RESUMO
Cryptophycins are microtubule-targeting agents (MTAs) that belong to the most potent antimitotic compounds known to date; however, their exact molecular mechanism of action remains unclear. Here, we present the 2.2 Å resolution X-ray crystal structure of a potent cryptophycin derivative bound to the αß-tubulin heterodimer. The structure addresses conformational issues present in a previous 3.3 Å resolution cryo-electron microscopy structure of cryptophycin-52 bound to the maytansine site of ß-tubulin. It further provides atomic details on interactions of cryptophycins, which had not been described previously, including ones that are in line with structure-activity relationship studies. Interestingly, we discovered a second cryptophycin-binding site that involves the T5-loop of ß-tubulin, a critical secondary structure element involved in the exchange of the guanosine nucleotide and in the formation of longitudinal tubulin contacts in microtubules. Cryptophycins are the first natural ligands found to bind to this new "ßT5-loop site" that bridges the maytansine and vinca sites. Our results offer unique avenues to rationally design novel MTAs with the capacity to modulate T5-loop dynamics and to simultaneously engage multiple ß-tubulin binding sites.
Assuntos
Maitansina , Tubulina (Proteína) , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Maitansina/química , Maitansina/análogos & derivados , Humanos , Cristalografia por Raios X , Sítios de Ligação , Microtúbulos/metabolismo , Microtúbulos/química , Alcaloides de Vinca/química , Alcaloides de Vinca/metabolismoRESUMO
The C-type lectin receptor DC-SIGN mediates interactions with envelope glycoproteins of many viruses such as SARS-CoV-2, ebola, and HIV and contributes to virus internalization and dissemination. In the context of the recent SARS-CoV-2 pandemic, involvement of DC-SIGN has been linked to severe cases of COVID-19. Inhibition of the interaction between DC-SIGN and viral glycoproteins has the potential to generate broad spectrum antiviral agents. Here, we demonstrate that mannose-functionalized poly-l-lysine glycoconjugates efficiently inhibit the attachment of viral glycoproteins to DC-SIGN-presenting cells with picomolar affinity. Treatment of these cells leads to prolonged receptor internalization and inhibition of virus binding for up to 6â h. Furthermore, the polymers are fully bio-compatible and readily cleared by target cells. The thermodynamic analysis of the multivalent interactions reveals enhanced enthalpy-driven affinities and promising perspectives for the future development of multivalent therapeutics.
Assuntos
Antivirais/farmacologia , Moléculas de Adesão Celular/antagonistas & inibidores , Glicoconjugados/farmacologia , Lectinas Tipo C/antagonistas & inibidores , Receptores de Superfície Celular/antagonistas & inibidores , Ligação Viral/efeitos dos fármacos , Antivirais/síntese química , Antivirais/metabolismo , Moléculas de Adesão Celular/metabolismo , Glicoconjugados/síntese química , Glicoconjugados/metabolismo , Humanos , Lectinas Tipo C/metabolismo , Manose/análogos & derivados , Manose/metabolismo , Manose/farmacologia , Testes de Sensibilidade Microbiana , Polilisina/análogos & derivados , Polilisina/metabolismo , Polilisina/farmacologia , Ligação Proteica/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo , SARS-CoV-2/efeitos dos fármacos , Células THP-1 , Termodinâmica , Proteínas do Envelope Viral/antagonistas & inibidores , Proteínas do Envelope Viral/metabolismoRESUMO
BRCA2 controls RAD51 recombinase during homologous DNA recombination (HDR) through eight evolutionarily conserved BRC repeats, which individually engage RAD51 via the motif Phe-x-x-Ala. Using structure-guided molecular design, templated on a monomeric thermostable chimera between human RAD51 and archaeal RadA, we identify CAM833, a 529 Da orthosteric inhibitor of RAD51:BRC with a Kd of 366 nM. The quinoline of CAM833 occupies a hotspot, the Phe-binding pocket on RAD51 and the methyl of the substituted α-methylbenzyl group occupies the Ala-binding pocket. In cells, CAM833 diminishes formation of damage-induced RAD51 nuclear foci; inhibits RAD51 molecular clustering, suppressing extended RAD51 filament assembly; potentiates cytotoxicity by ionizing radiation, augmenting 4N cell-cycle arrest and apoptotic cell death and works with poly-ADP ribose polymerase (PARP)1 inhibitors to suppress growth in BRCA2-wildtype cells. Thus, chemical inhibition of the protein-protein interaction between BRCA2 and RAD51 disrupts HDR and potentiates DNA damage-induced cell death, with implications for cancer therapy.
Assuntos
Proteína BRCA2/antagonistas & inibidores , Rad51 Recombinase/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Proteína BRCA2/química , Proteína BRCA2/metabolismo , Morte Celular/efeitos dos fármacos , Cristalografia por Raios X , Dano ao DNA , Humanos , Modelos Moleculares , Conformação Molecular , Ligação Proteica/efeitos dos fármacos , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Células Tumorais CultivadasRESUMO
The import of nonnatural molecules is a recurring problem in fundamental and applied aspects of microbiology. The dipeptide permease (Dpp) of Escherichia coli is an ABC-type multicomponent transporter system located in the cytoplasmic membrane, which is capable of transporting a wide range of di- and tripeptides with structurally and chemically diverse amino acid side chains into the cell. Given this low degree of specificity, Dpp was previously used as an entry gate to deliver natural and nonnatural cargo molecules into the cell by attaching them to amino acid side chains of peptides, in particular, the γ-carboxyl group of glutamate residues. However, the binding affinity of the substrate-binding protein dipeptide permease A (DppA), which is responsible for the initial binding of peptides in the periplasmic space, is significantly higher for peptides consisting of standard amino acids than for peptides containing side-chain modifications. Here, we used adaptive laboratory evolution to identify strains that utilize dipeptides containing γ-substituted glutamate residues more efficiently and linked this phenotype to different mutations in DppA. In vitro characterization of these mutants by thermal denaturation midpoint shift assays and isothermal titration calorimetry revealed significantly higher binding affinities of these variants toward peptides containing γ-glutamyl amides, presumably resulting in improved uptake and therefore faster growth in media supplemented with these nonstandard peptides.IMPORTANCE Fundamental and synthetic biology frequently suffer from insufficient delivery of unnatural building blocks or substrates for metabolic pathways into bacterial cells. The use of peptide-based transport vectors represents an established strategy to enable the uptake of such molecules as a cargo. We expand the scope of peptide-based uptake and characterize in detail the obtained DppA mutant variants. Furthermore, we highlight the potential of adaptive laboratory evolution to identify beneficial insertion mutations that are unlikely to be identified with existing directed evolution strategies.
Assuntos
Proteínas de Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Mutação , Peptídeos/metabolismo , Proteínas Periplásmicas de Ligação/genética , Proteínas Periplásmicas de Ligação/metabolismo , Amidas/metabolismo , Proteínas de Bactérias/genética , Transporte Biológico , Dipeptídeos , Escherichia coli/enzimologia , Proteínas de Escherichia coli/metabolismo , Ácido Glutâmico/metabolismo , Glutationa/metabolismo , Cinética , Proteínas de Membrana Transportadoras/genética , Redes e Vias Metabólicas , Especificidade por Substrato , gama-Glutamiltransferase/genética , gama-Glutamiltransferase/metabolismoRESUMO
CDK16 (also known as PCTAIRE1 or PCTK1) is an atypical member of the cyclin-dependent kinase (CDK) family that has emerged as a key regulator of neurite outgrowth, vesicle trafficking and cancer cell proliferation. CDK16 is activated through binding to cyclin Y via a phosphorylation-dependent 14-3-3 interaction and has a unique consensus substrate phosphorylation motif compared with conventional CDKs. To elucidate the structure and inhibitor-binding properties of this atypical CDK, we screened the CDK16 kinase domain against different inhibitor libraries and determined the co-structures of identified hits. We discovered that the ATP-binding pocket of CDK16 can accommodate both type I and type II kinase inhibitors. The most potent CDK16 inhibitors revealed by cell-free and cell-based assays were the multitargeted cancer drugs dabrafenib and rebastinib. An inactive DFG-out binding conformation was confirmed by the first crystal structures of CDK16 in separate complexes with the inhibitors indirubin E804 and rebastinib, respectively. The structures revealed considerable conformational plasticity, suggesting that the isolated CDK16 kinase domain was relatively unstable in the absence of a cyclin partner. The unusual structural features and chemical scaffolds identified here hold promise for the development of more selective CDK16 inhibitors and provide opportunity to better characterise the role of CDK16 and its related CDK family members in various physiological and pathological contexts.
Assuntos
Trifosfato de Adenosina/química , Antineoplásicos/química , Quinases Ciclina-Dependentes/química , Imidazóis/química , Oximas/química , Inibidores de Proteínas Quinases/química , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Proteínas 14-3-3/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Clonagem Molecular , Cristalografia por Raios X , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/química , Ciclinas/genética , Ciclinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Indóis/química , Cinética , Ligantes , Fosforilação , Ligação Proteica , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
We report that the anticancer activity of the widely used diabetic drug metformin is strongly potentiated by syrosingopine. Synthetic lethality elicited by combining the two drugs is synergistic and specific to transformed cells. This effect is unrelated to syrosingopine's known role as an inhibitor of the vesicular monoamine transporters. Syrosingopine binds to the glycolytic enzyme α-enolase in vitro, and the expression of the γ-enolase isoform correlates with nonresponsiveness to the drug combination. Syrosingopine sensitized cancer cells to metformin and its more potent derivative phenformin far below the individual toxic threshold of each compound. Thus, combining syrosingopine and codrugs is a promising therapeutic strategy for clinical application for the treatment of cancer.
Assuntos
Metformina/farmacologia , Reserpina/análogos & derivados , Animais , Linhagem Celular Tumoral , Sinergismo Farmacológico , Glicólise , Humanos , Camundongos , Camundongos Knockout , Fenformin/farmacologia , Fosfopiruvato Hidratase/química , Reserpina/farmacologiaRESUMO
Protein-protein interactions (PPIs) are increasingly important targets for drug discovery. Efficient fragment-based drug discovery approaches to tackle PPIs are often stymied by difficulties in the production of stable, unliganded target proteins. Here, we report an approach that exploits protein engineering to "humanise" thermophilic archeal surrogate proteins as targets for small-molecule inhibitor discovery and to exemplify this approach in the development of inhibitors against the PPI between the recombinase RAD51 and tumour suppressor BRCA2. As human RAD51 has proved impossible to produce in a form that is compatible with the requirements of fragment-based drug discovery, we have developed a surrogate protein system using RadA from Pyrococcus furiosus. Using a monomerised RadA as our starting point, we have adopted two parallel and mutually instructive approaches to mimic the human enzyme: firstly by mutating RadA to increase sequence identity with RAD51 in the BRC repeat binding sites, and secondly by generating a chimeric archaeal human protein. Both approaches generate proteins that interact with a fourth BRC repeat with affinity and stoichiometry comparable to human RAD51. Stepwise humanisation has also allowed us to elucidate the determinants of RAD51 binding to BRC repeats and the contributions of key interacting residues to this interaction. These surrogate proteins have enabled the development of biochemical and biophysical assays in our ongoing fragment-based small-molecule inhibitor programme and they have allowed us to determine hundreds of liganded structures in support of our structure-guided design process, demonstrating the feasibility and advantages of using archeal surrogates to overcome difficulties in handling human proteins.
Assuntos
Proteína BRCA2/metabolismo , Inibidores Enzimáticos/isolamento & purificação , Ligação Proteica/efeitos dos fármacos , Engenharia de Proteínas/métodos , Rad51 Recombinase/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Descoberta de Drogas/métodos , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Pyrococcus/enzimologia , Rad51 Recombinase/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Filamentation induced by cyclic AMP (FIC)-domain enzymes catalyze adenylylation or other posttranslational modifications of target proteins to control their function. Recently, we have shown that Fic enzymes are autoinhibited by an α-helix (αinh) that partly obstructs the active site. For the single-domain class III Fic proteins, the αinh is located at the C terminus and its deletion relieves autoinhibition. However, it has remained unclear how activation occurs naturally. Here, we show by structural, biophysical, and enzymatic analyses combined with in vivo data that the class III Fic protein NmFic from Neisseria meningitidis gets autoadenylylated in cis, thereby autonomously relieving autoinhibition and thus allowing subsequent adenylylation of its target, the DNA gyrase subunit GyrB. Furthermore, we show that NmFic activation is antagonized by tetramerization. The combination of autoadenylylation and tetramerization results in nonmonotonic concentration dependence of NmFic activity and a pronounced lag phase in the progress of target adenylylation. Bioinformatic analyses indicate that this elaborate dual-control mechanism is conserved throughout class III Fic proteins.
Assuntos
Proteínas de Bactérias/metabolismo , Biopolímeros/metabolismo , AMP Cíclico/metabolismo , Neisseria meningitidis/enzimologia , Nucleotidiltransferases/metabolismo , DNA Girase/metabolismo , Modelos MolecularesRESUMO
Molecular chaperones are essential in aiding client proteins to fold into their native structure and in maintaining cellular protein homeostasis. However, mechanistic aspects of chaperone function are still not well understood at the atomic level. We use nuclear magnetic resonance spectroscopy to elucidate the mechanism underlying client recognition by the adenosine triphosphate-independent chaperone Spy at the atomic level and derive a structural model for the chaperone-client complex. Spy interacts with its partially folded client Im7 by selective recognition of flexible, locally frustrated regions in a dynamic fashion. The interaction with Spy destabilizes a partially folded client but spatially compacts an unfolded client conformational ensemble. By increasing client backbone dynamics, the chaperone facilitates the search for the native structure. A comparison of the interaction of Im7 with two other chaperones suggests that the underlying principle of recognizing frustrated segments is of a fundamental nature.
Assuntos
Proteínas de Transporte/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Chaperonas Moleculares/química , Proteínas Periplásmicas/química , Ressonância Magnética Nuclear Biomolecular , Dobramento de ProteínaRESUMO
Activation of p53 target genes for tumor suppression depends on the stress-specific regulation of transcriptional coactivator complexes. Strap (stress-responsive activator of p300) is activated upon DNA damage by ataxia telangiectasia mutated (ATM) and Chk2 kinases and is a key regulator of the p53 response. In addition to antagonizing Mdm2, Strap facilitates the recruitment of p53 coactivators, including JMY and p300. Strap is a predicted TPR-repeat protein, but shows only limited sequence identity with any protein of known structure. To address this and to elucidate the molecular mechanism of Strap activity we determined the crystal structure of the full-length protein at 2.05 Å resolution. The structure of Strap reveals an atypical six tetratricopeptide repeat (TPR) protein that also contains an unexpected oligonucleotide/oligosaccharide-binding (OB)-fold domain. This previously unseen domain organization provides an extended superhelical scaffold allowing for protein-protein as well as protein-DNA interaction. We show that both of the TPR and OB-fold domains localize to the chromatin of p53 target genes and exhibit intrinsic regulatory activity necessary for the Strap-dependent p53 response.
Assuntos
Proteínas de Transporte/química , Cromatina/química , Genes p53 , Proteínas de Neoplasias/química , Oligonucleotídeos/química , Proteína Supressora de Tumor p53/química , Motivos de Aminoácidos , Animais , Proteínas de Transporte/metabolismo , Cristalografia por Raios X/métodos , Dano ao DNA , Proteína p300 Associada a E1A/metabolismo , Humanos , Camundongos , Modelos Moleculares , Proteínas de Neoplasias/metabolismo , Ligação Proteica , Conformação Proteica , Dobramento de Proteína , Estrutura Terciária de Proteína , Proteínas de Ligação a RNARESUMO
Classical protein folding invokes a cooperative transition between distinct thermodynamic states that are individually populated at equilibrium and separated by an energy barrier. It has been proposed, however, that the small protein, BBL, undergoes one-step downhill folding whereby it folds non-cooperatively to its native state without encountering an appreciable energy barrier. Only a single conformational ensemble is populated under given conditions, and so the denatured state ensemble progressively changes into the native structure. A wide dispersion of thermal denaturation midpoints that was observed for an extrinsically labelled fragment of BBL is proposed to be evidence for its one-state, downhill folding, a phenomenon that is also suggested to be functionally important for BBL and its homologues. We found, however, that thermal denaturation of unlabelled wild-type BBL was highly cooperative, with very similar transition midpoints for the melting of secondary and tertiary interactions, as well as for individual residues when monitored by NMR. Similar results were also observed for two other homologues, E3BD and POB. Further, the extrinsic fluorophores perturbed the unfolding energetics of labelled BBL, and complicated its equilibrium behaviour. One-step downhill folding may well occur for some proteins that do not have distinct folded states but not for BBL and its well-folded homologues.