolismo , Alanina Racemase/genética , Sequência de Aminoácidos , Antibióticos Antituberculose/metabolismo , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Ciclosserina/metabolismo , Escherichia coli , Isoxazóis/química , Ligases/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Oximas/química , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/genética
9.
Nucleic Acids Res ; 47(8): 4334-4348, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-30864660

RESUMO

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.


Assuntos
Proteínas Proto-Oncogênicas c-myc/genética , RNA Mensageiro/química , Proteínas de Ligação a RNA/química , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
10.
Nat Commun ; 9(1): 5061, 2018 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-30498216

RESUMO

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.


Assuntos
DNA Polimerase II/química , DNA Polimerase II/metabolismo , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , DNA/química , DNA/genética , DNA Polimerase II/genética , Replicação do DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Humanos , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína
12.
Sci Rep ; 7: 44313, 2017 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-28290497

RESUMO

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.


Assuntos
Acetiltransferases/química , Proteínas de Ciclo Celular/química , Proteínas Cromossômicas não Histona/química , Segregação de Cromossomos , Proteínas Nucleares/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Xenopus/química , Acetilação , Acetiltransferases/genética , Acetiltransferases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cristalografia por Raios X , Expressão Gênica , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/metabolismo
13.
Nat Commun ; 8: 13952, 2017 01 06.
Artigo em Inglês | MEDLINE | ID: mdl-28059076

RESUMO

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.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Sequência Conservada , Modelos Moleculares , Ligação Proteica , Subunidades Proteicas/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Coesinas
14.
Dev Cell ; 38(4): 384-98, 2016 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-27554858

RESUMO

Atypical protein kinase C (aPKC) is a key apical-basal polarity determinant and Par complex component. It is recruited by Par3/Baz (Bazooka in Drosophila) into epithelial apical domains through high-affinity interaction. Paradoxically, aPKC also phosphorylates Par3/Baz, provoking its relocalization to adherens junctions (AJs). We show that Par3 conserved region 3 (CR3) forms a tight inhibitory complex with a primed aPKC kinase domain, blocking substrate access. A CR3 motif flanking its PKC consensus site disrupts the aPKC kinase N lobe, separating P-loop/αB/αC contacts. A second CR3 motif provides a high-affinity anchor. Mutation of either motif switches CR3 to an efficient in vitro substrate by exposing its phospho-acceptor site. In vivo, mutation of either CR3 motif alters Par3/Baz localization from apical to AJs. Our results reveal how Par3/Baz CR3 can antagonize aPKC in stable apical Par complexes and suggests that modulation of CR3 inhibitory arms or opposing aPKC pockets would perturb the interaction, promoting Par3/Baz phosphorylation.


Assuntos
Junções Aderentes/metabolismo , Membrana Celular/metabolismo , Proteínas de Drosophila/metabolismo , Células Epiteliais/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Membrana/metabolismo , Proteína Quinase C/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Polaridade Celular/fisiologia , Drosophila , Proteínas de Drosophila/genética , Epitélio/crescimento & desenvolvimento , Células HCT116 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Membrana/genética , Fosforilação , Ligação Proteica/genética , Estrutura Terciária de Proteína
15.
Eur J Med Chem ; 112: 20-32, 2016 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-26874741

RESUMO

Deregulation of the receptor tyrosine kinase RET has been implicated in medullary thyroid cancer, a small percentage of lung adenocarcinomas, endocrine-resistant breast cancer and pancreatic cancer. There are several clinically approved multi-kinase inhibitors that target RET as a secondary pharmacology but additional activities, most notably inhibition of KDR, lead to dose-limiting toxicities. There is, therefore, a clinical need for more specific RET kinase inhibitors. Herein we report our efforts towards identifying a potent and selective RET inhibitor using vandetanib 1 as the starting point for structure-based drug design. Phenolic anilinoquinazolines exemplified by 6 showed improved affinities towards RET but, unsurprisingly, suffered from high metabolic clearance. Efforts to mitigate the metabolic liability of the phenol led to the discovery that a flanking substituent not only improved the hepatocyte stability, but could also impart a significant gain in selectivity. This culminated in the identification of 36; a potent RET inhibitor with much improved selectivity against KDR.


Assuntos
Piperidinas/química , Piperidinas/farmacologia , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-ret/antagonistas & inibidores , Quinazolinas/química , Quinazolinas/farmacologia , Animais , Linhagem Celular , Desenho de Fármacos , Humanos , Camundongos , Simulação de Acoplamento Molecular , Piperidinas/farmacocinética , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Proto-Oncogênicas c-ret/metabolismo , Quinazolinas/farmacocinética
16.
Acta Crystallogr D Biol Crystallogr ; 71(Pt 3): 555-64, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25760605

RESUMO

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.


Assuntos
Proteínas do Olho , Proteínas de Membrana , Proteínas do Tecido Nervoso , Núcleosídeo-Fosfato Quinase , Motivos de Aminoácidos , Animais , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Proteínas do Olho/química , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Guanilato Quinases/química , Guanilato Quinases/genética , Guanilato Quinases/metabolismo , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/química , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Núcleosídeo-Fosfato Quinase/química , Núcleosídeo-Fosfato Quinase/genética , Núcleosídeo-Fosfato Quinase/metabolismo , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína
17.
Cell Rep ; 8(6): 1894-1904, 2014 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-25242331

RESUMO

The RET receptor tyrosine kinase is essential to vertebrate development and implicated in multiple human diseases. RET binds a cell surface bipartite ligand comprising a GDNF family ligand and a GFRα coreceptor, resulting in RET transmembrane signaling. We present a hybrid structural model, derived from electron microscopy (EM) and low-angle X-ray scattering (SAXS) data, of the RET extracellular domain (RET(ECD)), GDNF, and GFRα1 ternary complex, defining the basis for ligand recognition. RET(ECD) envelopes the dimeric ligand complex through a composite binding site comprising four discrete contact sites. The GFRα1-mediated contacts are crucial, particularly close to the invariant RET calcium-binding site, whereas few direct contacts are made by GDNF, explaining how distinct ligand/coreceptor pairs are accommodated. The RET(ECD) cysteine-rich domain (CRD) contacts both ligand components and makes homotypic membrane-proximal interactions occluding three different antibody epitopes. Coupling of these CRD-mediated interactions suggests models for ligand-induced RET activation and ligand-independent oncogenic deregulation.


Assuntos
Membrana Celular/metabolismo , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Fator Neurotrófico Derivado de Linhagem de Célula Glial/metabolismo , Proteínas Proto-Oncogênicas c-ret/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos/imunologia , Sítios de Ligação , Células CHO , Cricetinae , Cricetulus , Epitopos/imunologia , Fator Neurotrófico Derivado de Linhagem de Célula Glial/química , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/química , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Humanos , Dados de Sequência Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-ret/química , Proteínas Proto-Oncogênicas c-ret/genética , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alinhamento de Sequência , Peixe-Zebra , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/genética
18.
Structure ; 22(2): 337-44, 2014 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-24389026

RESUMO

The combination of an E2 ubiquitin-conjugating enzyme with an E3 ubiquitin-ligase is essential for ubiquitin modification of a substrate. Moreover, the pairing dictates both the substrate choice and the modification type. The molecular details of generic E3-E2 interactions are well established. Nevertheless, the determinants of selective, specific E3-E2 recognition are not understood. There are ∼40 E2s and ∼600 E3s giving rise to a possible ∼24,000 E3-E2 pairs. Using the Fanconi Anemia pathway exclusive E3-E2 pair, FANCL-Ube2T, we report the atomic structure of the FANCL RING-Ube2T complex, revealing a specific and extensive network of additional electrostatic and hydrophobic interactions. Furthermore, we show that these specific interactions are required for selection of Ube2T over other E2s by FANCL.


Assuntos
Proteína do Grupo de Complementação L da Anemia de Fanconi/química , Sequência de Aminoácidos , Animais , Reparo do DNA , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Conformação Proteica , Homologia de Sequência de Aminoácidos , Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química , Xenopus laevis
19.
Sci Signal ; 6(293): ra82, 2013 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-24045153

RESUMO

Atypical protein kinase Cι (PKCι) has roles in cell growth, cellular polarity, and migration, and its abundance is frequently increased in cancer. We identified a protein interaction surface containing a dibasic motif (RIPR) that bound a distinct subset of PKCι substrates including lethal giant larvae 2 (LLGL2) and myosin X, but not other substrates such as Par3. Further characterization demonstrated that Arg471 in this motif was important for binding to LLGL2, whereas Arg474 was critical for interaction with myosin X, indicating that multiple complexes could be formed through this motif. A somatic mutation of the dibasic motif (R471C) was the most frequent mutation of PKCι in human cancer, and the intact dibasic motif was required for normal polarized epithelial morphogenesis in three-dimensional cysts. Thus, the R471C substitution is a change-of-function mutation acting at this substrate-specific recruitment site to selectively disrupt the polarizing activity of PKCι.


Assuntos
Polaridade Celular/genética , Proteínas do Citoesqueleto/metabolismo , Isoenzimas/química , Isoenzimas/genética , Modelos Moleculares , Proteínas de Neoplasias/genética , Conformação Proteica , Proteína Quinase C/química , Proteína Quinase C/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Diamino Aminoácidos/genética , Análise de Variância , Animais , Proteínas do Citoesqueleto/genética , Cães , Células HEK293 , Humanos , Immunoblotting , Células Madin Darby de Rim Canino , Espectrometria de Massas , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação/genética , Fosforilação , Alinhamento de Sequência , Especificidade por Substrato
20.
Nat Commun ; 4: 1983, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23770917

RESUMO

Mutations in the park2 gene, encoding the RING-inBetweenRING-RING E3 ubiquitin ligase parkin, cause 50% of autosomal recessive juvenile Parkinsonism cases. More than 70 known pathogenic mutations occur throughout parkin, many of which cluster in the inhibitory amino-terminal ubiquitin-like domain, and the carboxy-terminal RING2 domain that is indispensable for ubiquitin transfer. A structural rationale showing how autosomal recessive juvenile Parkinsonism mutations alter parkin function is still lacking. Here we show that the structure of parkin RING2 is distinct from canonical RING E3 ligases and lacks key elements required for E2-conjugating enzyme recruitment. Several pathogenic mutations in RING2 alter the environment of a single surface-exposed catalytic cysteine to inhibit ubiquitination. Native parkin adopts a globular inhibited conformation in solution facilitated by the association of the ubiquitin-like domain with the RING-inBetweenRING-RING C-terminus. Autosomal recessive juvenile Parkinsonism mutations disrupt this conformation. Finally, parkin autoubiquitinates only in cis, providing a molecular explanation for the recessive nature of autosomal recessive juvenile Parkinsonism.


Assuntos
Genes Recessivos/genética , Doença de Parkinson/genética , Ubiquitina-Proteína Ligases/genética , Sequência de Aminoácidos , Animais , Biocatálise , Drosophila melanogaster , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica , Estrutura Terciária de Proteína , Alinhamento de Sequência , Especificidade por Substrato , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/química
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