RESUMO
Photoaffinity labelling is a useful method for studying how proteins interact with ligands and biomolecules, and can help identify and characterise new targets for the development of new therapeutics. We present the design and synthesis of a novel multifunctional benzophenone linker that serves as both a photo-crosslinking motif and a peptide stapling reagent. Using double-click stapling, we attached the benzophenone to the peptide via the staple linker, rather than by modifying the peptide sequence with a photo-crosslinking amino acid. When applied to a p53-derived peptide, the resulting photoreactive stapled peptide was able to preferentially crosslink with MDM2 in the presence of competing protein. This multifunctional linker also features an extra alkyne handle for downstream applications such as pull-down assays, and can be used to investigate the target selectivity of stapled peptides.
Assuntos
Benzofenonas/química , Reagentes de Ligações Cruzadas/química , Peptídeos/química , Marcadores de Fotoafinidade , Proteínas Proto-Oncogênicas c-mdm2/química , Benzofenonas/síntese química , Química Click , Reagentes de Ligações Cruzadas/síntese química , Ligantes , Estrutura MolecularRESUMO
Glutamate-gated ion channels (ionotropic glutamate receptors, iGluRs) sense the extracellular milieu via an extensive extracellular portion, comprised of two clamshell-shaped segments. The distal, N-terminal domain (NTD) has allosteric potential in NMDA-type iGluRs, which has not been ascribed to the analogous domain in AMPA receptors (AMPARs). In this study, we present new structural data uncovering dynamic properties of the GluA2 and GluA3 AMPAR NTDs. GluA3 features a zipped-open dimer interface with unconstrained lower clamshell lobes, reminiscent of metabotropic GluRs (mGluRs). The resulting labile interface supports interprotomer rotations, which can be transmitted to downstream receptor segments. Normal mode analysis reveals two dominant mechanisms of AMPAR NTD motion: intraprotomer clamshell motions and interprotomer counter-rotations, as well as accessible interconversion between AMPAR and mGluR conformations. In addition, we detect electron density for a potential ligand in the GluA2 interlobe cleft, which may trigger lobe motions. Together, these data support a dynamic role for the AMPAR NTDs, which widens the allosteric landscape of the receptor and could provide a novel target for ligand development.
Assuntos
Regulação Alostérica , Cálcio/metabolismo , Membrana Celular/metabolismo , Receptores de AMPA/química , Receptores de AMPA/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Eletrofisiologia , Humanos , Canais Iônicos , Dados de Sequência Molecular , Conformação Proteica , Multimerização Proteica , Subunidades Proteicas , Transporte Proteico , Homologia de Sequência de Aminoácidos , UltracentrifugaçãoRESUMO
The assembly of AMPA-type glutamate receptors (AMPARs) into distinct ion channel tetramers ultimately governs the nature of information transfer at excitatory synapses. How cells regulate the formation of diverse homo- and heteromeric AMPARs is unknown. Using a sensitive biophysical approach, we show that the extracellular, membrane-distal AMPAR N-terminal domains (NTDs) orchestrate selective routes of heteromeric assembly via a surprisingly wide spectrum of subunit-specific association affinities. Heteromerization is dominant, occurs at the level of the dimer, and results in a preferential incorporation of the functionally critical GluA2 subunit. Using a combination of structure-guided mutagenesis and electrophysiology, we further map evolutionarily variable hotspots in the NTD dimer interface, which modulate heteromerization capacity. This 'flexibility' of the NTD not only explains why heteromers predominate but also how GluA2-lacking, Ca(2+)-permeable homomers could form, which are induced under specific physiological and pathological conditions. Our findings reveal that distinct NTD properties set the stage for the biogenesis of functionally diverse pools of homo- and heteromeric AMPAR tetramers.
Assuntos
Cálcio/metabolismo , Membrana Celular/metabolismo , Receptores de AMPA/química , Receptores de AMPA/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/metabolismo , Cristalografia por Raios X , Eletrofisiologia , Humanos , Canais Iônicos , Conformação Proteica , Multimerização Proteica , Subunidades Proteicas , Transporte Proteico , Sinapses , UltracentrifugaçãoRESUMO
Peptide stapling is a method for designing macrocyclic alpha-helical inhibitors of protein-protein interactions. However, obtaining a cell-active inhibitor can require significant optimization. We report a novel stapling technique based on a double strain-promoted azide-alkyne reaction, and exploit its biocompatibility to accelerate the discovery of cell-active stapled peptides. As a proof of concept, MDM2-binding peptides were stapled in parallel, directly in cell culture medium in 96-well plates, and simultaneously evaluated in a p53 reporter assay. This inâ situ stapling/screening process gave an optimal candidate that showed improved proteolytic stability and nanomolar binding to MDM2 in subsequent biophysical assays. α-Helicity was confirmed by a crystal structure of the MDM2-peptide complex. This work introduces inâ situ stapling as a versatile biocompatible technique with many other potential high-throughput biological applications.
Assuntos
Compostos Macrocíclicos/química , Peptídeos/química , Meios de Cultura , Ligação Proteica , Proteínas Proto-Oncogênicas c-mdm2/químicaRESUMO
Indirect somatic genetic rescue (SGR) of a germline mutation is thought to be rare in inherited Mendelian disorders. Here, we establish that acquired mutations in the EIF6 gene are a frequent mechanism of SGR in Shwachman-Diamond syndrome (SDS), a leukemia predisposition disorder caused by a germline defect in ribosome assembly. Biallelic mutations in the SBDS or EFL1 genes in SDS impair release of the anti-association factor eIF6 from the 60S ribosomal subunit, a key step in the translational activation of ribosomes. Here, we identify diverse mosaic somatic genetic events (point mutations, interstitial deletion, reciprocal chromosomal translocation) in SDS hematopoietic cells that reduce eIF6 expression or disrupt its interaction with the 60S subunit, thereby conferring a selective advantage over non-modified cells. SDS-related somatic EIF6 missense mutations that reduce eIF6 dosage or eIF6 binding to the 60S subunit suppress the defects in ribosome assembly and protein synthesis across multiple SBDS-deficient species including yeast, Dictyostelium and Drosophila. Our data suggest that SGR is a universal phenomenon that may influence the clinical evolution of diverse Mendelian disorders and support eIF6 suppressor mimics as a therapeutic strategy in SDS.
Assuntos
Mutação , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Ribossomos/genética , Ribossomos/patologia , Síndrome de Shwachman-Diamond/genética , Síndrome de Shwachman-Diamond/patologia , Adolescente , Adulto , Animais , Fenômenos Biológicos , Células Cultivadas , Criança , Pré-Escolar , Dictyostelium , Drosophila , Fatores de Iniciação em Eucariotos/genética , Fatores de Iniciação em Eucariotos/metabolismo , Células Germinativas , Humanos , Lactente , Simulação de Dinâmica Molecular , Fatores de Alongamento de Peptídeos/genética , Fatores de Alongamento de Peptídeos/metabolismo , Ligação Proteica , Biossíntese de Proteínas , Proteínas/genética , Proteínas/metabolismo , Ribonucleoproteína Nuclear Pequena U5/genética , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae , Homologia de Sequência de Aminoácidos , Síndrome de Shwachman-Diamond/metabolismo , Adulto JovemRESUMO
Human saposins are essential proteins required for degradation of sphingolipids and lipid antigen presentation. Despite the conserved structural organization of saposins, their distinct modes of interaction with biological membranes are not fully understood. We describe two crystal structures of human saposin C in an "open" configuration with unusual domain swapped homodimers. This form of SapC dimer supports the "clip-on" model for SapC-induced vesicle fusion. In addition, we present the crystal structure of SapD in two crystal forms. They reveal the monomer-monomer interface for the SapD dimer, which was confirmed in solution by analytical ultracentrifugation. The crystal structure of SapD suggests that side chains of Lys10 and Arg17 are involved in initial association with the preferred anionic biological membranes by forming salt bridges with sulfate or phosphate lipid headgroups.
Assuntos
Saposinas/química , Sequência de Aminoácidos , Membrana Celular/metabolismo , Clonagem Molecular , Sequência Conservada , Cristalografia por Raios X , Cisteína/química , Dimerização , Humanos , Interações Hidrofóbicas e Hidrofílicas , Metabolismo dos Lipídeos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese , Pichia/genética , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Saposinas/genética , Saposinas/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
The human polo-like kinase PLK1 coordinates mitotic chromosome segregation by phosphorylating multiple chromatin- and kinetochore-binding proteins. How PLK1 activity is directed to specific substrates via phosphopeptide recognition by its carboxyl-terminal polo-box domain (PBD) is poorly understood. Here, we combine molecular, structural and chemical biology to identify a determinant for PLK1 substrate recognition that is essential for proper chromosome segregation. We show that mutations ablating an evolutionarily conserved, Tyr-lined pocket in human PLK1 PBD trigger cellular anomalies in mitotic progression and timing. Tyr pocket mutations selectively impair PLK1 binding to the kinetochore phosphoprotein substrate PBIP1, but not to the centrosomal substrate NEDD1. Through a structure-guided approach, we develop a small-molecule inhibitor, Polotyrin, which occupies the Tyr pocket. Polotyrin recapitulates the mitotic defects caused by mutations in the Tyr pocket, further evidencing its essential function, and exemplifying a new approach for selective PLK1 inhibition. Thus, our findings support a model wherein substrate discrimination via the Tyr pocket in the human PLK1 PBD regulates mitotic chromosome segregation to preserve genome integrity.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Segregação de Cromossomos , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Regiões 3' não Traduzidas , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Sobrevivência Celular , Células HeLa , Histonas/metabolismo , Humanos , Cinetocoros/metabolismo , Mitose/efeitos dos fármacos , Mutagênese , Ligação Proteica , Domínios Proteicos , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Especificidade por Substrato , Quinase 1 Polo-LikeRESUMO
CK2 is a critical cell cycle regulator that also promotes various anti-apoptotic mechanisms. Development of ATP-non-competitive inhibitors of CK2 is a very attractive strategy considering that the ATP binding site is highly conserved among other kinases. We have previously utilised a pocket outside the active site to develop a novel CK2 inhibitor, CAM4066. Whilst CAM4066 bound to this new pocket it was also interacting with the ATP site: herein, we describe an example of a CK2α inhibitor that binds completely outside the active site. This second generation αD-site binding inhibitor, compound CAM4712 (IC50 = 7 µM, GI50 = 10.0 ± 3.6 µM), has numerous advantages over the previously reported CAM4066, including a reduction in the number of rotatable bonds, the absence of amide groups susceptible to the action of proteases and improved cellular permeability. Unlike with CAM4066, there was no need to facilitate cellular uptake by making a prodrug. Moreover, CAM4712 displayed no drop off between its ability to inhibit the kinase in vitro (IC50) and the ability to inhibit cell proliferation (GI50).
RESUMO
Protein-protein interactions (PPIs) determine a wide range of biological processes and analysis of these dynamic networks is increasingly becoming a mandatory tool for studying protein function. Using the globular ATPase domain of recombinase RadA as a scaffold, we have developed a peptide display system (RAD display), which allows for the presentation of target peptides, protein domains or full-length proteins and their rapid recombinant production in bacteria. The design of the RAD display system includes differently tagged versions of the scaffold, which allows for flexibility in the protein purification method, and chemical coupling for small molecule labeling or surface immobilization. When combined with the significant thermal stability of the RadA protein, these features create a versatile multipurpose scaffold system. Using various orthogonal biophysical techniques, we show that peptides displayed on the scaffold bind to their natural targets in a fashion similar to linear parent peptides. We use the examples of CK2ß/CK2α kinase and TPX2/Aurora A kinase protein complexes to demonstrate that the peptide displayed by the RAD scaffold can be used in PPI studies with the same binding efficacy but at lower costs compared with their linear synthetic counterparts.
Assuntos
Peptídeos/química , Peptídeos/genética , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Peptídeos/metabolismo , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Free energy perturbation theory, in combination with enhanced sampling of protein-ligand binding modes, is evaluated in the context of fragment-based drug design, and used to design two new small-molecule inhibitors of the Aurora A kinase-TPX2 protein-protein interaction.
Assuntos
Aurora Quinase A/antagonistas & inibidores , Proteínas de Ciclo Celular/antagonistas & inibidores , Proteínas Associadas aos Microtúbulos/antagonistas & inibidores , Simulação de Dinâmica Molecular , Proteínas Nucleares/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Aurora Quinase A/química , Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Estrutura Molecular , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/química , Bibliotecas de Moléculas Pequenas/químicaRESUMO
The essential mitotic kinase Aurora A (AURKA) is controlled during cell cycle progression via two distinct mechanisms. Following activation loop autophosphorylation early in mitosis when it localizes to centrosomes, AURKA is allosterically activated on the mitotic spindle via binding to the microtubule-associated protein, TPX2. Here, we report the discovery of AurkinA, a novel chemical inhibitor of the AURKA-TPX2 interaction, which acts via an unexpected structural mechanism to inhibit AURKA activity and mitotic localization. In crystal structures, AurkinA binds to a hydrophobic pocket (the 'Y pocket') that normally accommodates a conserved Tyr-Ser-Tyr motif from TPX2, blocking the AURKA-TPX2 interaction. AurkinA binding to the Y- pocket induces structural changes in AURKA that inhibit catalytic activity in vitro and in cells, without affecting ATP binding to the active site, defining a novel mechanism of allosteric inhibition. Consistent with this mechanism, cells exposed to AurkinA mislocalise AURKA from mitotic spindle microtubules. Thus, our findings provide fresh insight into the catalytic mechanism of AURKA, and identify a key structural feature as the target for a new class of dual-mode AURKA inhibitors, with implications for the chemical biology and selective therapeutic targeting of structurally related kinases.
Assuntos
Aurora Quinase A/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Proteínas Quinases/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Linhagem Celular Tumoral , Células HeLa , Humanos , Mitose/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Fosfoproteínas/metabolismo , Ligação Proteica/efeitos dos fármacos , Fuso Acromático/efeitos dos fármacosRESUMO
Ionotropic glutamate receptor (iGluR) family members are integrated into supramolecular complexes that modulate their location and function at excitatory synapses. However, a lack of structural information beyond isolated receptors or fragments thereof currently limits the mechanistic understanding of physiological iGluR signaling. Here, we report structural and functional analyses of the prototypical molecular bridge linking postsynaptic iGluR δ2 (GluD2) and presynaptic ß-neurexin 1 (ß-NRX1) via Cbln1, a C1q-like synaptic organizer. We show how Cbln1 hexamers "anchor" GluD2 amino-terminal domain dimers to monomeric ß-NRX1. This arrangement promotes synaptogenesis and is essential for D: -serine-dependent GluD2 signaling in vivo, which underlies long-term depression of cerebellar parallel fiber-Purkinje cell (PF-PC) synapses and motor coordination in developing mice. These results lead to a model where protein and small-molecule ligands synergistically control synaptic iGluR function.
Assuntos
Depressão Sináptica de Longo Prazo , Proteínas do Tecido Nervoso/química , Neurogênese , Precursores de Proteínas/química , Receptores de Glutamato/química , Sinapses/fisiologia , Animais , Ligantes , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Multimerização Proteica , Precursores de Proteínas/metabolismo , Estrutura Terciária de Proteína , Células de Purkinje/metabolismo , Células de Purkinje/fisiologia , Receptores de Glutamato/metabolismo , Transdução de Sinais , Sinapses/metabolismoRESUMO
Protein-protein interactions (PPI) have become increasingly popular drug targets, with a number of promising compounds currently in clinical trials. Recent research shows, that PPIs can be modulated in more ways than direct inhibition, where novel non-competitive modes of action promise a solution for the difficult nature of PPI drug discovery. Here, we review recently discovered PPI modulators in light of their mode of action and categorise them as disrupting versus stabilising, orthosteric versus allosteric and by their ability to affect the proteins' dynamics. We also give recent examples of compounds successful in the clinic, analyse their physicochemical properties and discuss how to overcome the hurdles in discovering alternative modes of modulation.
Assuntos
Descoberta de Drogas , Regulação Alostérica , Animais , Sistemas de Liberação de Medicamentos , Humanos , Ligação Proteica , Bibliotecas de Moléculas Pequenas/metabolismoRESUMO
The manganese-stabilizing protein PsbO is associated with the luminal side of thylakoids close to the redox-active Mn(4)Ca cluster at the catalytically active site of photosystem II (PSII). PsbO is believed to increase the efficiency of oxygen evolution and to stabilize the Mn(4)Ca cluster against photoinhibition. Using small-angle X-ray scattering, we investigated the low-resolution structure of wild-type spinach PsbO and that of chimeric spinach PsbO fused with maltose-binding protein. Small-angle X-ray scattering data revealed that both proteins are monomeric in solution, and that plant and cyanobacterial PsbO have similar structures. We show a highly efficient expression system that allows recombinant production of the active wild type and the chimeric PsbO from spinach and cyanobacteria, with yields compatible with biophysical and structural studies. The binding of spinach PsbO fused with maltose-binding protein to PSII depleted of extrinsic subunits (PSII-ΔpsbO,P,Q) was confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The reconstituted PSII was shown to have similar oxygen evolution rates as obtained with wild-type spinach PsbO.
Assuntos
Cianobactérias/metabolismo , Proteínas Ligantes de Maltose/metabolismo , Complexo de Proteína do Fotossistema II/química , Proteínas de Plantas/química , Spinacia oleracea/metabolismo , Western Blotting , Domínio Catalítico , Dicroísmo Circular , Cianobactérias/crescimento & desenvolvimento , Manganês/metabolismo , Modelos Moleculares , Oxigênio/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas de Plantas/metabolismo , Ligação Proteica , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrofotometria Ultravioleta , Spinacia oleracea/crescimento & desenvolvimentoRESUMO
Inflammatory processes are accompanied by the posttranslational modification of certain arginine residues within proteins to yield citrulline, although it is largely unknown how this modification influences antigen presentation. We employed crystallographic and functional studies to investigate whether the exchange of arginine to citrulline affects the display of a peptide by two human major histocompatibility antigen class I subtypes, HLA-B(*)2705 and HLA-B(*)2709. Both differ only in residue 116 within the peptide binding groove despite their differential association with ankylosing spondylitis, an inflammatory rheumatic disorder. The crystal structures described here show that a modified self-peptide, pVIPR-U5 (RRKWURWHL; U = citrulline), is presented by the two HLA-B27 molecules in distinct conformations. These binding modes differ not only drastically from each other but also from the conformations exhibited by the non-citrullinated peptide in a given subtype. The differential reactivity of HLA-B27-restricted cytotoxic T cells with modified or unmodified pVIPR supports the structural findings and shows that the presentation of citrullinated peptides has the potential to influence immune responses.