RESUMEN
The activation of N-methyl-d-aspartate receptor (NMDAR) is triggered by the closure of bilobed (D1 and D2) clamshell-like clefts upon binding glycine (Gly) and glutamate. There is evidence that cholinergic compounds modulate NMDAR-mediated currents via direct receptor-ligand interactions; however, molecular bases are unknown. Here, we first propose a mechanistic structure-based explanation for the observed ACh-induced submaximal potentiation of NMDA-elicited currents in striatal neurons by predicting competitive inhibition with Gly. Then, the model was validated, in principle, by confirming that the coapplication of Gly and ACh significantly reduces these neuronal currents. Finally, we delineate the interplay of ACh with the NMDAR by a combination of computational strategies. Crystallographic ACh-bound complexes were studied, revealing a similar ACh binding environment on the GluN1 subunit of the NMDAR. We illustrate how ACh can occupy X-ray monomeric open, dimeric "semiopen" cleft conformations obtained by molecular dynamics and a full-active cryo-EM NMDAR structure, explaining the suboptimal NMDAR electrophysiological activity under the "Venus Flytrap model". At an evolutionary biology level, the binding mode of ACh coincides with that of the homologous ornithine-bound periplasmic LAO binding protein complex. Our computed results indicate an analogous mechanism of action, inasmuch as ACh may stabilize the GluN1 subunit "semiclosed" conformations by inducing direct and indirect D1-to-D2 interdomain bonds. Additionally, an alternative binding site was detected, shared by the known NMDAR allosteric modulators. Experimental and computed results strongly suggest that ACh acts as a Gly-competitive, submaximal potentiating agent of the NMDAR, possibly constituting a novel chemotype for multitarget-directed drug development, e.g., to treat Alzheimer's, and it may lead to a new understanding of glutamatergic neurotransmission.
Asunto(s)
Acetilcolina , Receptores de N-Metil-D-Aspartato , Glicina/farmacología , N-Metilaspartato , NeuronasRESUMEN
Activation of T cells triggers the expression of regulatory molecules like the programmed cell death 1 (PD1) protein. The association of PD1 with the natural ligands PDL1 and PDL2 induces an inhibitory signal that prevents T cells from proliferating and exerting effector functions. However, little is known about how the binding of the ligands induce the PD1 inhibitory signal over T cells effector functions. Here, we explore the dynamics of PD1 free, and in complex with different PDL1 variants as well as the therapeutic antibodies nivolumab and pembrolizumab in order to assess the conformational changes in PD1 related to the signaling process. Our simulations suggest a pre-conformational selection mechanism for the binding of the different PDL1 variants, while an induced-fit model fits better for the molecular recognition process of the therapeutic antibodies. A deep analysis of the changes on PD1 movement upon the binding to different ligands revealed that as larger is the difference in the conformation adopted by loop C'D with respect to the complex with PDL1 is higher the ligand ability to reduce the PD1 inhibitory signaling. This behavior suggests that targeting specific conformations of this loop can be useful for designing therapies able to recover T cells effector functions.
Asunto(s)
Anticuerpos Monoclonales Humanizados/química , Antígeno B7-H1/química , Nivolumab/química , Receptor de Muerte Celular Programada 1/química , Anticuerpos Monoclonales Humanizados/inmunología , Anticuerpos Monoclonales Humanizados/metabolismo , Antineoplásicos Inmunológicos , Antígeno B7-H1/genética , Antígeno B7-H1/inmunología , Sitios de Unión , Expresión Génica , Humanos , Ligandos , Simulación de Dinámica Molecular , Nivolumab/inmunología , Nivolumab/metabolismo , Análisis de Componente Principal , Receptor de Muerte Celular Programada 1/genética , Receptor de Muerte Celular Programada 1/inmunología , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Transducción de SeñalRESUMEN
In this work, we investigated how activity and oligomeric state are related in a purified GH1 ß-glucosidase from Spodoptera frugiperda (Sfßgly). Gel filtration chromatography coupled to a multiple angle light scattering detector allowed separation of the homodimer and monomer states and determination of the dimer dissociation constant (KD ), which was in the micromolar range. Enzyme kinetic parameters showed that the dimer is on average 2.5-fold more active. Later, we evaluated the kinetics of homodimerization, scanning the changes in the Sfßgly intrinsic fluorescence over time when the dimer dissociates into the monomer after a large dilution. We described how the rate constant of monomerization (koff ) is affected by temperature, revealing the enthalpic and entropic contributions to the process. We also evaluated how the rate constant (kobs ) by which equilibrium is reached after dimer dilution behaves when varying the initial Sfßgly concentration. These data indicated that Sfßgly dimerizes through the conformational selection mechanism, in which the monomer undergoes a conformational exchange and then binds to a similar monomer, forming a more active homodimer. Finally, we noted that conformational selection reports and experiments usually rely on a ligand whose concentration is in excess, but for homodimerization, this approach does not hold. Hence, since our approach overcomes this limitation, this study not only is a new contribution to the comprehension of GH1 ß-glucosidases, but it can also help to elucidate protein interaction pathways.
Asunto(s)
Glicósido Hidrolasas/química , Proteínas de Insectos/química , Multimerización de Proteína , Spodoptera/enzimología , Animales , Glicósido Hidrolasas/genética , Proteínas de Insectos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Spodoptera/genéticaRESUMEN
The present study aims at numerically describing to what extent substrate - enzyme complexes in solution may change over time as a natural process of conformational changes for a liganded enzyme in comparison to those movements which occur independently from substrate interaction, i.e. without a ligand. To this end, we selected structurally known pairs of liganded / unliganded CYP450 3A4 enzymes with different geometries hinting at induced fit events. We carried out molecular dynamics simulations (MD) comparing the trajectories in a "cross-over" protocol: (i) we added the ligand to the unliganded crystal form which should adopt geometries similar to the known geometry of the liganded crystal structure during MD, and - conversely - (ii) we removed the bound ligand form the known liganded complex to test if a geometry similar to the known unliganded (apo-) form can be adopted during MD. To compare continues changes we measured root means square deviations and frequencies. Results for case (i) hint at larger conformational changes required for accepting the substrate during its approach to final position - in contrast to case (ii) when mobility is fairly reduced by ligand binding (strain energy). In conclusion, a larger conformational sampling prior to ligand binding and the freezing-in (rigidity) of conformations for bound ligands can be interpreted as two conditions linked to induced-fit.
RESUMEN
Conformational rearrangements in antibody·antigen recognition are essential events where kinetic discrimination of isomers expands the universe of combinations. We investigated the interaction mechanism of a monoclonal antibody, M1, raised against E7 from human papillomavirus, a prototypic viral oncoprotein and a model intrinsically disordered protein. The mapped 12-amino acid immunodominant epitope lies within a "hinge" region between the N-terminal intrinsically disordered and the C-terminal globular domains. Kinetic experiments show that despite being within an intrinsically disordered region, the hinge E7 epitope has at least two populations separated by a high energy barrier. Nuclear magnetic resonance traced the origin of this barrier to a very slow (t(1/2)â¼4 min) trans-cis prolyl isomerization event involving changes in secondary structure. The less populated (10%) cis isomer is the binding-competent species, thus requiring the 90% of molecules in the trans configuration to isomerize before binding. The association rate for the cis isomer approaches 6 × 10(7) M(-1) s(-1), a ceiling for antigen-antibody interactions. Mutagenesis experiments showed that Pro-41 in E7Ep was required for both binding and isomerization. After a slow postbinding unimolecular rearrangement, a consolidated complex with K(D) = 1.2 × 10(-7) M is reached. Our results suggest that presentation of this viral epitope by the antigen-presenting cells would have to be "locked" in the cis conformation, in opposition to the most populated trans isomer, in order to select the specific antibody clone that goes through affinity and kinetic maturation.