RESUMEN
Magnaporthe AVRs and ToxB-like (MAX) effectors constitute a family of secreted virulence proteins in the fungus Pyricularia oryzae (syn. Magnaporthe oryzae), which causes blast disease on numerous cereals and grasses. In spite of high sequence divergence, MAX effectors share a common fold characterized by a ß-sandwich core stabilized by a conserved disulfide bond. In this study, we investigated the structural landscape and diversity within the MAX effector repertoire of P. oryzae. Combining experimental protein structure determination and in silico structure modeling we validated the presence of the conserved MAX effector core domain in 77 out of 94 groups of orthologs (OG) identified in a previous population genomic study. Four novel MAX effector structures determined by NMR were in remarkably good agreement with AlphaFold2 (AF2) predictions. Based on the comparison of the AF2-generated 3D models we propose a classification of the MAX effectors superfamily in 20 structural groups that vary in the canonical MAX fold, disulfide bond patterns, and additional secondary structures in N- and C-terminal extensions. About one-third of the MAX family members remain singletons, without strong structural relationship to other MAX effectors. Analysis of the surface properties of the AF2 MAX models also highlights the high variability within the MAX family at the structural level, potentially reflecting the wide diversity of their virulence functions and host targets.
Asunto(s)
Ascomicetos , Proteínas Fúngicas , Enfermedades de las Plantas , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Ascomicetos/genética , Ascomicetos/patogenicidad , Ascomicetos/metabolismo , Enfermedades de las Plantas/microbiología , Modelos Moleculares , Conformación Proteica , Virulencia , Factores de Virulencia/genética , Factores de Virulencia/química , Factores de Virulencia/metabolismo , Secuencia de AminoácidosRESUMEN
The polyhistidine (6XHis) motif is one of the most ubiquitous protein purification tags. The 6XHis motif enables the binding of tagged proteins to various metals, which can be advantageously used for purification with immobilized metal affinity chromatography. Despite its popularity, protein structures encompassing metal-bound 6XHis are rare. Here, we obtained a 2.5 Å resolution crystal structure of a single chain Fv antibody (scFv) bearing a C-terminal sortase motif, 6XHis and TwinStrep tags (LPETGHHHHHHWSHPQFEK[G3S]3WSHPQFEK). The structure, obtained in the presence of cobalt, reveals a unique tetramerization motif (TetrHis) stabilized by 8 Co2+ ions. The TetrHis motif contains four 6 residues-long ß-strands, and each metal center coordinates 3 to 5 residues, including all 6XHis histidines. By combining dynamic light scattering, small angle x-ray scattering and molecular dynamics simulations, We investigated the influence of Co2+ on the conformational dynamics of scFv 2A2, observing an open/close equilibrium of the monomer and the formation of cobalt-stabilized tetramers. By using a similar scFv design, we demonstrate the transferability of the tetramerization property. This novel metal-dependent tetramerization motif might be used as a fiducial marker for cryoelectron microscopy of scFv complexes, or even provide a starting point for designing metal-loaded biomaterials.
RESUMEN
Nanoviruses are plant multipartite viruses with a genome composed of six to eight circular single-stranded DNA segments. The distinct genome segments are encapsidated individually in icosahedral particles that measure ≈18 nm in diameter. Recent studies on the model species Faba bean necrotic stunt virus (FBNSV) revealed that complete sets of genomic segments rarely occur in infected plant cells and that the function encoded by a given viral segment can complement the others across neighbouring cells, presumably by translocation of the gene products through unknown molecular processes. This allows the viral genome to replicate, assemble into viral particles and infect anew, even with the distinct genome segments scattered in different cells. Here, we question the form under which the FBNSV genetic material propagates long distance within the vasculature of host plants and, in particular, whether viral particle assembly is required. Using structure-guided mutagenesis based on a 3.2 Å resolution cryogenic-electron-microscopy reconstruction of the FBNSV particles, we demonstrate that specific site-directed mutations preventing capsid formation systematically suppress FBNSV long-distance movement, and thus systemic infection of host plants, despite positive detection of the mutated coat protein when the corresponding segment is agroinfiltrated into plant leaves. These results strongly suggest that the viral genome does not propagate within the plant vascular system under the form of uncoated DNA molecules or DNA:coat-protein complexes, but rather moves long distance as assembled viral particles.
Asunto(s)
Nanovirus , Vicia faba , Nanovirus/genética , Proteínas de la Cápside/genética , Vicia faba/genética , ADN Viral/genética , Virión/genética , Genoma Viral , MutagénesisRESUMEN
The septin collar of budding yeast is an ordered array of septin filaments that serves a scaffolding function for the cytokinetic machinery at the bud neck and compartmentalizes the membrane between mother and daughter cell. How septin architecture is aided by septin-binding proteins is largely unknown. Syp1 is an endocytic protein that was implicated in the timely recruitment of septins to the newly forming collar through an unknown mechanism. Using advanced microscopy and in vitro reconstitution assays, we show that Syp1 is able to align laterally and tightly pack septin filaments, thereby forming flat bundles or sheets. This property is shared by the Syp1 mammalian counterpart FCHo2, thus emphasizing conserved protein functions. Interestingly, the septin-bundling activity of Syp1 resides mainly in its intrinsically disordered region. Our data uncover the mechanism through which Syp1 promotes septin collar assembly and offer another example of functional diversity of unstructured protein domains.
Asunto(s)
Microscopía , SeptinasRESUMEN
Life is thought to have appeared in the depth of the sea under high hydrostatic pressure. Nowadays, it is known that the deep biosphere hosts a myriad of life forms thriving under high-pressure conditions. However, the evolutionary mechanisms leading to their adaptation are still not known. Here, we show the molecular bases of these mechanisms through a joint structural and dynamical study of two orthologous proteins. We observed that pressure adaptation involves the decoupling of protein-water dynamics and the elimination of cavities in the protein core. This is achieved by rearranging the charged residues on the protein surface and using bulkier hydrophobic residues in the core. These findings will be the starting point in the search for a complete genomic model explaining high-pressure adaptation.
Asunto(s)
Aclimatación , Adaptación Fisiológica , Presión HidrostáticaRESUMEN
Viruses of the sobemovirus genus are plant viruses, most of which generate very important agricultural and financial losses. Among them, the rice yellow mottle virus (RYMV) is one of the most damaging pathogens devastating rice fields in Africa. RYMV infectivity and propagation rely on its protein P1, identified as a key movement and potential long-distance RNA silencing suppressor. Here we describe P1's complete 3D structure and dynamics obtained by an integrative approach combining X-Ray crystallography and NMR spectroscopy. We show that P1 is organized in two semi-independent and topologically unrelated domains, each harboring an original zinc finger. The two domains exhibit different affinities for zinc and sensitivities to oxidoreduction conditions, making the C-terminal P1 region a potential labile sensor of the plant redox status. An additional level of regulation resides on the capacity of P1 to oligomerize through its N-terminal domain. Coupling P1 structure information with site-directed mutagenesis and plant functional assays, we identified key residues in each zinc domain essential for infectivity and spread in rice tissues. Altogether, our results provide the first complete structure of a sobemoviral P1 movement protein and highlight structural and dynamical properties that may serve RYMV functions to infect and invade its host plant.
Asunto(s)
Oryza , Virus de Plantas , Proteínas Virales , Dedos de Zinc , Cristalografía por Rayos X , Resonancia Magnética Nuclear Biomolecular , Oryza/virología , Virus de Plantas/patogenicidad , Dominios Proteicos , Proteínas Virales/química , Proteínas Virales/genética , Zinc/metabolismoRESUMEN
Staphylococcus aureus (SA) leukocidin ED (LukED) belongs to a family of bicomponent pore forming toxins that play important roles in SA immune evasion and nutrient acquisition. LukED targets specific G protein-coupled chemokine receptors to lyse human erythrocytes (red blood cells) and leukocytes (white blood cells). The first recognition step of receptors is critical for specific cell targeting and lysis. The structural and molecular bases for this mechanism are not well understood but could constitute essential information to guide antibiotic development. Here, we characterized the interaction of LukE with chemokine receptors ACKR1, CCR2, and CCR5 using a combination of structural, pharmacological, and computational approaches. First, crystal structures of LukE in complex with a small molecule mimicking sulfotyrosine side chain (p-cresyl sulfate) and with peptides containing sulfotyrosines issued from receptor sequences revealed the location of receptor sulfotyrosine binding sites in the toxins. Then, by combining previous and novel experimental data with protein docking, classical and accelerated weight histogram (AWH) molecular dynamics we propose models of the ACKR1-LukE and CCR5-LukE complexes. This work provides novel insights into chemokine receptor recognition by leukotoxins and suggests that the conserved sulfotyrosine binding pocket could be a target of choice for future drug development.
Asunto(s)
Infecciones Estafilocócicas , Staphylococcus aureus , Humanos , Evasión Inmune , Leucocidinas/metabolismo , Receptores de Quimiocina/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Staphylococcus aureus/genéticaRESUMEN
Metabotropic glutamate receptors (mGluRs) are dimeric G-protein-coupled receptors activated by the main excitatory neurotransmitter, L-glutamate. mGluR activation by agonists binding in the venus flytrap domain is regulated by positive (PAM) or negative (NAM) allosteric modulators binding to the 7-transmembrane domain (7TM). We report the cryo-electron microscopy structures of fully inactive and intermediate-active conformations of mGlu5 receptor bound to an antagonist and a NAM or an agonist and a PAM, respectively, as well as the crystal structure of the 7TM bound to a photoswitchable NAM. The agonist induces a large movement between the subunits, bringing the 7TMs together and stabilizing a 7TM conformation structurally similar to the inactive state. Using functional approaches, we demonstrate that the PAM stabilizes a 7TM active conformation independent of the conformational changes induced by agonists, representing an alternative mode of mGlu activation. These findings provide a structural basis for different mGluR activation modes.
Asunto(s)
Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Receptor del Glutamato Metabotropico 5/agonistas , Receptor del Glutamato Metabotropico 5/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacos , Microscopía por Crioelectrón , Cristalografía por Rayos X , Agonistas de Aminoácidos Excitadores/metabolismo , Antagonistas de Aminoácidos Excitadores/metabolismo , Células HEK293 , Humanos , Modelos Moleculares , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estabilidad Proteica , Subunidades de Proteína , Receptor del Glutamato Metabotropico 5/metabolismo , Receptor del Glutamato Metabotropico 5/ultraestructura , Relación Estructura-ActividadRESUMEN
Alkaline ceramidases (ACERs) are a class of poorly understood transmembrane enzymes controlling the homeostasis of ceramides. They are implicated in human pathophysiology, including progressive leukodystrophy, colon cancer as well as acute myeloid leukemia. We report here the crystal structure of the human ACER type 3 (ACER3). Together with computational studies, the structure reveals that ACER3 is an intramembrane enzyme with a seven transmembrane domain architecture and a catalytic Zn2+ binding site in its core, similar to adiponectin receptors. Interestingly, we uncover a Ca2+ binding site physically and functionally connected to the Zn2+ providing a structural explanation for the known regulatory role of Ca2+ on ACER3 enzymatic activity and for the loss of function in E33G-ACER3 mutant found in leukodystrophic patients.
Asunto(s)
Ceramidasa Alcalina/metabolismo , Enfermedades Desmielinizantes del Sistema Nervioso Central Hereditarias/genética , Ceramidasa Alcalina/química , Ceramidasa Alcalina/genética , Animales , Sitios de Unión/genética , Calcio/metabolismo , Cristalografía por Rayos X , Células HEK293 , Humanos , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Mutación Puntual , Conformación Proteica , Receptores de Adiponectina/química , Células Sf9 , SpodopteraRESUMEN
The development of the malaria parasite, Plasmodium falciparum, in the human erythrocyte, relies on phospholipid metabolism to fulfil the massive need for membrane biogenesis. Phosphatidylcholine (PC) is the most abundant phospholipid in Plasmodium membranes. PC biosynthesis is mainly ensured by the de novo Kennedy pathway that is considered as an antimalarial drug target. The CTP:phosphocholine cytidylyltransferase (CCT) catalyses the rate-limiting step of the Kennedy pathway. Here we report a series of structural snapshots of the PfCCT catalytic domain in its free, substrate- and product-complexed states that demonstrate the conformational changes during the catalytic mechanism. Structural data show the ligand-dependent conformational variations of a flexible lysine. Combined kinetic and ligand-binding analyses confirm the catalytic roles of this lysine and of two threonine residues of the helix αE. Finally, we assessed the variations in active site residues between Plasmodium and mammalian CCT which could be exploited for future antimalarial drug design.
Asunto(s)
Citidililtransferasa de Colina-Fosfato/química , Lipogénesis/genética , Malaria Falciparum/genética , Plasmodium falciparum/química , Secuencia de Aminoácidos/genética , Animales , Antimaláricos/química , Antimaláricos/uso terapéutico , Catálisis , Dominio Catalítico/genética , Citidililtransferasa de Colina-Fosfato/genética , Humanos , Cinética , Ligandos , Lípidos/biosíntesis , Lípidos/química , Lípidos/genética , Malaria Falciparum/enzimología , Malaria Falciparum/parasitología , Plasmodium falciparum/enzimología , Plasmodium falciparum/genética , Plasmodium falciparum/patogenicidad , Unión Proteica , Especificidad por SustratoRESUMEN
According to their restricted conformational freedom, heterocyclic γ-amino acids are usually considered to be related to Z-vinylogous γ-amino acids. In this context, oligomers alternating α-amino acids and thiazole-based γ-amino acids (ATCs) were expected to fold into a canonical 12-helical shape as described for α/γ-hybrid peptides composed of cis-α/ß-unsaturated γ-amino acids. However, through a combination of X-ray crystallography, NMR spectroscopy, FTIR experiments, and DFT calculations, it was determined that the folding behavior of ATC-containing hybrid peptides is much more complex. The homochiral α/(S)-ATC sequences were unable to adopt a stable conformation, whereas the heterochiral α/(R)-ATC peptides displayed novel ribbon structures stabilized by unusual C9/12 -bifurcated hydrogen bonds. These ribbon structures could be considered as a succession of pre-organized γ/α dipeptides and may provide the basis for designing original α-helix mimics.
Asunto(s)
Aminoácidos/química , Péptidos/química , Tiazoles/química , Dicroismo Circular , Cristalografía por Rayos X , Enlace de Hidrógeno , Espectroscopía de Resonancia Magnética , Péptidos/síntesis química , Estructura Secundaria de Proteína , Espectroscopía Infrarroja por Transformada de Fourier , EstereoisomerismoRESUMEN
Adiponectin receptors (ADIPORs) are integral membrane proteins that control glucose and lipid metabolism by mediating, at least in part, a cellular ceramidase activity that catalyses the hydrolysis of ceramide to produce sphingosine and a free fatty acid (FFA). The crystal structures of the two receptor subtypes, ADIPOR1 and ADIPOR2, show a similar overall seven-transmembrane-domain architecture with large unoccupied cavities and a zinc binding site within the seven transmembrane domain. However, the molecular mechanisms by which ADIPORs function are not known. Here we describe the crystal structure of ADIPOR2 bound to a FFA molecule and show that ADIPOR2 possesses intrinsic basal ceramidase activity that is enhanced by adiponectin. We also identify a ceramide binding pose and propose a possible mechanism for the hydrolytic activity of ADIPOR2 using computational approaches. In molecular dynamics simulations, the side chains of residues coordinating the zinc rearrange quickly to promote the nucleophilic attack of a zinc-bound hydroxide ion onto the ceramide amide carbonyl. Furthermore, we present a revised ADIPOR1 crystal structure exhibiting a seven-transmembrane-domain architecture that is clearly distinct from that of ADIPOR2. In this structure, no FFA is observed and the ceramide binding pocket and putative zinc catalytic site are exposed to the inner membrane leaflet. ADIPOR1 also possesses intrinsic ceramidase activity, so we suspect that the two distinct structures may represent key steps in the enzymatic activity of ADIPORs. The ceramidase activity is low, however, and further studies will be required to characterize fully the enzymatic parameters and substrate specificity of ADIPORs. These insights into ADIPOR function will enable the structure-based design of potent modulators of these clinically relevant enzymes.
Asunto(s)
Ceramidas/química , Ceramidas/metabolismo , Receptores de Adiponectina/química , Receptores de Adiponectina/metabolismo , Adiponectina/metabolismo , Adiponectina/farmacología , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Ácidos Grasos no Esterificados/química , Ácidos Grasos no Esterificados/metabolismo , Humanos , Hidrólisis/efectos de los fármacos , Hidróxidos/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Dominios Proteicos , Zinc/metabolismoRESUMEN
X-ray crystallography is an established technique for ligand screening in fragment-based drug-design projects, but the required manual handling steps - soaking crystals with ligand and the subsequent harvesting - are tedious and limit the throughput of the process. Here, an alternative approach is reported: crystallization plates are pre-coated with potential binders prior to protein crystallization and X-ray diffraction is performed directly 'in situ' (or in-plate). Its performance is demonstrated on distinct and relevant therapeutic targets currently being studied for ligand screening by X-ray crystallography using either a bending-magnet beamline or a rotating-anode generator. The possibility of using DMSO stock solutions of the ligands to be coated opens up a route to screening most chemical libraries.
Asunto(s)
Cristalización/métodos , Cristalografía por Rayos X/métodos , Descubrimiento de Drogas/métodos , Proteínas/química , Animales , Pollos , Peptidil-Prolil Isomerasa F , Ciclofilinas/química , Ciclofilinas/metabolismo , Humanos , Ligandos , Proteína Quinasa 1 Activada por Mitógenos/química , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Muramidasa/química , Muramidasa/metabolismo , PPAR gamma/química , PPAR gamma/metabolismo , Proteínas/metabolismo , RatasRESUMEN
Multidrug resistance, which is acquired by both Gram-positive and Gram-negative bacteria, causes infections that are associated with significant morbidity and mortality in many clinical settings around the world. Because of the rapidly increasing incidence of pathogens that have become resistant to all or nearly all available antibiotics, there is a need for a new generation of antimicrobials with a broad therapeutic range for specific applications against infections. Aedesin is a cecropin-like anti-microbial peptide that was recently isolated from dengue virus-infected salivary glands of the Aedes aegypti mosquito. In the present study, we have refined the analysis of its structural characteristics and have determined its antimicrobial effects against a large panel of multidrug resistant bacterial strains, directly isolated from infected patients. Based the results from nuclear magnetic resonance spectroscopy analysis, Aedesin has a helix-bend-helix structure typical for a member of the family of α-helix anti-microbial peptides. Aedesin efficiently killed Gram-negative bacterial strains that display the most worrisome resistance mechanisms encountered in the clinic, including resistance to carbapenems, aminoglycosides, cephalosporins, 4th generation fluoroquinolones, folate inhibitors and monobactams. In contrast, Gram-positive strains were insensitive to the lytic effects of the peptide. The anti-bacterial activity of Aedesin was found to be salt-resistant, indicating that it is active under physiological conditions encountered in body fluids characterized by ionic salt concentrations. In conclusion, because of its strong lytic activity against multidrug resistant Gram-negative bacterial strains displaying all types of clinically relevant resistance mechanisms known today, Aedesin might be an interesting candidate for the development of alternative treatment for infections caused by these types of bacteria.
Asunto(s)
Aedes/química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Farmacorresistencia Bacteriana Múltiple/efectos de los fármacos , Bacterias Gramnegativas/efectos de los fármacos , Proteínas de Insectos/farmacología , Aedes/inmunología , Secuencia de Aminoácidos , Animales , Antibacterianos/síntesis química , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/síntesis química , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Bacterias Gramnegativas/crecimiento & desarrollo , Proteínas de Insectos/síntesis química , Proteínas de Insectos/química , Proteínas de Insectos/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Glándulas Salivales/química , Glándulas Salivales/inmunología , Tolerancia a la SalRESUMEN
The Cop9 signalosome complex (CSN) regulates the functional cycle of the major E3 ubiquitin ligase family, the cullin RING E3 ubiquitin ligases (CRLs). Activated CRLs are covalently modified by the ubiquitin-like protein Nedd8 (neural precursor cell expressed developmentally down-regulated protein 8). CSN serves an essential role in myriad cellular processes by reversing this modification through the isopeptidase activity of its CSN5 subunit. CSN5 alone is inactive due to an auto-inhibited conformation of its catalytic domain. Here we report the molecular basis of CSN5 catalytic domain activation and unravel a molecular hierarchy in CSN deneddylation activity. The association of CSN5 and CSN6 MPN (for Mpr1/Pad1 N-terminal) domains activates its isopeptidase activity. The CSN5/CSN6 module, however, is inefficient in CRL deneddylation, indicating a requirement of further elements in this reaction such as other CSN subunits. A hybrid molecular model of CSN5/CSN6 provides a structural framework to explain these functional observations. Docking this model into a published CSN electron density map and using distance constraints obtained from cross-linking coupled to mass-spectrometry, we find that the C-termini of the CSN subunits could form a helical bundle in the centre of the structure. They likely play a key scaffolding role in the spatial organization of CSN and precise positioning of the dimeric MPN catalytic core.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Péptidos y Proteínas de Señalización Intracelular/química , Complejos Multiproteicos/química , Péptido Hidrolasas/química , Multimerización de Proteína , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Complejo del Señalosoma COP9 , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Modelos Moleculares , Complejos Multiproteicos/metabolismo , Proteína NEDD8 , Péptido Hidrolasas/metabolismo , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína , Ubiquitinas/metabolismoRESUMEN
By virtual screening using a fragment-based drug design (FBDD) approach, 33 fragments were selected within small pockets around interaction hot spots on the Sec7 surface of the nucleotide exchange factor Arno, and then their ability to interfere with the Arno-catalyzed nucleotide exchange on the G-protein Arf1 was evaluated. By use of SPR, NMR, and fluorescence assays, the direct binding of three of the identified fragments to Arno Sec7 domain was demonstrated and the promiscuous aggregate behavior evaluated. Then the binding mode of one fragment and of a more active analogue was solved by X-ray crystallography. This highlighted the role of stable and transient pockets at the Sec7 domain surface in the discovery and binding of interfering compounds. These results provide structural information on how small organic compounds can interfere with the Arf1-Arno Sec7 domain interaction and may guide the rational drug design of competitive inhibitors of Arno enzymatic activity.
Asunto(s)
Factor 1 de Ribosilacion-ADP/antagonistas & inhibidores , Diseño de Fármacos , Factores de Intercambio de Guanina Nucleótido/antagonistas & inhibidores , Sulfonamidas/farmacología , Factor 1 de Ribosilacion-ADP/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Factores de Intercambio de Guanina Nucleótido/química , Ensayos Analíticos de Alto Rendimiento , Concentración de Iones de Hidrógeno , Modelos Moleculares , Estructura Molecular , Unión Proteica/efectos de los fármacos , Relación Estructura-Actividad , Sulfonamidas/químicaRESUMEN
The COP9 (Constitutive photomorphogenesis 9) signalosome (CSN), a large multiprotein complex that resembles the 19S lid of the 26S proteasome, plays a central role in the regulation of the E3-cullin RING ubiquitin ligases (CRLs). The catalytic activity of the CSN complex, carried by subunit 5 (CSN5/Jab1), resides in the deneddylation of the CRLs that is the hydrolysis of the cullin-neural precursor cell expressed developmentally downregulated gene 8 (Nedd8)isopeptide bond. Whereas CSN-dependent CSN5 displays isopeptidase activity, it is intrinsically inactive in other physiologically relevant forms. Here we analyze the crystal structure of CSN5 in its catalytically inactive form to illuminate the molecular basis for its activation state. We show that CSN5 presents a catalytic domain that brings essential elements to understand its activity control. Although the CSN5 active site is catalytically competent and compatible with di-isopeptide binding, the Ins-1 segment obstructs access to its substrate-binding site, and structural rearrangements are necessary for the Nedd8-binding pocket formation. Detailed study of CSN5 by molecular dynamics unveils signs of flexibility and plasticity of the Ins-1 segment. These analyses led to the identification of a molecular trigger implicated in the active/inactive switch that is sufficient to impose on CSN5 an active isopeptidase state. We show that a single mutation in the Ins-1 segment restores biologically relevant deneddylase activity. This study presents detailed insights into CSN5 regulation. Additionally, a dynamic monomer-dimer equilibrium exists both in vitro and in vivo and may be functionally relevant.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Péptido Hidrolasas/química , Péptido Hidrolasas/metabolismo , Secuencia de Aminoácidos , Arginina/química , Complejo del Señalosoma COP9 , Dominio Catalítico , Cristalografía por Rayos X , Activación Enzimática , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteína NEDD8 , Péptido Hidrolasas/genética , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Subunidades de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Ubiquitinas/metabolismo , Zinc/metabolismoRESUMEN
Cdk2 promotes DNA replication and is a promising cancer therapeutic target, but its functions appear redundant with Cdk1, an essential Cdk affected by most Cdk2 inhibitors. Here, we present an integrated multidisciplinary approach to address Cdk redundancy. Mathematical modeling of enzymology data predicted conditions allowing selective chemical Cdk2 inhibition. Together with experiments in Xenopus egg extracts, this supports a rate-limiting role for Cdk2 in DNA replication. To confirm this we designed inhibitor-resistant (ir)-Cdk2 mutants using a novel bioinformatics approach. Bypassing inhibition with ir-Cdk2 or with Cdk1 shows that Cdk2 is rate-limiting for replication in this system because Cdk1 is insufficiently active. Additionally, crystal structures and kinetics reveal alternative binding modes of Cdk1-selective and Cdk2-selective inhibitors and mechanisms of Cdk2 inhibitor resistance. Our approach thus provides insight into structure, functions, and biochemistry of a cyclin-dependent kinase.
Asunto(s)
Quinasa 2 Dependiente de la Ciclina/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Proteína Quinasa CDC2/antagonistas & inhibidores , Proteína Quinasa CDC2/metabolismo , Cristalografía por Rayos X , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Ciclinas/metabolismo , Replicación del ADN/efectos de los fármacos , Humanos , Interfase , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Óvulo/metabolismo , Unión Proteica , Inhibidores de Proteínas Quinasas/farmacología , Estructura Terciaria de Proteína , Proteínas Recombinantes/antagonistas & inhibidores , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Xenopus/crecimiento & desarrollo , Xenopus/metabolismoRESUMEN
X-ray crystallography is now a recognized technique for ligand screening, especially for fragment-based drug design. However, protein crystal handling is still tedious and limits further automation. An alternative method for the solution of crystal structures of proteins in complex with small ligands is proposed. Crystallization drops are directly exposed to an X-ray beam after cocrystallization or soaking with the desired ligands. The use of dedicated plates in connection with an optimal parametrization of the G-rob robot allows efficient data collection. Three proteins currently under study in our laboratory for ligand screening by X-ray crystallography were used as validation test cases. The protein crystals belonged to different space groups, including a challenging monoclinic case. The resulting diffraction data can lead to clear ligand recognition, including indication of alternating conformations. These results demonstrate a possible method for automation of ligand screening by X-ray crystallography.
Asunto(s)
Cristalografía por Rayos X/métodos , Proteínas/química , Difracción de Rayos X/métodos , Diseño de FármacosRESUMEN
Cauliflower mosaic virus (CaMV) is transmitted from plant to plant through a seemingly simple interaction with insect vectors. This process involves an aphid receptor and two viral proteins, P2 and P3. P2 binds to both the aphid receptor and P3, itself tightly associated with the virus particle, with the ensemble forming a transmissible viral complex. Here, we describe the conformations of both unliganded CaMV P3 protein and its virion-associated form. X-ray crystallography revealed that the N-terminal domain of unliganded P3 is a tetrameric parallel coiled coil with a unique organization showing two successive four-stranded subdomains with opposite supercoiling handedness stabilized by a ring of interchain disulfide bridges. A structural model of virus-liganded P3 proteins, folding as an antiparallel coiled-coil network coating the virus surface, was derived from molecular modeling. Our results highlight the structural and biological versatility of this coiled-coil structure and provide new insights into the molecular mechanisms involved in CaMV acquisition and transmission by the insect vector.