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1.
Science ; 374(6573): eabm4805, 2021 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-34762488

RESUMO

Protein-protein interactions play critical roles in biology, but the structures of many eukaryotic protein complexes are unknown, and there are likely many interactions not yet identified. We take advantage of advances in proteome-wide amino acid coevolution analysis and deep-learning­based structure modeling to systematically identify and build accurate models of core eukaryotic protein complexes within the Saccharomyces cerevisiae proteome. We use a combination of RoseTTAFold and AlphaFold to screen through paired multiple sequence alignments for 8.3 million pairs of yeast proteins, identify 1505 likely to interact, and build structure models for 106 previously unidentified assemblies and 806 that have not been structurally characterized. These complexes, which have as many as five subunits, play roles in almost all key processes in eukaryotic cells and provide broad insights into biological function.


Assuntos
Aprendizado Profundo , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Mapeamento de Interação de Proteínas , Proteoma/química , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Aciltransferases/química , Aciltransferases/metabolismo , Segregação de Cromossomos , Biologia Computacional , Simulação por Computador , Reparo do DNA , Evolução Molecular , Recombinação Homóloga , Ligases/química , Ligases/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Moleculares , Biossíntese de Proteínas , Conformação Proteica , Mapas de Interação de Proteínas , Proteoma/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/química , Ubiquitina/química , Ubiquitina/metabolismo
2.
Sci Rep ; 11(1): 17038, 2021 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-34426585

RESUMO

Over the last decades the phase problem in macromolecular x-ray crystallography has become more controllable as methods and approaches have diversified and improved. However, solving the phase problem is still one of the biggest obstacles on the way of successfully determining a crystal structure. To overcome this caveat, we have utilized the anomalous scattering properties of the heavy alkali metal cesium. We investigated the introduction of cesium in form of cesium chloride during the three major steps of protein treatment in crystallography: purification, crystallization, and cryo-protection. We derived a step-wise procedure encompassing a "quick-soak"-only approach and a combined approach of CsCl supplement during purification and cryo-protection. This procedure was successfully applied on two different proteins: (i) Lysozyme and (ii) as a proof of principle, a construct consisting of the PH domain of the TFIIH subunit p62 from Chaetomium thermophilum for de novo structure determination. Usage of CsCl thus provides a versatile, general, easy to use, and low cost phasing strategy.

3.
DNA Repair (Amst) ; 105: 103143, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34144487

RESUMO

The general transcription factor II H (TFIIH) plays an essential role in transcription and nucleotide excision DNA repair (NER). TFIIH is a complex 10 subunit containing molecular machine that harbors three enzymatic activities while the remaining subunits assume regulatory and/or structural functions. Intriguingly, the three enzymatic activities of the CDK7 kinase, the XPB translocase, and the XPD helicase exert different impacts on the overall activities of TFIIH. While the enzymatic function of the XPD helicase is exclusively required in NER, the CDK7 kinase is deeply involved in transcription, whereas XPB is essential to both processes. Recent structural and biochemical endeavors enabled unprecedented details towards the molecular basis of these different TFIIH functions and how the enzymatic activities are regulated within the entire complex. Due to its involvement in two fundamental processes, TFIIH has become increasingly important as a target in cancer therapy and two of the three enzymes have already been addressed successfully. Here we explore the possibilities of recent high resolution structures in the context of TFIIH druggability and shed light on the functional consequences of the different approaches towards TFIIH inhibition.


Assuntos
Antineoplásicos/farmacologia , Reparo do DNA , Neoplasias/metabolismo , Fator de Transcrição TFIIH/antagonistas & inibidores , Fator de Transcrição TFIIH/metabolismo , Antineoplásicos/uso terapêutico , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , DNA/metabolismo , DNA Helicases/antagonistas & inibidores , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/metabolismo , Humanos , Neoplasias/tratamento farmacológico , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo
4.
ACS Infect Dis ; 7(4): 746-758, 2021 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-33710875

RESUMO

The enoyl-acyl carrier protein (ACP) reductase (ENR) is a key enzyme within the bacterial fatty-acid synthesis pathway. It has been demonstrated that small-molecule inhibitors carrying the diphenylether (DPE) scaffold bear a great potential for the development of highly specific and effective drugs against this enzyme class. Interestingly, different substitution patterns of the DPE scaffold have been shown to lead to varying effects on the kinetic and thermodynamic behavior toward ENRs from different organisms. Here, we investigated the effect of a 4'-pyridone substituent in the context of the slow tight-binding inhibitor SKTS1 on the inhibition of the Staphylococcus aureus enoyl-ACP-reductase saFabI and the closely related isoenzyme from Mycobacterium tuberculosis, InhA, and explored a new interaction site of DPE inhibitors within the substrate-binding pocket. Using high-resolution crystal structures of both complexes in combination with molecular dynamics (MD) simulations, kinetic measurements, and quantum mechanical (QM) calculations, we provide evidence that the 4'-pyridone substituent adopts different tautomeric forms when bound to the two ENRs. We furthermore elucidate the structural determinants leading to significant differences in the residence time of SKTS1 on both enzymes.


Assuntos
Inibidores Enzimáticos/farmacologia , Isoenzimas , Oxirredutases/antagonistas & inibidores , Isomerismo , Mycobacterium tuberculosis/enzimologia , Staphylococcus aureus/enzimologia
5.
Front Cell Dev Biol ; 9: 617160, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33777931

RESUMO

The successful elimination of bulky DNA damages via the nucleotide excision repair (NER) system is largely determined by the damage recognition step. This step consists of primary recognition and verification of the damage. The TFIIH helicase XPD plays a key role in the verification step during NER. To date, the mechanism of damage verification is not sufficiently understood and requires further detailed research. This study is a systematic investigation of the interaction of ctXPD (Chaetomium thermophilum) as well as ctXPD-ctp44 with model DNAs, which contain structurally different bulky lesions with previously estimated NER repair efficiencies. We have used ATPase and DNA binding studies to assess the interaction of ctXPD with damaged DNA. The result of the analysis of ctXPD-ctp44 binding to DNA containing fluorescent and photoactivatable lesions demonstrates the relationship between the affinity of XPD for DNAs containing bulky damages and the ability of the NER system to eliminate the damage. Photo-cross-linking of ctXPD with DNA probes containing repairable and unrepairable photoactivatable damages reveals differences in the DNA interaction efficiency in the presence and absence of ctp44. In general, the results obtained indicate the ability of ctXPD-ctp44 to interact with a damage and suggest a significant role for ctp44 subunit in the verification process.

6.
Nucleic Acids Res ; 48(21): 12282-12296, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33196848

RESUMO

The superfamily 2 helicase XPB is an integral part of the general transcription factor TFIIH and assumes essential catalytic functions in transcription initiation and nucleotide excision repair. The ATPase activity of XPB is required in both processes. We investigated the interaction network that regulates XPB via the p52 and p8 subunits with functional mutagenesis based on our crystal structure of the p52/p8 complex and current cryo-EM structures. Importantly, we show that XPB's ATPase can be activated either by DNA or by the interaction with the p52/p8 proteins. Intriguingly, we observe that the ATPase activation by p52/p8 is significantly weaker than the activation by DNA and when both p52/p8 and DNA are present, p52/p8 dominates the maximum activation. We therefore define p52/p8 as the master regulator of XPB acting as an activator and speed limiter at the same time. A correlative analysis of the ATPase and translocase activities of XPB shows that XPB only acts as a translocase within the context of complete core TFIIH and that XPA increases the processivity of the translocase complex without altering XPB's ATPase activity. Our data define an intricate network that tightly controls the activity of XPB during transcription and nucleotide excision repair.


Assuntos
Adenosina Trifosfatases/química , Chaetomium/química , DNA/genética , Proteínas Fúngicas/química , Subunidades Proteicas/química , Fator de Transcrição TFIIH/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sítios de Ligação , Chaetomium/genética , Chaetomium/metabolismo , Clonagem Molecular , Cristalografia por Raios X , DNA/metabolismo , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/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 , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Fator de Transcrição TFIIH/genética , Fator de Transcrição TFIIH/metabolismo , Transcrição Genética
7.
Nucleic Acids Res ; 48(22): 12689-12696, 2020 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-33166411

RESUMO

Nucleotide excision repair (NER) in eukaryotes is orchestrated by the core form of the general transcription factor TFIIH, containing the helicases XPB, XPD and five 'structural' subunits, p62, p44, p34, p52 and p8. Recent cryo-EM structures show that p62 makes extensive contacts with p44 and in part occupies XPD's DNA binding site. While p44 is known to regulate the helicase activity of XPD during NER, p62 is thought to be purely structural. Here, using helicase and adenosine triphosphatase assays we show that a complex containing p44 and p62 enhances XPD's affinity for dsDNA 3-fold over p44 alone. Remarkably, the relative affinity is further increased to 60-fold by dsDNA damage. Direct binding studies show this preference derives from p44/p62's high affinity (20 nM) for damaged ssDNA. Single molecule imaging of p44/p62 complexes without XPD reveals they bind to and randomly diffuse on DNA, however, in the presence of UV-induced DNA lesions these complexes stall. Combined with the analysis of a recent cryo-EM structure, we suggest that p44/p62 acts as a novel DNA-binding entity that enhances damage recognition in TFIIH. This revises our understanding of TFIIH and prompts investigation into the core subunits for an active role during DNA repair and/or transcription.


Assuntos
Reparo do DNA/genética , Proteínas de Ligação a RNA/ultraestrutura , Fator de Transcrição TFIIH/ultraestrutura , Sítios de Ligação/efeitos da radiação , Microscopia Crioeletrônica , Dano ao DNA/efeitos da radiação , DNA Helicases/genética , DNA Helicases/ultraestrutura , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/efeitos da radiação , DNA de Cadeia Simples/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Complexos Multiproteicos/genética , Complexos Multiproteicos/ultraestrutura , Proteínas de Ligação a RNA/genética , Imagem Individual de Molécula , Fator de Transcrição TFIIH/genética , Transcrição Genética/efeitos da radiação , Raios Ultravioleta/efeitos adversos , Proteína Grupo D do Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma Pigmentoso/ultraestrutura
8.
Proc Natl Acad Sci U S A ; 117(43): 26739-26748, 2020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33055219

RESUMO

Cyclin-dependent kinase 7 (CDK7), Cyclin H, and the RING-finger protein MAT1 form the heterotrimeric CDK-activating kinase (CAK) complex which is vital for transcription and cell-cycle control. When associated with the general transcription factor II H (TFIIH) it activates RNA polymerase II by hyperphosphorylation of its C-terminal domain (CTD). In the absence of TFIIH the trimeric complex phosphorylates the T-loop of CDKs that control cell-cycle progression. CAK holds a special position among the CDK branch due to this dual activity and the dependence on two proteins for activation. We solved the structure of the CAK complex from the model organism Chaetomium thermophilum at 2.6-Å resolution. Our structure reveals an intricate network of interactions between CDK7 and its two binding partners MAT1 and Cyclin H, providing a structural basis for the mechanism of CDK7 activation and CAK activity regulation. In vitro activity measurements and functional mutagenesis show that CDK7 activation can occur independent of T-loop phosphorylation and is thus exclusively MAT1-dependent by positioning the CDK7 T-loop in its active conformation.


Assuntos
Ciclina H , Quinases Ciclina-Dependentes , Ciclo Celular , Chaetomium/química , Chaetomium/enzimologia , Ciclina H/química , Ciclina H/metabolismo , Quinases Ciclina-Dependentes/química , Quinases Ciclina-Dependentes/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Fosforilação , Transcrição Genética
9.
RNA ; 26(10): 1448-1463, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32646969

RESUMO

RNA-binding proteins (RBPs) play important roles in bacterial gene expression and physiology but their true number and functional scope remain little understood even in model microbes. To advance global RBP discovery in bacteria, we here establish glycerol gradient sedimentation with RNase treatment and mass spectrometry (GradR). Applied to Salmonella enterica, GradR confirms many known RBPs such as CsrA, Hfq, and ProQ by their RNase-sensitive sedimentation profiles, and discovers the FopA protein as a new member of the emerging family of FinO/ProQ-like RBPs. FopA, encoded on resistance plasmid pCol1B9, primarily targets a small RNA associated with plasmid replication. The target suite of FopA dramatically differs from the related global RBP ProQ, revealing context-dependent selective RNA recognition by FinO-domain RBPs. Numerous other unexpected RNase-induced changes in gradient profiles suggest that cellular RNA helps to organize macromolecular complexes in bacteria. By enabling poly(A)-independent generic RBP discovery, GradR provides an important element in the quest to build a comprehensive catalog of microbial RBPs.


Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribonucleases/metabolismo , Poli A/metabolismo , Domínios Proteicos/fisiologia , RNA Bacteriano/metabolismo , Proteínas Repressoras/metabolismo , Salmonella enterica/metabolismo
10.
Nat Commun ; 11(1): 1667, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-32245994

RESUMO

The XPD helicase is a central component of the general transcription factor TFIIH which plays major roles in transcription and nucleotide excision repair (NER). Here we present the high-resolution crystal structure of the Arch domain of XPD with its interaction partner MAT1, a central component of the CDK activating kinase complex. The analysis of the interface led to the identification of amino acid residues that are crucial for the MAT1-XPD interaction. More importantly, mutagenesis of the Arch domain revealed that these residues are essential for the regulation of (i) NER activity by either impairing XPD helicase activity or the interaction of XPD with XPG; (ii) the phosphorylation of the RNA polymerase II and RNA synthesis. Our results reveal how MAT1 shields these functionally important residues thereby providing insights into how XPD is regulated by MAT1 and defining the Arch domain as a major mechanistic player within the XPD scaffold.


Assuntos
Proteínas de Ciclo Celular/ultraestrutura , Domínios Proteicos/fisiologia , Fatores de Transcrição/ultraestrutura , Proteína Grupo D do Xeroderma Pigmentoso/ultraestrutura , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cristalografia por Raios X , Reparo do DNA , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica/genética , RNA Polimerase II/metabolismo , Relação Estrutura-Atividade , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína Grupo D do Xeroderma Pigmentoso/genética , Proteína Grupo D do Xeroderma Pigmentoso/metabolismo
11.
Nat Commun ; 11(1): 1356, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170071

RESUMO

Nucleotide excision repair (NER) removes a wide range of DNA lesions, including UV-induced photoproducts and bulky base adducts. XPA is an essential protein in eukaryotic NER, although reports about its stoichiometry and role in damage recognition are controversial. Here, by PeakForce Tapping atomic force microscopy, we show that human XPA binds and bends DNA by ∼60° as a monomer. Furthermore, we observe XPA specificity for the helix-distorting base adduct N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene over non-damaged dsDNA. Moreover, single molecule fluorescence microscopy reveals that DNA-bound XPA exhibits multiple modes of linear diffusion between paused phases. The presence of DNA damage increases the frequency of pausing. Truncated XPA, lacking the intrinsically disordered N- and C-termini, loses specificity for DNA lesions and shows less pausing on damaged DNA. Our data are consistent with a working model in which monomeric XPA bends DNA, displays episodic phases of linear diffusion along DNA, and pauses in response to DNA damage.


Assuntos
DNA/química , DNA/metabolismo , Imagem Individual de Molécula/métodos , Proteína de Xeroderma Pigmentoso Grupo A/química , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Biofísica/métodos , Adutos de DNA/química , Adutos de DNA/metabolismo , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Humanos , Microscopia de Força Atômica , Ligação Proteica , Raios Ultravioleta
12.
Mol Cell ; 74(3): 421-435.e10, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30926243

RESUMO

Deubiquitinases have emerged as promising drug targets for cancer therapy. The two DUBs USP25 and USP28 share high similarity but vary in their cellular functions. USP28 is known for its tumor-promoting role, whereas USP25 is a regulator of the innate immune system and, recently, a role in tumorigenesis was proposed. We solved the structures of the catalytic domains of both proteins and established substantial differences in their activities. While USP28 is a constitutively active dimer, USP25 presents an auto-inhibited tetramer. Our data indicate that the activation of USP25 is not achieved through substrate or ubiquitin binding. USP25 cancer-associated mutations lead to activation in vitro and in vivo, thereby providing a functional link between auto-inhibition and the cancer-promoting role of the enzyme. Our work led to the identification of significant differences between USP25 and USP28 and provided the molecular basis for the development of new and highly specific anti-cancer drugs.


Assuntos
Carcinogênese/genética , Neoplasias/genética , Ubiquitina Tiolesterase/genética , Sequência de Aminoácidos/genética , Domínio Catalítico/genética , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/genética , Humanos , Mutação/genética , Neoplasias/tratamento farmacológico , Ligação Proteica/genética , Conformação Proteica , Multimerização Proteica/genética , Ubiquitina/genética , Ubiquitina Tiolesterase/química
13.
DNA Repair (Amst) ; 74: 17-25, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30641156

RESUMO

DNA replication fidelity maintains low mutation rates in bacteria. The ε-subunit of a replisome generally acts as the main proofreader during replication, using its 3'-5' exonuclease activity to excise misincorporated bases thereby maintaining faithful replication. In Mycobacterium tuberculosis (Mtb), however, the polymerase and histidinol phosphatase (PHP) domain of the DNA polymerase DnaE1 is the primary proofreader. This domain thus maintains low mutation rates during replication and is an attractive target for drug development. Even though the structures of DnaE polymerases are available from various organisms, including Mtb, the mechanism of exonuclease activity remains elusive. In this study, we sought to unravel the mechanism and also to identify scaffolds that can specifically inhibit the exonuclease activity. To gain insight into the mode of action, we also characterized the PHP domain of the Mtb error-prone polymerase DnaE2 which shares a nearly identical active site with DnaE1-PHP. Kinetic and mutational studies allowed us to identify the critical residue involved in catalysis. Combined inhibition and computational studies also revealed a specific mode of inhibition of DnaE1-PHP by nucleoside diphosphates. Thus, this study lays the foundation for the rational design of novel inhibitors which target the Mtb replicative proofreader.


Assuntos
DNA Polimerase III/antagonistas & inibidores , DNA Polimerase III/metabolismo , Replicação do DNA/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Exonucleases/metabolismo , Mycobacterium tuberculosis/enzimologia , Sequência de Aminoácidos , Domínio Catalítico , DNA Polimerase III/química , Desenho de Fármacos , Cinética , Modelos Moleculares , Mycobacterium tuberculosis/genética
14.
ChemMedChem ; 13(19): 2014-2023, 2018 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-30028574

RESUMO

Based on the similarity between the active sites of the deubiquitylating and deneddylating enzyme ChlaDub1 (Cdu1) and the evolutionarily related protease adenain, a target-hopping screening approach on a focused set of adenain inhibitors was investigated. The cyanopyrimidine-based inhibitors identified represent the first active-site-directed small-molecule inhibitors of Cdu1. High-resolution crystal structures of Cdu1 in complex with two covalently bound cyanopyrimidines, as well as with its substrate ubiquitin, were obtained. These structural data were complemented by enzymatic assays and covalent docking studies to provide insight into the substrate recognition of Cdu1, active-site pocket flexibility and potential hotspots for ligand interaction. Combined, these data provide a strong basis for future structure-guided medicinal chemistry optimization of this cyanopyrimidine scaffold into more potent and selective Cdu1 inhibitors.


Assuntos
Chlamydia trachomatis/enzimologia , Enzimas Desubiquitinantes/antagonistas & inibidores , Inibidores Enzimáticos/química , Proteínas Fúngicas/antagonistas & inibidores , Pirimidinas/química , Sequência de Aminoácidos , Domínio Catalítico , Chlamydia trachomatis/química , Cisteína Endopeptidases/química , Enzimas Desubiquitinantes/química , Proteínas Fúngicas/química , Humanos , Simulação de Acoplamento Molecular , Oligopeptídeos/química , Saccharomyces cerevisiae/enzimologia , Alinhamento de Sequência , Especificidade por Substrato
15.
J Med Chem ; 61(8): 3350-3369, 2018 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-29590750

RESUMO

Rhodesain (RD) is a parasitic, human cathepsin L (hCatL) like cysteine protease produced by Trypanosoma brucei ( T. b.) species and a potential drug target for the treatment of human African trypanosomiasis (HAT). A library of hCatL inhibitors was screened, and macrocyclic lactams were identified as potent RD inhibitors ( Ki < 10 nM), preventing the cell-growth of Trypanosoma brucei rhodesiense (IC50 < 400 nM). SARs addressing the S2 and S3 pockets of RD were established. Three cocrystal structures with RD revealed a noncovalent binding mode of this ligand class due to oxidation of the catalytic Cys25 to a sulfenic acid (Cys-SOH) during crystallization. The P-glycoprotein efflux ratio was measured and the in vivo brain penetration in rats determined. When tested in vivo in acute HAT model, the compounds permitted up to 16.25 (vs 13.0 for untreated controls) mean days of survival.


Assuntos
Catepsina L/antagonistas & inibidores , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Lactamas Macrocíclicas/farmacologia , Tripanossomicidas/farmacologia , Trypanosoma brucei rhodesiense/efeitos dos fármacos , Animais , Sítios de Ligação , Barreira Hematoencefálica/metabolismo , Linhagem Celular , Cisteína Endopeptidases/química , Inibidores de Cisteína Proteinase/síntese química , Inibidores de Cisteína Proteinase/química , Inibidores de Cisteína Proteinase/farmacocinética , Reposicionamento de Medicamentos , Humanos , Lactamas Macrocíclicas/síntese química , Lactamas Macrocíclicas/química , Lactamas Macrocíclicas/farmacocinética , Ligantes , Masculino , Camundongos Endogâmicos C57BL , Estrutura Molecular , Ratos , Relação Estrutura-Atividade , Suínos , Tripanossomicidas/síntese química , Tripanossomicidas/química , Tripanossomicidas/farmacocinética
16.
Structure ; 26(1): 137-144.e3, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29225079

RESUMO

Ctf18-RFC is an alternative PCNA loader which plays important but poorly understood roles in multiple DNA replication-associated processes. To fulfill its specialist roles, the Ctf18-RFC clamp loader contains a unique module in which the Dcc1-Ctf8 complex is bound to the C terminus of Ctf18 (the Ctf18-1-8 module). Here, we report the structural and functional characterization of the heterotetrameric complex formed between Ctf18-1-8 and a 63 kDa fragment of DNA polymerase ɛ. Our data reveal that Ctf18-1-8 binds stably to the polymerase and far from its other functional sites, suggesting that Ctf18-RFC could be associated with Pol ɛ throughout normal replication as the leading strand clamp loader. We also show that Pol ɛ and double-stranded DNA compete to bind the same winged-helix domain on Dcc1, with Pol ɛ being the preferred binding partner, thus suggesting that there are two alternative pathways to recruit Ctf18-RFC to sites of replication.


Assuntos
Proteínas Cromossômicas não Histona/química , DNA Polimerase II/química , Replicação do DNA , Proteínas de Ligação a DNA/química , DNA/química , Proteína de Replicação C/química , Proteínas de Saccharomyces cerevisiae/química , Sítios de Ligação , Ligação Competitiva , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Clonagem Molecular , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteína de Replicação C/genética , Proteína de Replicação C/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato
17.
Proc Natl Acad Sci U S A ; 114(51): 13453-13458, 2017 12 19.
Artigo em Inglês | MEDLINE | ID: mdl-29208709

RESUMO

Phosphorylation is a major regulator of protein interactions; however, the mechanisms by which regulation occurs are not well understood. Here we identify a salt-bridge competition or "theft" mechanism that enables a phospho-triggered swap of protein partners by Raf Kinase Inhibitory Protein (RKIP). RKIP transitions from inhibiting Raf-1 to inhibiting G-protein-coupled receptor kinase 2 upon phosphorylation, thereby bridging MAP kinase and G-Protein-Coupled Receptor signaling. NMR and crystallography indicate that a phosphoserine, but not a phosphomimetic, competes for a lysine from a preexisting salt bridge, initiating a partial unfolding event and promoting new protein interactions. Structural elements underlying the theft occurred early in evolution and are found in 10% of homo-oligomers and 30% of hetero-oligomers including Bax, Troponin C, and Early Endosome Antigen 1. In contrast to a direct recognition of phosphorylated residues by binding partners, the salt-bridge theft mechanism represents a facile strategy for promoting or disrupting protein interactions using solvent-accessible residues, and it can provide additional specificity at protein interfaces through local unfolding or conformational change.


Assuntos
Sequência Conservada , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Substituição de Aminoácidos , Animais , Evolução Molecular , Humanos , Lisina/genética , Lisina/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/química , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Fosforilação , Ligação Proteica , Serina/genética , Serina/metabolismo , Troponina C/química , Troponina C/genética , Troponina C/metabolismo , Proteínas de Transporte Vesicular/química , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genética , Proteína X Associada a bcl-2/metabolismo
18.
Nucleic Acids Res ; 45(18): 10872-10883, 2017 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-28977422

RESUMO

The general transcription factor IIH (TFIIH) is a multi-protein complex and its 10 subunits are engaged in an intricate protein-protein interaction network critical for the regulation of its transcription and DNA repair activities that are so far little understood on a molecular level. In this study, we focused on the p44 and the p34 subunits, which are central for the structural integrity of core-TFIIH. We solved crystal structures of a complex formed by the p34 N-terminal vWA and p44 C-terminal zinc binding domains from Chaetomium thermophilum and from Homo sapiens. Intriguingly, our functional analyses clearly revealed the presence of a second interface located in the C-terminal zinc binding region of p34, which can rescue a disrupted interaction between the p34 vWA and the p44 RING domain. In addition, we demonstrate that the C-terminal zinc binding domain of p34 assumes a central role with respect to the stability and function of TFIIH. Our data reveal a redundant interaction network within core-TFIIH, which may serve to minimize the susceptibility to mutational impairment. This provides first insights why so far no mutations in the p34 or p44 TFIIH-core subunits have been identified that would lead to the hallmark nucleotide excision repair syndromes xeroderma pigmentosum or trichothiodystrophy.


Assuntos
Fator de Transcrição TFIIH/química , Chaetomium/enzimologia , Proteínas Fúngicas/química , Humanos , Modelos Moleculares , Mutação , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/química , Fator de Transcrição TFIIH/genética
19.
Nat Microbiol ; 2(11): 1523-1532, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28894098

RESUMO

The most prominent defence of the unicellular parasite Trypanosoma brucei against the host immune system is a dense coat that comprises a variant surface glycoprotein (VSG). Despite the importance of the VSG family, no complete structure of a VSG has been reported. Making use of high-resolution structures of individual VSG domains, we employed small-angle X-ray scattering to elucidate the first two complete VSG structures. The resulting models imply that the linker regions confer great flexibility between domains, which suggests that VSGs can adopt two main conformations to respond to obstacles and changes of protein density, while maintaining a protective barrier at all times. Single-molecule diffusion measurements of VSG in supported lipid bilayers substantiate this possibility, as two freely diffusing populations could be detected. This translates into a highly flexible overall topology of the surface VSG coat, which displays both lateral movement in the plane of the membrane and variation in the overall thickness of the coat.


Assuntos
Trypanosoma brucei brucei/química , Glicoproteínas Variantes de Superfície de Trypanosoma/química , Cristalografia por Raios X , Modelos Moleculares , Conformação Proteica , Multimerização Proteica , Espalhamento a Baixo Ângulo , Trypanosoma brucei brucei/metabolismo , Glicoproteínas Variantes de Superfície de Trypanosoma/metabolismo
20.
ACS Infect Dis ; 3(9): 666-675, 2017 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-28786661

RESUMO

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), is a highly successful human pathogen and has infected approximately one-third of the world's population. Multiple drug resistant (MDR) and extensively drug resistant (XDR) TB strains and coinfection with HIV have increased the challenges of successfully treating this disease pandemic. The metabolism of host cholesterol by Mtb is an important factor for both its virulence and pathogenesis. In Mtb, the cholesterol side chain is degraded through multiple cycles of ß-oxidation and FadA5 (Rv3546) catalyzes side chain thiolysis in the first two cycles. Moreover, FadA5 is important during the chronic stage of infection in a mouse model of Mtb infection. Here, we report the redox control of FadA5 catalytic activity that results from reversible disulfide bond formation between Cys59-Cys91 and Cys93-Cys377. Cys93 is the thiolytic nucleophile, and Cys377 is the general acid catalyst for cleavage of the ß-keto-acyl-CoA substrate. The disulfide bond formed between the two catalytic residues Cys93 and Cys377 blocks catalysis. The formation of the disulfide bonds is accompanied by a large domain swap at the FadA5 dimer interface that serves to bring Cys93 and Cys377 in close proximity for disulfide bond formation. The catalytic activity of FadA5 has a midpoint potential of -220 mV, which is close to the Mtb mycothiol potential in the activated macrophage. The redox profile of FadA5 suggests that FadA5 is fully active when Mtb resides in the unactivated macrophage to maximize flux into cholesterol catabolism. Upon activation of the macrophage, the oxidative shift in the mycothiol potential will decrease the thiolytic activity by 50%. Thus, the FadA5 midpoint potential is poised to rapidly restrict cholesterol side chain degradation in response to oxidative stress from the host macrophage environment.


Assuntos
Acetil-CoA C-Acetiltransferase/química , Acetil-CoA C-Acetiltransferase/metabolismo , Proteínas de Bactérias/química , Colesterol/metabolismo , Mycobacterium tuberculosis/patogenicidade , Compostos de Sulfidrila/metabolismo , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Dicroísmo Circular , Cisteína/metabolismo , Humanos , Ativação de Macrófagos , Modelos Moleculares , Oxirredução , Conformação Proteica
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