RESUMEN
SCOPE: Brown adipose tissue (BAT) is the main regulator of thermogenesis by increasing energy expenditure through the uncoupling of oxidative metabolism from ATP synthesis. There is a growing body of evidence for BAT being the key responsible organ in combating obesity and its related disorders. Herein we propose the fungal protein ostreolysin (Oly), which has been previously shown to bind to cholesterol-enriched raft-like membrane domains (lipid rafts) of mammalian cells, as a suitable candidate for interaction with brown preadipocytes. The aim of the present study was therefore to characterize the mechanism by which a recombinant version of ostreolysin (rOly) induces brown adipocyte differentiation. METHODS AND RESULTS: Primary isolated brown preadipocytes or HIB-1B brown preadipocyte cells were treated with rOly and the effects on morphology, lipid accumulation, respiration rate, and associated gene and protein expression were measured. rOly upregulated mRNA and protein levels of factors related to brown adipocyte differentiation, induced lipid droplet formation, and increased cellular respiration rate due to expression of uncoupling protein 1. rOly also upregulated ß-tubulin expression, and therefore microtubules might be involved in its mechanism of action. CONCLUSION: rOly promotes brown adipocyte differentiation, suggesting a new mechanism for rOly's contribution to the battle against obesity.
Asunto(s)
Adipocitos Marrones/efectos de los fármacos , Proteínas Hemolisinas/farmacología , Adipocitos Marrones/citología , Adipocitos Marrones/metabolismo , Animales , Proteínas Potenciadoras de Unión a CCAAT/genética , Diferenciación Celular/efectos de los fármacos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas Hemolisinas/química , Proteínas Hemolisinas/metabolismo , Metabolismo de los Lípidos/efectos de los fármacos , Ratones , PPAR gamma/genética , Fenotipo , Estructura Secundaria de Proteína , Proteínas Recombinantes/farmacología , Tubulina (Proteína)/química , Proteína Desacopladora 1/genéticaRESUMEN
Due to increasing interest in peptides as signaling modulators and drug candidates, several methods for peptide docking to their target proteins are under active development. The "blind" docking problem, where the peptide-binding site on the protein surface is unknown, presents one of the current challenges in the field. AnchorDock protocol was developed by Ben-Shimon and Niv to address this challenge.This protocol narrows the docking search to the most relevant parts of the conformational space. This is achieved by pre-folding the free peptide and by computationally detecting anchoring spots on the surface of the unbound protein. Multiple flexible simulated annealing molecular dynamics (SAMD) simulations are subsequently carried out, starting from pre-folded peptide conformations, constrained to the various precomputed anchoring spots.Here, AnchorDock is demonstrated using two known protein-peptide complexes. A PDZ-peptide complex provides a relatively easy case due to the relatively small size of the protein, and a typical peptide conformation and binding region; a more challenging example is a complex between USP7N-term and a p53-derived peptide, where the protein is larger, and the peptide conformation and a binding site are generally assumed to be unknown. AnchorDock returned native-like solutions ranked first and third for the PDZ and USP7 complexes, respectively. We describe the procedure step by step and discuss possible modifications where applicable.
Asunto(s)
Bases de Datos de Proteínas , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Proteínas/metabolismo , Programas Informáticos , Sitios de Unión , Humanos , Modelos Moleculares , Simulación del Acoplamiento Molecular , Unión Proteica , Conformación Proteica , Proteínas/química , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/metabolismo , Peptidasa Específica de Ubiquitina 7/química , Peptidasa Específica de Ubiquitina 7/metabolismo , Navegador WebRESUMEN
Protein-protein interactions play a vital role in cellular processes as exemplified by assembly of the intricate multi-enzyme cellulosome complex. Cellulosomes are assembled by selective high-affinity binding of enzyme-borne dockerin modules to repeated cohesin modules of structural proteins termed scaffoldins. Recent sequencing of the fiber-degrading Ruminococcus flavefaciens FD-1 genome revealed a particularly elaborate cellulosome system. In total, 223 dockerin-bearing ORFs potentially involved in cellulosome assembly and a variety of multi-modular scaffoldins were identified, and the dockerins were classified into six major groups. Here, extensive screening employing three complementary medium- to high-throughput platforms was used to characterize the different cohesin-dockerin specificities. The platforms included (i) cellulose-coated microarray assay, (ii) enzyme-linked immunosorbent assay (ELISA) and (iii) in-vivo co-expression and screening in Escherichia coli. The data revealed a collection of unique cohesin-dockerin interactions and support the functional relevance of dockerin classification into groups. In contrast to observations reported previously, a dual-binding mode is involved in cellulosome cell-surface attachment, whereas single-binding interactions operate for cellulosome integration of enzymes. This sui generis cellulosome model enhances our understanding of the mechanisms governing the remarkable ability of R. flavefaciens to degrade carbohydrates in the bovine rumen and provides a basis for constructing efficient nano-machines applied to biological processes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Celulosomas/metabolismo , Mapas de Interacción de Proteínas , Ruminococcus/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas de Ciclo Celular/metabolismo , Celulosa/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Modelos Biológicos , Filogenia , Análisis por Matrices de Proteínas , CohesinasRESUMEN
G protein-coupled receptors (GPCRs) are seven transmembrane (TM) proteins that play a key role in human physiology. The GPCR superfamily comprises about 800 members, classified into several classes, with rhodopsin-like Class A being the largest and most studied thus far. A huge component of the human repertoire consists of the chemosensory GPCRs, including â¼400 odorant receptors, 25 bitter taste receptors (TAS2Rs), which are thought to guard the organism from consuming poisons, and sweet and umami TAS1R heteromers, which indicate the nutritive value of food. The location of the binding site of TAS2Rs is similar to that of Class A GPCRs. However, most of the known bitter ligands are agonists, with only a few antagonists documented thus far. The agonist-to-antagonist ratios of Class A GPCRs vary, but in general are much lower than for TAS2Rs. For a set of well-studied GPCRs, a gradual change in agonists-to-antagonists ratios is observed when comparing low (10 µM)- and high (10 nM)-affinity ligand sets from ChEMBL and the DrugBank set of drugs. This shift reflects pharmaceutical bias toward the therapeutically desirable pharmacology for each of these GPCRs, while the 10 µM sets possibly represent the native tendency of the receptors toward either agonists or antagonists. Analyzing ligand-GPCR interactions in 56 X-ray structures representative of currently available structural data, we find that the N-terminus, TM1 and TM2 are more involved in binding of antagonists than of agonists. On the other hand, ECL2 tends to be more involved in binding of agonists. This is of interest, since TAS2Rs harbor variations on the typical Class A sequence motifs, including the absence of the ECL2-TM3 disulfide bridge. This suggests an alternative mode of regulation of conformational states for TAS2Rs, with potentially less stabilized inactive state. The comparison of TAS2Rs and Class A GPCRs structural features and the pharmacology of the their ligands highlights the intricacies of GPCR architecture and provides a framework for rational design of new ligands.
Asunto(s)
Receptores Acoplados a Proteínas G/química , Sitios de Unión , Humanos , Ligandos , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Gusto/efectos de los fármacosRESUMEN
The cellulolytic bacterium Ruminococcus flavefaciens of the herbivore rumen produces an elaborate cellulosome system, anchored to the bacterial cell wall via the covalently bound scaffoldin ScaE. Dockerin-bearing scaffoldins also bind to an autonomous cohesin of unknown function, called cohesin G (CohG). Here, we demonstrate that CohG binds to the scaffoldin-borne dockerin in opposite orientation on a distinct site, relative to that of ScaE. Based on these structural data, we propose that the complexed dockerin is still available to bind ScaE on the cell surface. CohG may thus serve as a molecular shuttle for delivery of scaffoldins to the bacterial cell surface.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Celulosomas/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas Cromosómicas no Histona/química , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , CohesinasRESUMEN
Interactions between cohesin and dockerin modules play a crucial role in the assembly of multienzyme cellulosome complexes. Although intraspecies cohesin and dockerin modules bind in general with high affinity but indiscriminately, cross-species binding is rare. Here, we combined ELISA-based experiments with Rosetta-based computational design to evaluate the contribution of distinct residues at the Clostridium thermocellum cohesin-dockerin interface to binding affinity, specificity, and promiscuity. We found that single mutations can show distinct and significant effects on binding affinity and specificity. In particular, mutations at cohesin position Asn(37) show dramatic variability in their effect on dockerin binding affinity and specificity: the N37A mutant binds promiscuously both to cognate (C. thermocellum) as well as to non-cognate Clostridium cellulolyticum dockerin. N37L in turn switches binding specificity: compared with the wild-type C. thermocellum cohesin, this mutant shows significantly increased preference for C. cellulolyticum dockerin combined with strongly reduced binding to its cognate C. thermocellum dockerin. The observation that a single mutation can overcome the naturally observed specificity barrier provides insights into the evolutionary dynamics of this system that allows rapid modulation of binding specificity within a high affinity background.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Proteínas de Ciclo Celular/química , Proteínas Cromosómicas no Histona/química , Carbohidratos/química , Celulosa/metabolismo , Clostridium cellulolyticum/metabolismo , Clostridium thermocellum/metabolismo , Biología Computacional , Ensayo de Inmunoadsorción Enzimática , Concentración 50 Inhibidora , Mutación , Análisis por Matrices de Proteínas , Unión Proteica , Estructura Terciaria de Proteína , Programas Informáticos , Especificidad de la Especie , Especificidad por Sustrato , Termodinámica , CohesinasRESUMEN
Cellulosomes are large multicomponent cellulose-degrading assemblies found on the surfaces of cellulolytic microorganisms. Often containing hundreds of components, the self-assembly of cellulosomes is mediated by the ultra-high-affinity cohesin-dockerin interaction, which allows them to adopt the complex architectures necessary for degrading recalcitrant cellulose. Better understanding of how the cellulosome assembles and functions and what kinds of structures it adopts will further effort to develop industrial applications of cellulosome components, including their use in bioenergy production. Ruminococcus flavefaciens is a well-studied anaerobic cellulolytic bacteria found in the intestinal tracts of ruminants and other herbivores. Key to cellulosomal self-assembly in this bacterium is the dockerin ScaADoc, found on the non-catalytic structural subunit scaffoldin ScaA, which is responsible for assembling arrays of cellulose-degrading enzymes. This work expands on previous efforts by conducting a series of binding studies on ScaADoc constructs that contain mutations in their cohesin recognition interface, in order to identify which residues play important roles in binding. Molecular dynamics simulations were employed to gain insight into the structural basis for our findings. A specific residue pair in the first helix of ScaADoc, as well as a glutamate near the C-terminus, was identified to be essential for cohesin binding. By advancing our understanding of the cohesin binding of ScaADoc, this study serves as a foundation for future work to more fully understand the structural basis of cellulosome assembly in R. flavefaciens.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Ácido Glutámico/metabolismo , Ruminococcus/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Celulosa/metabolismo , Celulosomas/química , Celulosomas/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Mutación , Estructura Secundaria de Proteína , CohesinasRESUMEN
The cellulosome is a large extracellular multi-enzyme complex that facilitates the efficient hydrolysis and degradation of crystalline cellulosic substrates. During the course of our studies on the cellulosome of the rumen bacterium Ruminococcus flavefaciens, we focused on the critical ScaA dockerin (ScaADoc), the unique dockerin that incorporates the primary enzyme-integrating ScaA scaffoldin into the cohesin-bearing ScaB adaptor scaffoldin. In the absence of a high-resolution structure of the ScaADoc module, we generated a computational model, and, upon its analysis, we were surprised to discover a putative stacking interaction between an N-terminal Trp and a C-terminal Pro, which we termed intramolecular clasp. In order to verify the existence of such an interaction, these residues were mutated to alanine. Circular dichroism spectroscopy, intrinsic tryptophan and ANS fluorescence, and NMR spectroscopy indicated that mutation of these residues has a destabilizing effect on the functional integrity of the Ca(2+)-bound form of ScaADoc. Analysis of recently determined dockerin structures from other species revealed the presence of other well-defined intramolecular clasps, which consist of different types of interactions between selected residues at the dockerin termini. We propose that this thematic interaction may represent a major distinctive structural feature of the dockerin module.
RESUMEN
Phylogenetic analysis of known dockerins in Ruminococcus flavefaciens revealed a novel subtype, type-III, in the scaffoldin proteins, ScaA, ScaB, ScaC and ScaE. In this study, we explored the Ca²âº-binding properties of the type-III dockerin from the ScaA scaffoldin (ScaADoc) using a battery of structural and biophysical approaches including circular dichroism spectroscopy, isothermal titration calorimetry, differential scanning calorimetry, and nuclear magnetic resonance spectroscopy. Despite the lack of a second canonical Ca²âº-binding loop, the behaviour of ScaADoc is similar with respect to other dockerin protein modules in terms of its responsiveness to Ca²âº and affinity for the cohesin from the ScaB scaffoldin. Our results highlight the robustness of dockerin modules and how their Ca²âº-binding properties can be exploited in the construction of designer cellulosomes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al Calcio/metabolismo , Dominios y Motivos de Interacción de Proteínas , Ruminococcus/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Sitios de Unión , Calcio/metabolismo , Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/aislamiento & purificación , Rastreo Diferencial de Calorimetría , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/aislamiento & purificación , Proteínas de Ciclo Celular/metabolismo , Celulosomas/química , Celulosomas/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/aislamiento & purificación , Proteínas Cromosómicas no Histona/metabolismo , Dicroismo Circular , Motivos EF Hand , Interacciones Hidrofóbicas e Hidrofílicas , Complejos Multiproteicos/química , Complejos Multiproteicos/genética , Complejos Multiproteicos/aislamiento & purificación , Complejos Multiproteicos/metabolismo , Resonancia Magnética Nuclear Biomolecular , Filogenia , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Espectrometría de Fluorescencia , Propiedades de Superficie , CohesinasRESUMEN
Cellulose-degrading enzyme systems are of significant interest from both a scientific and technological perspective due to the diversity of cellulase families, their unique assembly and substrate binding mechanisms, and their potential applications in several key industrial sectors, notably cellulose hydrolysis for second-generation biofuel production. Particularly fascinating are cellulosomes, the multimodular extracellular complexes produced by numerous anaerobic bacteria. Using single-molecule force spectroscopy, we analyzed the mechanical stability of the intermolecular interfaces between the cohesin and the dockerin modules responsible for self-assembly of the cellulosomal components into the multienzyme complex. The observed cohesin-dockerin rupture forces (>120 pN) are among the highest reported for a receptor-ligand system to date. Using an atomic force microscope protocol that quantified single-molecule binding activity, we observed force-induced dissociation of calcium ions from the duplicated loop-helix F-hand motif located within the dockerin module, which in the presence of EDTA resulted in loss of affinity to the cohesin partner. A cohesin amino acid mutation (D39A) that eliminated hydrogen bonding with the dockerin's critically conserved serine residues reduced the observed rupture forces. Consequently, no calcium loss occurred and dockerin activity was maintained throughout multiple forced dissociation events. These results offer insights at the single-molecule level into the stability and folding of an exquisite class of high-affinity protein-protein interactions that dictate fabrication and architecture of cellulose-degrading molecular machines.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Ciclo Celular/química , Proteínas Cromosómicas no Histona/química , Sustitución de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Biofisica , Calcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Clostridium thermocellum/genética , Clostridium thermocellum/metabolismo , Cristalografía por Rayos X , Enlace de Hidrógeno , Microscopía de Fuerza Atómica , Modelos Moleculares , Complejos Multiproteicos/química , Mutagénesis Sitio-Dirigida , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estabilidad Proteica , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Termodinámica , Respuesta de Proteína Desplegada , CohesinasRESUMEN
The interaction between the cohesin and dockerin modules serves to attach cellulolytic enzymes (carrying dockerins) to non-catalytic scaffoldin units (carrying multiple cohesins) in cellulosome, a multienzyme plant cell-wall degrading complex. This interaction is species-specific, for example, the enzyme-borne dockerin from Clostridium thermocellum bacteria binds to scaffoldin cohesins from the same bacteria but not to cohesins from Clostridium cellulolyticum and vice versa. We studied the role of interface residues, contributing either to affinity or specificity, by mutating these residues on the cohesin counterpart from C. thermocellum. The high affinity of the cognate interactions makes it difficult to evaluate the effect of these mutations by common methods used for measuring protein-protein interactions, especially when subtle discrimination between the mutants is needed. We described in this article an approach based on indirect enzyme-linked immunosorbent assay (ELISA) that is able to detect differences in binding between the various cohesin mutants, whereas surface plasmon resonance and standard ELISA failed to distinguish between high-affinity interactions. To be able to calculate changes in energy of binding (ΔΔG) and dissociation constants (K(d)) of mutants relative to wild type, a pre-equilibrium step was added to the standard indirect ELISA procedure. Thus, the cohesin-dockerin interaction under investigation occurs in solution rather than between soluble and immobilized proteins. Unbound dockerins are then detected through their interaction with immobilized cohesins. Because our method allows us to assess the effect of mutations on particularly tenacious protein-protein interactions much more accurately than do other prevalent methods used to measure binding affinity, we therefore suggest this approach as a method of choice for comparing relative binding in high-affinity interactions.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Proteínas de Ciclo Celular/química , Celulasa/química , Celulosomas/química , Proteínas Cromosómicas no Histona/química , Clostridium cellulolyticum/química , Clostridium thermocellum/química , Proteínas Bacterianas/genética , Sitios de Unión , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Celulasa/genética , Proteínas Cromosómicas no Histona/genética , Pruebas de Enzimas , Ensayo de Inmunoadsorción Enzimática , Cinética , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Resonancia por Plasmón de Superficie , Termodinámica , CohesinasRESUMEN
The cellulosome is a large bacterial extracellular multienzyme complex able to degrade crystalline cellulosic substrates. The complex contains catalytic and noncatalytic subunits, interconnected by high-affinity cohesin-dockerin interactions. In this chapter, we introduce an optimized method for comparative binding among different cohesins or cohesin mutants to the dockerin partner. This assay offers advantages over other methods (such as ELISA, cELIA, SPR, and ITC) for particularly high-affinity binding interactions. In this approach, the high-affinity interaction of interest occurs in the liquid phase during the equilibrated binding step, whereas the interaction with the immobilized phase is used only for detection of the unbound dockerins that remain in the solution phase. Once equilibrium conditions are reached, the change in free energy of binding (ΔΔG(binding)), as well as the affinity constant of mutants, can be estimated against the known affinity constant of the wild-type interaction. In light of the above, we propose this method as a preferred alternative for the relative quantification of high-affinity protein interactions.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Celulosomas/enzimología , Proteínas Cromosómicas no Histona/metabolismo , Clostridium thermocellum/enzimología , Ensayo de Inmunoadsorción Enzimática/métodos , Geobacillus stearothermophilus/enzimología , Mapeo de Interacción de Proteínas/métodos , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/aislamiento & purificación , Celulosomas/genética , Celulosomas/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/aislamiento & purificación , Clonación Molecular/métodos , Clostridium thermocellum/genética , Clostridium thermocellum/metabolismo , Escherichia coli/genética , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/metabolismo , Mutación , Unión Proteica , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , CohesinasRESUMEN
The specificity of cohesin-dockerin interactions is critically important for the assembly of cellulosomal enzymes into the multienzyme cellulolytic complex (cellulosome). In order to investigate the origins of the observed specificity, a variety of selected amino acid positions at the cohesin-dockerin interface can be subjected to mutagenesis, and a library of mutants can be constructed. In this chapter, we describe a protein-protein microarray technique based on the high affinity of a carbohydrate-binding module (CBM), attached to mutant cohesins. Using cellulose-coated glass slides, libraries of mutants can be screened for binding to complementary partners. The advantages of this tool are that crude cell lysate can be used without additional purification, and the microarray can be used for screening both large libraries as initial scanning for "positive" plates, and for small libraries, wherein individual colonies are printed on the slide. Since the time-consuming step of purifying proteins can be circumvented, the approach is also appropriate for providing molecular insight into the multicomponent organization of complex cellulosomes.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Celulosa/metabolismo , Celulosomas/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Clostridium thermocellum/enzimología , Ensayos Analíticos de Alto Rendimiento/métodos , Análisis por Matrices de Proteínas/métodos , Proteínas Bacterianas/genética , Proteínas de Ciclo Celular/genética , Celulosomas/enzimología , Celulosomas/genética , Proteínas Cromosómicas no Histona/genética , Clostridium thermocellum/genética , Clostridium thermocellum/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Diseño de Equipo , Ensayos Analíticos de Alto Rendimiento/instrumentación , Mutación , Análisis por Matrices de Proteínas/instrumentación , CohesinasRESUMEN
Type I interferons (IFNs) elicit antiviral, antiproliferative and immunomodulatory properties in cells. All of them bind to the same receptor proteins, IFNAR1 and IFNAR2, with different affinities. While the 13 known IFNalphas are highly conserved, the C-terminal unstructured tail was found to have large variation in its net charge, from neutral to +4. This led us to speculate that the tail may have a role in modulation of the IFN biological activity, through fine-tuning the binding to IFNAR2. To evaluate this hypothesis, we replaced the tail of IFNalpha2 with that of IFNalpha8 and IFNbeta tails, or deleted the last five residues of this segment. Mutations to the more positively charged tail of IFNalpha8 resulted in a 20-fold higher affinity to IFNAR2, which results in a higher antiviral and antiproliferative activity. Double and multiple mutant cycle analysis placed the tail near a negatively charged loop on IFNAR2, comprising of residues Glu 132-134. Deleting the tail resulted in only twofold reduction in binding compared to the wild-type. Next, we modeled the location of the tail using a two-step procedure: first we generated 200 models of the tail docked on IFNAR2 using HADDOCK, second the models were scored according to the fit between experimentally determined rates of association of nine mutant complexes, and their calculated rates using the PARE software. From the results we suggest that the unstructured tail of IFNalpha is gaining a specific structure in the bound state, binding to a groove below the 132-134 loop in IFNAR2.