RESUMEN
ErbB2 (or HER2) is a receptor tyrosine kinase overexpressed in some breast cancers and associated with poor prognosis. Treatments targeting the receptor extracellular and kinase domains have greatly improved disease outcome in the last 20 years. In parallel, the structures of these domains have been described, enabling better mechanistic understanding of the receptor function and targeted inhibition. However, the ErbB2 disordered C-terminal cytoplasmic tail (CtErbB2) remains very poorly characterized in terms of structure, dynamics, and detailed functional mechanism. Yet, it is where signal transduction is triggered via phosphorylation of tyrosine residues and carried out via interaction with adaptor proteins. Here, we report the first description, to our knowledge, of the ErbB2 disordered tail at atomic resolution using NMR, complemented by small-angle x-ray scattering. We show that although no part of CtErbB2 has any fully populated secondary or tertiary structure, it contains several transient α-helices and numerous transient polyproline II helices, populated up to 20 and 40%, respectively, and low but significant compaction. The presence of some structural elements suggests, along the lines of the results obtained for EGFR (ErbB1), that they may have a functional role in ErbB2's autoregulation processes. In addition, the transient formation of polyproline II helices is compliant with previously suggested interactions with SH3 domains. All in all, our in-depth structural study opens perspectives in the mechanistic understanding of ErbB2.
Asunto(s)
Neoplasias de la Mama , Receptor ErbB-2 , Proteínas Adaptadoras Transductoras de Señales , Femenino , Humanos , Fosforilación , Receptor ErbB-2/metabolismo , Transducción de Señal , Dominios Homologos srcRESUMEN
Immunotoxins are emerging candidates for cancer therapeutics. These biomolecules consist of a cell-targeting protein combined to a polypeptide toxin. Associations of both entities can be achieved either chemically by covalent bonds or genetically creating fusion proteins. However, chemical agents can affect the activity and/or stability of the conjugate proteins, and additional purification steps are often required to isolate the final conjugate from unwanted byproducts. As for fusion proteins, they often suffer from low solubility and yield. In this report, we describe a straightforward conjugation process to generate an immunotoxin using coassociating peptides (named K3 and E3), originating from the tetramerization domain of p53. To that end, a nanobody targeting the human epidermal growth factor receptor 2 (nano-HER2) and a protein toxin fragment from Pseudomonas aeruginosa exotoxin A (TOX) were genetically fused to the E3 and K3 peptides. Entities were produced separately in Escherichia coli in soluble forms and at high yields. The nano-HER2 fused to the E3 or K3 helixes (nano-HER2-E3 and nano-HER2-K3) and the coassembled immunotoxins (nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX) presented binding specificity on HER2-overexpressing cells with relative binding constants in the low nanomolar to picomolar range. Both toxin modules (E3-TOX and K3-TOX) and the combined immunotoxins exhibited similar cytotoxicity levels compared to the toxin alone (TOX). Finally, nano-HER2-K3E3-TOX and nano-HER2-E3K3-TOX evaluated on various breast cancer cells were highly potent and specific to killing HER2-overexpressing breast cancer cells with IC50 values in the picomolar range. Altogether, we demonstrate that this noncovalent conjugation method using two coassembling peptides can be easily implemented for the modular engineering of immunotoxins targeting different types of cancers.
Asunto(s)
ADP Ribosa Transferasas/farmacología , Antineoplásicos/farmacología , Toxinas Bacterianas/farmacología , Exotoxinas/farmacología , Inmunotoxinas/farmacología , Receptor ErbB-2/antagonistas & inhibidores , Anticuerpos de Dominio Único/farmacología , Factores de Virulencia/farmacología , ADP Ribosa Transferasas/química , ADP Ribosa Transferasas/genética , Antineoplásicos/química , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Neoplasias de la Mama/tratamiento farmacológico , Línea Celular Tumoral , Exotoxinas/química , Exotoxinas/genética , Femenino , Humanos , Inmunotoxinas/química , Inmunotoxinas/genética , Modelos Moleculares , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Anticuerpos de Dominio Único/química , Anticuerpos de Dominio Único/genética , Factores de Virulencia/química , Factores de Virulencia/genética , Exotoxina A de Pseudomonas aeruginosaRESUMEN
Because of their large conformational heterogeneity, structural characterization of intrinsically disordered proteins (IDPs) is very challenging using classical experimental methods alone. In this study, we use NMR and small-angle x-ray scattering (SAXS) data with multiple molecular dynamics (MD) simulations to describe the conformational ensemble of the fully disordered verprolin homology domain of the neural Aldrich syndrome protein involved in the regulation of actin polymerization. First, we studied several back-calculation software of SAXS scattering intensity and optimized the adjustable parameters to accurately calculate the SAXS intensity from an atomic structure. We also identified the most appropriate force fields for MD simulations of this IDP. Then, we analyzed four conformational ensembles of neural Aldrich syndrome protein verprolin homology domain, two generated with the program flexible-meccano with or without NMR-derived information as input and two others generated by MD simulations with two different force fields. These four conformational ensembles were compared to available NMR and SAXS data for validation. We found that MD simulations with the AMBER-03w force field and the TIP4P/2005s water model are able to correctly describe the conformational ensemble of this 67-residue IDP at both local and global level.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Proteína del Síndrome de Wiskott-Aldrich/química , Humanos , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Dominios Proteicos , Dispersión del Ángulo Pequeño , Difracción de Rayos XRESUMEN
Nuclear magnetic resonance spectroscopy is a powerful tool to study structural and functional properties of proteins, provided that they can be enriched in stable isotopes such as (15)N, (13)C and (2)H. This is usually easy and inexpensive when the proteins are expressed in Escherichiacoli, but many eukaryotic (human in particular) proteins cannot be produced this way. An alternative is to express them in insect cells. Labeled insect cell growth media are commercially available but at prohibitive prices, limiting the NMR studies to only a subset of biologically important proteins. Non-commercial solutions from academic institutions have been proposed, but none of them is really satisfying. We have developed a (15)N-labeling procedure based on the use of a commercial medium depleted of all amino acids and supplemented with a (15)N-labeled yeast autolysate for a total cost about five times lower than that of the currently available solutions. We have applied our procedure to the production of a non-polymerizable mutant of actin in Sf9 cells and of fragments of eukaryotic and viral membrane fusion proteins in S2 cells, which typically cannot be produced in E. coli, with production yields comparable to those obtained with standard commercial media. Our results support, in particular, the putative limits of a self-folding domain within a viral glycoprotein of unknown structure.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Imagen por Resonancia Magnética , Biosíntesis de Proteínas , Proteínas de la Matriz Viral/química , Aminoácidos/química , Animales , Drosophila/química , Drosophila/genética , Humanos , Radioisótopos de Nitrógeno/química , Células Sf9 , SpodopteraRESUMEN
Cargo adaptors are crucial in coupling motor proteins with their respective cargos and regulatory proteins. BicD2 is a prominent example within the cargo adaptor family. BicD2 is able to recruit the microtubule motor dynein to RNA, viral particles, and nuclei. The BicD2-mediated interaction between the nucleus and dynein is implicated in mitosis, interkinetic nuclear migration (INM) in radial glial progenitor cells, and neuron precursor migration during embryonic neocortex development. In vitro studies involving full-length cargo adaptors are difficult to perform due to the hydrophobic character, low-expression levels, and intrinsic flexibility of cargo adaptors. Here, we report the recombinant production of full-length human BicD2 and confirm its biochemical activity by interaction studies with RanBP2. We also describe pH-dependent conformational changes of BicD2 using cryoelectron microscopy (cryo-EM), template-free structure predictions, and biophysical tools. Our results will help define the biochemical parameters for the in vitro reconstitution of higher-order BicD2 protein complexes.
Asunto(s)
Dineínas , Proteínas Asociadas a Microtúbulos , Humanos , Dineínas/metabolismo , Complejo Dinactina/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Microscopía por Crioelectrón , Microtúbulos/metabolismoRESUMEN
BAG1 is a family of polypeptides with a conserved C-terminal BAG domain that functions as a nucleotide exchange factor for the molecular chaperone HSP70. BAG1 proteins also control several signaling processes including proteostasis, apoptosis, and transcription. The largest isoform, BAG1L, controls the activity of the androgen receptor (AR) and is upregulated in prostate cancer. Here, we show that BAG1L regulates AR dynamics in the nucleus and its ablation attenuates AR target gene expression especially those involved in oxidative stress and metabolism. We show that a small molecule, A4B17, that targets the BAG domain downregulates AR target genes similar to a complete BAG1L knockout and upregulates the expression of oxidative stress-induced genes involved in cell death. Furthermore, A4B17 outperformed the clinically approved antagonist enzalutamide in inhibiting cell proliferation and prostate tumor development in a mouse xenograft model. BAG1 inhibitors therefore offer unique opportunities for antagonizing AR action and prostate cancer growth.
RESUMEN
AAA+ proteins form asymmetric hexameric rings that hydrolyze ATP and thread substrate proteins through a central channel via mobile substrate-binding pore loops. Understanding how ATPase and threading activities are regulated and intertwined is key to understanding the AAA+ protein mechanism. We studied the disaggregase ClpB, which contains tandem ATPase domains (AAA1, AAA2) and shifts between low and high ATPase and threading activities. Coiled-coil M-domains repress ClpB activity by encircling the AAA1 ring. Here, we determine the mechanism of ClpB activation by comparing ATPase mechanisms and cryo-EM structures of ClpB wild-type and a constitutively active ClpB M-domain mutant. We show that ClpB activation reduces ATPase cooperativity and induces a sequential mode of ATP hydrolysis in the AAA2 ring, the main ATPase motor. AAA1 and AAA2 rings do not work synchronously but in alternating cycles. This ensures high grip, enabling substrate threading via a processive, rope-climbing mechanism.
Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Adenosina Trifosfato/metabolismo , Endopeptidasa Clp/química , Endopeptidasa Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Dominio AAA/genética , ATPasas Asociadas con Actividades Celulares Diversas/química , Microscopía por Crioelectrón , Endopeptidasa Clp/genética , Endopeptidasa Clp/ultraestructura , Escherichia coli/química , Escherichia coli/enzimología , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/ultraestructura , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/ultraestructura , Modelos Moleculares , Mutación , Unión Proteica , Dominios Proteicos/genéticaRESUMEN
Several bacterial pathogens produce nucleotidyl cyclase toxins to manipulate eukaryotic host cells. Inside host cells they are activated by endogenous cofactors to produce high levels of cyclic nucleotides (cNMPs). The ExoY toxin from Pseudomonas aeruginosa (PaExoY) and the ExoY-like module (VnExoY) found in the MARTX (Multifunctional-Autoprocessing Repeats-in-ToXin) toxin of Vibrio nigripulchritudo share modest sequence similarity (~38%) but were both recently shown to be activated by actin after their delivery to the eukaryotic host cell. Here, we further characterized the ExoY-like cyclase of V. nigripulchritudo. We show that, in contrast to PaExoY that requires polymerized actin (F-actin) for maximum activation, VnExoY is selectively activated by monomeric actin (G-actin). These two enzymes also display different nucleotide substrate and divalent cation specificities. In vitro in presence of the cation Mg2+, the F-actin activated PaExoY exhibits a promiscuous nucleotidyl cyclase activity with the substrate preference GTP>ATP≥UTP>CTP, while the G-actin activated VnExoY shows a strong preference for ATP as substrate, as it is the case for the well-known calmodulin-activated adenylate cyclase toxins from Bordetella pertussis or Bacillus anthracis. These results suggest that the actin-activated nucleotidyl cyclase virulence factors despite sharing a common activator may actually display a greater variability of biological effects in infected cells than initially anticipated.
Asunto(s)
Citoesqueleto de Actina/genética , Toxina de Adenilato Ciclasa/química , Células Eucariotas/efectos de los fármacos , Pseudomonas aeruginosa/química , Citoesqueleto de Actina/química , Adenosina Trifosfato/química , Toxina de Adenilato Ciclasa/genética , Bacillus anthracis/efectos de los fármacos , Bacillus anthracis/patogenicidad , Proteínas Bacterianas/genética , Bordetella pertussis/efectos de los fármacos , Bordetella pertussis/patogenicidad , Glucosiltransferasas/genética , Interacciones Huésped-Patógeno/genética , Humanos , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/patogenicidad , Especificidad por Sustrato , Toxinas Biológicas/química , Toxinas Biológicas/genética , Vibrio/efectos de los fármacos , Vibrio/genética , Vibrio/patogenicidad , Factores de Virulencia/química , Factores de Virulencia/genéticaRESUMEN
Refolding aggregated proteins is essential in combating cellular proteotoxic stress. Together with Hsp70, Hsp100 chaperones, including Escherichia coli ClpB, form a powerful disaggregation machine that threads aggregated polypeptides through the central pore of tandem adenosine triphosphatase (ATPase) rings. To visualize protein disaggregation, we determined cryo-electron microscopy structures of inactive and substrate-bound ClpB in the presence of adenosine 5'-O-(3-thiotriphosphate), revealing closed AAA+ rings with a pronounced seam. In the substrate-free state, a marked gradient of resolution, likely corresponding to mobility, spans across the AAA+ rings with a dynamic hotspot at the seam. On the seam side, the coiled-coil regulatory domains are locked in a horizontal, inactive orientation. On the opposite side, the regulatory domains are accessible for Hsp70 binding, substrate targeting, and activation. In the presence of the model substrate casein, the polypeptide threads through the entire pore channel and increased nucleotide occupancy correlates with higher ATPase activity. Substrate-induced domain displacements indicate a pathway of regulated substrate transfer from Hsp70 to the ClpB pore, inside which a spiral of loops contacts the substrate. The seam pore loops undergo marked displacements, along with ordering of the regulatory domains. These asymmetric movements suggest a mechanism for ATPase activation and substrate threading during disaggregation.
Asunto(s)
Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/metabolismo , Microscopía por Crioelectrón/métodos , Modelos Moleculares , Regiones Promotoras Genéticas , Agregado de Proteínas , Unión Proteica , Conformación Proteica , Dominios y Motivos de Interacción de Proteínas , Replegamiento Proteico , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Understanding the structural basis of actin cytoskeleton remodeling requires stabilization of actin monomers, oligomers, and filaments in complex with partner proteins, using various biochemical strategies. Here, we report a dramatic destabilization of the dynamic interaction with a model ß-thymosin/WH2 domain induced by mutations in actin. This result underlines that mutant actins should be used with prudence to characterize interactions with intrinsically disordered partners as destabilization of dynamic interactions, although identifiable by NMR, may be invisible to other structural techniques. It also highlights how both ß-thymosin/WH2 domains and actin tune local structure and dynamics in regulatory processes involving intrinsically disordered domains.
Asunto(s)
Actinas/genética , Mutación , Timosina/química , Actinas/química , Animales , Cristalografía por Rayos X , Modelos Moleculares , Unión Proteica , Conformación Proteica , Conejos , Homología de Secuencia de AminoácidoRESUMEN
Many actin-binding proteins (ABPs) use complex multidomain architectures to integrate and coordinate multiple signals and interactions with the dynamic remodeling of actin cytoskeleton. In these proteins, small segments that are intrinsically disordered in their unbound native state can be functionally as important as identifiable folded units. These functional intrinsically disordered regions (IDRs) are however difficult to identify and characterize in vitro. Here, we try to summarize the state of the art in understanding the structural features and interfacial properties of IDRs involved in actin self-assembly dynamics. Recent structural and functional insights into the regulation of widespread, multifunctional WH2/ß-thymosin domains, and of other IDRs such as those associated with WASP/WAVE, formin or capping proteins are examined. Understanding the functional versatility of IDRs in actin assembly requires apprehending by multiple structural and functional approaches their large conformational plasticity and dynamics in their interactions. In many modular ABPs, IDRs relay labile interactions with multiple partners and act as interaction hubs in interdomain and protein-protein interfaces. They thus control multiple conformational transitions between the inactive and active states or between various active states of multidomain ABPs, and play an important role to coordinate the high turnover of interactions in actin self-assembly dynamics.