RESUMEN
Many disordered proteins conserve essential functions in the face of extensive sequence variation, making it challenging to identify the mechanisms responsible for functional selection. Here we identify the molecular mechanism of functional selection for the disordered adenovirus early gene 1A (E1A) protein. E1A competes with host factors to bind the retinoblastoma (Rb) protein, subverting cell cycle regulation. We show that two binding motifs tethered by a hypervariable disordered linker drive picomolar affinity Rb binding and host factor displacement. Compensatory changes in amino acid sequence composition and sequence length lead to conservation of optimal tethering across a large family of E1A linkers. We refer to this compensatory mechanism as conformational buffering. We also detect coevolution of the motifs and linker, which can preserve or eliminate the tethering mechanism. Conformational buffering and motif-linker coevolution explain robust functional encoding within hypervariable disordered linkers and could underlie functional selection of many disordered protein regions.
Asunto(s)
Proteínas Intrínsecamente Desordenadas , Proteínas E1A de Adenovirus/química , Proteínas E1A de Adenovirus/genética , Proteínas E1A de Adenovirus/metabolismo , Secuencia de Aminoácidos , Proteínas Intrínsecamente Desordenadas/química , Unión Proteica , Dominios Proteicos , Proteína de Retinoblastoma/metabolismoRESUMEN
The rapid advances of 3D techniques for the structural determination of proteins and the development of numerous computational methods and strategies have led to identifying highly active compounds in computer drug design. Molecular docking is a method widely used in high-throughput virtual screening campaigns to filter potential ligands targeted to proteins. A great variety of docking programs are currently available, which differ in the algorithms and approaches used to predict the binding mode and the affinity of the ligand. All programs heavily rely on scoring functions to accurately predict ligand binding affinity, and despite differences in performance, none of these docking programs is preferable to the others. To overcome this problem, consensus scoring methods improve the outcome of virtual screening by averaging the rank or score of individual molecules obtained from different docking programs. The successful application of consensus docking in high-throughput virtual screening highlights the need to optimize the predictive power of molecular docking methods.
Asunto(s)
Proteínas , Simulación del Acoplamiento Molecular , Unión Proteica , Ligandos , Consenso , Proteínas/químicaRESUMEN
Botulinum neurotoxin A (BoNT A) is a substrate of the Src family of tyrosine kinases. Here, we report that the BoNT A light chain (LC) is phosphorylated in the tyrosine-71 located at N-terminus. Covalent modification of this residue notably increases the thermal stability of the endopeptidase activity, without affecting its catalytic efficacy. Similarly, mutation of this residue specifically affected the protein stability but not its endopeptidase function. Fusion of the Tat-translocating domain to the N-terminus of the enzyme produced a cell permeable, functional enzyme, as evidenced by immunocytochemistry and by the cleavage of cytosolic SNAP25 in intact PC12 cells. Noteworthy, truncation of cellular SNAP25 was reduced in cells when the Src kinase activity was inhibited with a specific antagonist, implying that tyrosine phosphorylation of BoNT A LC modulates the in vivo proteolytic activity of the neurotoxin. Taken together, these findings substantiate the tenet that tyrosine phosphorylation of BoNT A LC could be an important modulatory strategy of the neurotoxin stability and suggest that the phosphorylated neurotoxin may be a relevant molecule in vivo.
Asunto(s)
Toxinas Botulínicas Tipo A/metabolismo , Fármacos Neuromusculares/metabolismo , Tirosina/metabolismo , Animales , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/genética , Dominio Catalítico , Proteínas de la Membrana/metabolismo , Modelos Moleculares , Proteínas del Tejido Nervioso/metabolismo , Fármacos Neuromusculares/química , Células PC12 , Fosforilación , Ratas , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteína 25 Asociada a Sinaptosomas , Temperatura , Familia-src Quinasas/metabolismoRESUMEN
Synthetic peptides patterned after the C-terminus of synaptosomal associated protein of 25 kDa (SNAP25) efficiently abrogate regulated exocytosis. In contrast, the use of SNAP25 N-terminal-derived peptides to modulate SNAP receptors (SNARE) complex assembly and neurosecretion has not been explored. Here, we show that the N-terminus of SNAP25, specially the segment that encompasses 22Ala-44Ile, is essential for the formation of the SNARE complex. Peptides patterned after this protein domain are potent inhibitors of SNARE complex formation. The inhibitory activity correlated with their propensity to adopt an alpha-helical secondary structure. These peptides abrogated SNARE complex formation only when added previous to the onset of aggregate assembly. Analysis of the mechanism of action revealed that these peptides disrupted the binary complex formed by SNAP25 and syntaxin. The identified peptides inhibited Ca2+-dependent exocytosis from detergent-permeabilized excitable cells. Noteworthy, these amino acid sequences markedly protected intact hippocampal neurones against hypoglycaemia-induced, glutamate-mediated excitotoxicity with a potency that rivalled that displayed by botulinum neurotoxins. Our findings indicate that peptides patterned after the N-terminus of SNAP25 are potent inhibitors of SNARE complex formation and neuronal exocytosis. Because of their activity in intact neurones, these cell permeable peptides may be hits for antispasmodic and analgesic drug development.
Asunto(s)
Exocitosis/fisiología , Proteínas de la Membrana/química , Proteínas del Tejido Nervioso/química , Péptidos/química , Proteínas de Transporte Vesicular , Secuencia de Aminoácidos , Animales , Calcio/farmacología , Catecolaminas/metabolismo , Bovinos , Muerte Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Cultivadas , Células Cromafines/efectos de los fármacos , Células Cromafines/metabolismo , Detergentes/química , Detergentes/farmacología , Relación Dosis-Respuesta a Droga , Exocitosis/efectos de los fármacos , Glucosa/deficiencia , Glucosa/metabolismo , Glucosa/farmacología , Sustancias Macromoleculares , Proteínas de la Membrana/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/metabolismo , Neuronas/efectos de los fármacos , Péptidos/metabolismo , Péptidos/farmacología , Estructura Terciaria de Proteína/fisiología , Proteínas SNARE , Relación Estructura-Actividad , Proteína 25 Asociada a SinaptosomasRESUMEN
The ability of certain Src homology 3 (SH3) domains to bind specifically both type I and type II polyproline ligands is perhaps the best characterized, but also the worst understood, example in the family of protein-interaction modules. A detailed analysis of the structural variations in SH3 domains, with respect to ligand-binding specificity, together with mutagenesis of SH3 Fyn tyrosine kinase, reveal the structural basis for types I and II binding specificity by SH3 domains. The conserved Trp in the SH3 binding pocket can adopt two different orientations that, in turn, determine the type of ligand (I or II) able to bind to the domain. The only exceptions are ligands with Leu at positions P(-1) and P(2), that deviate from standard poly-Pro angles. The motion of the conserved Trp depends on the presence of certain residues located in a key position (132 for Fyn), near the binding pocket. SH3 domains placing aromatic residues in this key position are promiscuous. By contrast, those presenting beta-branched or long aliphatic residues block the conserved Trp in one of the two possible orientations, preventing binding in a type I orientation. This is experimentally demonstrated by a single mutation in Fyn SH3 (Y132I) that abolishes type I ligand binding, while preserving binding to type II ligands. Thus, simple conformational changes, governed by simple rules, can have profound effects on protein-protein interactions, highlighting the importance of structural details to predict protein-protein interactions.
Asunto(s)
Proteínas Tirosina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Triptófano/química , Dominios Homologos src/fisiología , Secuencia de Aminoácidos , Escherichia coli/enzimología , Humanos , Leucina/química , Leucina/metabolismo , Ligandos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Péptidos/química , Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Proteínas Tirosina Quinasas/química , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-fyn , Homología de Secuencia de Aminoácido , Triptófano/metabolismoRESUMEN
Synthetic peptides patterned after the proteins involved in vesicle fusion [the so-called SNARE (soluble N -ethylmaleimide-sensitive fusion protein attachment protein receptor) proteins] are potent inhibitors of SNARE complex assembly and neuronal exocytosis. It is noteworthy that the identification of peptide sequences not related to the SNARE proteins has not been accomplished yet; this is due, in part, to the structural constraints and the specificity of the protein interactions that govern the formation of the SNARE complex. Here we have addressed this question and used a combinatorial approach to identify peptides that modulate the assembly of the SNARE core complex and inhibit neuronal exocytosis. An alpha-helix-constrained, mixture-based, 17-mer combinatorial peptide library composed of 137180 sequences was synthesized in a positional scanning format. Peptide mixtures were assayed for their ability to prevent the formation of the in vitro -reconstituted SDS-resistant SNARE core complex. Library deconvolution identified eight peptides that inhibited the assembly of the SNARE core complex. Notably, the most potent 17-mer peptide (acetyl-SAAEAFAKLYAEAFAKG-NH2) abolished both Ca2+-evoked catecholamine secretion from detergent-permeabilized chromaffin cells and L-glutamate release from intact hippocampal primary cultures. Collectively, these findings indicate that amino acid sequences that prevent SNARE complex formation are not restricted to those that mimic domains of SNARE proteins, thus expanding the diversity of molecules that target neuronal exocytosis. Because of the implication of neurosecretion in the aetiology of several human neurological disorders, these newly identified peptides may be considered hits for the development of novel anti-spasmodic drugs.