An Ensemble-Based Protocol for the Computational Prediction of Helix-Helix Interactions in G Protein-Coupled Receptors using Coarse-Grained Molecular Dynamics

The accurate identification of the specific points of interaction between G protein-coupled receptor (GPCR) oligomers is essential for the design of receptor ligands targeting oligomeric receptor targets. A coarse-grained molecular dynamics computer simulation approach would provide a compelling means of identifying these specific protein–protein interactions and could be applied both for known oligomers of interest and as a high-throughput screen to identify novel oligomeric targets. However, to be effective, this in silico modeling must provide accurate, precise, and reproducible information. This has been achieved recently in numerous biological systems using an ensemble-based all-atom molecular dynamics approach. In this study, we describe an equivalent methodology for ensemble-based coarse-grained simulations. We report the performance of this method when applied to four different GPCRs known to oligomerize using error analysis to determine the ensemble size and individual replica simulation time required. Our measurements of distance between residues shown to be involved in oligomerization of the fifth transmembrane domain from the adenosine A2A receptor are in very good agreement with the existing biophysical data and provide information about the nature of the contact interface that cannot be determined experimentally. Calculations of distance between rhodopsin, CXCR4, and β1AR transmembrane domains reported to form contact points in homodimers correlate well with the corresponding measurements obtained from experimental structural data, providing an ability to predict contact interfaces computationally. Interestingly, error analysis enables identification of noninteracting regions. Our results confirm that GPCR interactions can be reliably predicted using this novel methodology

Transmembrane peptide 4 and 5 of APJ are essential for its heterodimerization with OX1R

Increasing evidence indicates some G protein-coupled receptors function as a heterodimer, which provide a novel target for therapeutics investigation. However, study on the receptor-receptor interaction interface, a potent target on interfering dimer formation, are still limited. Here, using bioluminescence resonance energy transfer (BRET) combined with co-immunoprecipitation (Co-IP), we found a new constitutive GPCR heterodimer, apelin receptor (APJ)-orexin receptor type 1 (OX1R). Both APJ and OX1R co-internalized when constantly subjected to cognate agonist (apelin-13 or orexin-A) specific to either protomer. Combined with BRET and immunostaining, the in vitro synthesized transmembrane peptides (TMs) interfering experiments suggests that TM4 and 5 of APJ act as the interaction interface of the APJ-OX1R heterodimer, and co-internalization could be disrupted by these peptides as well. Our study not only provide new evidence on GPCR heterodimerization, but address a novel heterodimerization interface, which can be severed as a potential pharmacological target

A novel coarse-grained molecular dynamics method for the accurate prediction of helix-helix interactions in GPCRs

This thesis describes a novel computational method developed to identify and characterise points of protein-protein interaction between two G protein-coupled receptors (GPCRs). An ensemble-based coarse-grained molecular dynamics (eCG-MD) approach was applied to GPCR oligomers with experimentally-determined contact interfaces (adenosine A2A receptor, rhodopsin, CXCR4 and β1AR). Error analysis was used to determine 1) the number of replicas in an ensemble and 2) the simulation time for each replica that were needed to obtain convergence with experimental results. Error analysis also enabled identification of non-interacting regions. This novel method yielded calculations of distance between rhodopsin, CXCR4 and β1AR transmembrane domains reported to form contact points in homodimers that correlated well with the corresponding measurements obtained from the structural data, demonstrating an ability to predict contact interfaces computationally. The method gave distance measurements between residues shown to be involved in oligomerisation of the fifth transmembrane domain from the adenosine A2A receptor that were in very good agreement with the existing biophysical data. Further, the method provided information about the nature of the contact interface that could not be determined experimentally. This CG-MD method was then used as a high-throughput screen to identify novel sites of interaction in the adenosine A2A receptor, informing the design of future experimental work. Experimental methods to investigate interactions are also described in this thesis. These were less successful in identifying contact points, however, the present computational method will enable novel interaction points between GPCRs to be predicted and tested experimentally using assays of ligand binding and receptor signaling. In conclusion, this work provides an accurate, reproducible and reliable method for determining the specific points of interaction between GPCR dimers. The eCG-MD method discriminates between residues in TM helices that form specific interactions and residues that are in close proximity but do not interact

Diferents estats d’oligomerització entre els receptors 5-HT1A, GALR1 I GPR39 com a noves dianes terapèutiques en depressió

The type of serotonin receptor 1A (5-HT1A), the galanin type 1 (GalR1) and the orphan receptor 39 (GPR39) belong to the superfamily The serotonin receptor type 1A (5-HT1A), the galanin receptor type 1 (GalR1) and the orphan receptor 39 (GPR39) belong to the superfamily of G-protein-coupled receptors. All three GPCRs share, among other features, a relationship with the pathophysiology of unipolar depression associated with presence of zinc. The current paradigm with respect to these functional unities is that these receptors are not acting as monomeric forms but through complexs involving specific interactions with themselves or other receptors from the same family. This allows them to increase their functional possibilities exponentially, allowing versatility from a fixed number of receptors, and consequently also increases the number of pharmacological approachmes. The heterodimerization between 5-HT1A receptor and GalR1 has been previously described as an antagonistic interaction that could lead to unipolar depression. In this thesis we have shown that this interaction is avoided in the presence of zinc, giving a rational explanation for the antidepressant effect widely described for this cation. Although there was no published evidence regarding the interaction of these two receptors with GPR39, a receptor that is activated by zinc and whose expression depends on the concentration of this cation, in this thesis we have demonstrated the ability to interact of these three receptors in both forms GPR39-5-HT1A, 5-HT1A-GalR1 and the trimer GPR39-5-HT1A-GalR1. It has also been found that the functional capacity of the receptors is modified according the type of interaction in which these receptors are involved. So that monomeric and oligomeric forms have different signalling capacities. This would suggest that in the human brain all putative receptor configurations could be present, and that the presence of one or other would be regulated by zinc concentration. Deepening in the detailed molecular mechanism of the effect of zinc on these interactions should allow the development of new drugs for a disease with high prevalence in the nowadays society.El receptor de serotonina del tipus 1A (5-HT1A), el de galanina de tipus 1 (GalR1) i el receptor orfe 39 (GPR39) pertanyen a la superfamília dels receptors acoblats a proteïna G. Tots tres receptors comparteixen, entre d'altres característiques, una relació amb la fisiopatologia de la depressió unipolar associada amb la presència de zinc. El paradigma actual, respecte a la unitat funcional d'aquests receptors, és que no actuen en forma monomèrica sinó mitjançant complexos que involucren interaccions específiques amb ells mateixos o amb altres receptors de la mateixa família. Això els hi permet augmentar les seves possibilitats funcionals exponencialment, permetent una gran versatilitat a partir d'un nombre fix de receptors, i en conseqüència també augmenta el nombre de possibilitats d'abordatge farmacològic. L'heterodimerització entre el receptor 5-HT1A i el GalR1 s'ha descrit prèviament com una interacció antagònica que podria donar lloc a la depressió unipolar. En aquesta tesi s'ha demostrat que aquesta interacció s'evita en presència de zinc, donant una explicació racional a l'efecte antidepressiu àmpliament descrit per a aquest catió. Tot i que no hi havia cap evidència publicada respecte a la interacció d'aquests dos receptors amb el GPR39, un receptor que és activat pel zinc i l'expressió del qual depèn de la concentració del mateix, en aquesta tesi s'ha demostrat la capacitat d'interacció d'aquests tres receptors, tant en les formes GPR39-5-HT1A i 5-HT1A-GalR1 com en el trímer GPR39-5-HT1A-GalR1. A més, s'ha trobat que la capacitat funcional dels receptors es veu modificada segons el tipus d'interacció en la que aquests receptors participen, de manera que les formes monomèriques i oligomèriques presenten una capacitat de senyalització diferent. Això faria pensar que en el cervell humà podrien trobar-se totes les configuracions potencials de receptors, i que la presència d'unes o altres estaria regulada per la concentració de zinc. L'aprofundiment en el mecanisme molecular detallat de l'efecte del zinc en aquestes interaccions hauria de permetre el desenvolupament de nous fàrmacs per a una malaltia d'alta prevalença en la població mundial

Homodimerization of neurotensin 1 receptor involves helices 1, 2, and 4: insights from quaternary structure predictions and dimerization free energy estimations.

A computational approach based upon rigid-body docking, ad hoc filtering, and cluster analysis has been combined with a protocol for dimerization free energy estimations to predict likely interfaces in the neurotensin 1 receptor (NTS1) homodimers. The results of this study suggest that the likely intermonomer interfaces compatible with in vitro binding affinity constants essentially involve helices 1, 2, and 4 and do not include disulfide bridges. The correlative model initially developed on Glycophorin A and herein extended to a G protein-Coupled Receptor (GPCR) appears to be a useful tool for estimating the association free energies of transmembrane proteins independent of the size and shape of the interface. In the desirable future cases, in which in vitro intermonomer binding affinities will be available for other GPCRs, such a correlative model will work as an additional criterion for helping in the selection of the most likely dimers

Diseño y evaluación de inhibidores peptídicos dirigidos a la interfaz de Dimerización de la Tripanotjón Reductasa de Leishmania Infantum

Entre las muchas peculiaridades que se encuentran al estudiar los organismos tripanosomátidos de interés clínico, una de las más interesantes desde el punto de vista de la identificación de nuevas dianas terapéuticas es la forma como este parásito destoxifica su ambiente intracelular de especies oxidantes. Al carecer del ubicuo sistema glutatión/glutatión reductasa, en su lugar, el parásito cuenta con un sistema análogo modificado que emplea el tripanotión (T[SH]2), que no es más que dos moléculas de glutatión unidas por un puente espermidina. Esta unión permite que los tioles activos de la molécula se encuentren en más proximidad haciéndola mejor reductor que el glutatión. En el centro del metabolismo del T(SH)2 se encuentra la tripanotión reductasa (TryR), una enzima esencial para la supervivencia del parásito que está encargada de mantener en todo momento un pool de tripanotión reducido dentro de la célula. Esta enzima es un homodímero que cuenta con dos centros activos en los que participan residuos de ambas subunidades, haciendo de su configuración dimérica una característica fundamental para su actividad. Al sintetizar un péptido que emula una de las hélices que hacen parte de la interfaz de dimerización ubicada donde ambas subunidades hacen contacto, hemos sido capaces de desestabilizar el dímero de la enzima y minimizar su actividad. Se han desarrollado distintas variantes de este péptido con el fin de mejorar su estabilidad y potenciar su actividad. Entre las modificaciones realizadas se encuentran (1) la sustitución por alanina de cada uno de sus aminoácidos para evaluar su relevancia, (2) el acortamiento del péptido para determinar la secuencia mínima que conserva la actividad deseada y (3) la creación de péptidos estructuralmente restringidos en los que se intentan mantener las características estructurales de la hélice original. A partir de las exploraciones realizadas se obtuvieron dos péptidos especialmente relevantes: TRL35, (Ac-PKIIQSVGISNLEKNLE-NH2) y TRL38 (Ac-PKIIQSVGI-NH2), de 13 y 9 aminoácidos, respectivamente. Además, basándonos en las interacciones que se dan entre la enzima y el péptido corto deducidas de las simulaciones de dinámica molecular, se desarrolló un modelo farmacofórico que permitió la identificación de moléculas pequeñas con capacidad de inhibir la actividad enzimática. Para finalizar, hemos caracterizado bioquímicamente la interacción entre TRL35 y TryR, logrando identificar el modelo mediante el cual se da esta inhibición y las implicaciones de esta en la estabilidad de la estructura globular de la enzima

The HBP1 tumor suppressor is a negative epigenetic regulator of MYCN driven neuroblastoma through interaction with the PRC2 complex

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