RESUMO
The serotonin transporter (SERT) is the primary target for the selective serotonin reuptake inhibitors (SSRIs). However, the structural basis for the extraordinarily high binding affinity of the widely prescribed SSRI, paroxetine, to human SERT (hSERT) has not yet been fully elucidated. Our previous findings unveiled a plausible ambiguity in paroxetine's binding orientations that may constitute an integral component of this SSRI's high affinity for hSERT. Herein, we investigate factors contributing to paroxetine's high affinity by modifying both the ligand and the protein. We generated a series of bromine (Br)-containing derivatives and found that the one in which the 4-F of paroxetine had been replaced with the chemically similar but more electron-rich Br atom (13) had the highest affinity. By comparatively characterizing the binding of paroxetine and 13 to both wild type (WT) and a construct harboring a paroxetine-sensitive mutation in the binding cavity, we identified a mechanistic determinant responsible for the pose ambiguity of paroxetine, which can guide future drug design.
Assuntos
Bromo/metabolismo , Paroxetina/análogos & derivados , Paroxetina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Bromo/química , Cristalografia por Raios X/métodos , Células HEK293 , Células HeLa , Humanos , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Inibidores Seletivos de Recaptação de Serotonina/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/farmacologiaRESUMO
The serotonin transporter (SERT) is one of the primary targets for medications to treat neuropsychiatric disorders and functions by exploiting pre-existing ion gradients of Na+, Cl-, and K+ to translocate serotonin from the synaptic cleft into the presynaptic neuron. Although recent hSERT crystal structures represent a milestone for structure-function analyses of mammalian neurotransmitter:sodium symporters, they are all derived from thermostabilized but transport-deficient constructs. Two of these structures are in complex with paroxetine, the most potent selective serotonin reuptake inhibitor known. In this study, by carrying out and analyzing the results of extensive and comparative molecular dynamics simulations while also re-evaluating the transport and binding properties of the thermostabilized constructs, we identified functionally important structural elements that are perturbed by these mutations, revealed unexpected dynamics in the central primary binding site of SERT, and uncovered a conceivable ambiguity in paroxetine's binding orientation. We propose that the favored entropy contribution plays a significant role in paroxetine's extraordinarily high affinity for SERT. Our findings lay the foundation for future mechanistic studies and rational design of high-affinity SERT inhibitors. This article is part of the issue entitled 'Special Issue on Neurotransmitter Transporters'.
Assuntos
Paroxetina/metabolismo , Inibidores Seletivos de Recaptação de Serotonina/metabolismo , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo , Transporte Biológico Ativo , Entropia , Humanos , Cinética , Modelos Moleculares , Simulação de Dinâmica Molecular , Mutação/genética , Ligação Proteica , Conformação Proteica , Proteínas da Membrana Plasmática de Transporte de Serotonina/químicaRESUMO
Transporters essential for neurotransmission in mammalian organisms and bacterial multidrug transporters involved in antibiotic resistance are evolutionarily related. To understand in more detail the evolutionary aspects of the transformation of a bacterial multidrug transporter to a mammalian neurotransporter and to learn about mechanisms in a milieu amenable for structural and biochemical studies, we identified, cloned, and partially characterized bacterial homologues of the rat vesicular monoamine transporter (rVMAT2). We performed preliminary biochemical characterization of one of them, Brevibacillus brevis monoamine transporter (BbMAT), from the bacterium B. brevis. BbMAT shares substrates with rVMAT2 and transports them in exchange with >1H(+), like the mammalian transporter. Here we present a homology model of BbMAT that has the standard major facilitator superfamily fold; that is, with two domains of six transmembrane helices each, related by 2-fold pseudosymmetry whose axis runs normal to the membrane and between the two halves. The model predicts that four carboxyl residues, a histidine, and an arginine are located in the transmembrane segments. We show here that two of the carboxyls are conserved, equivalent to the corresponding ones in rVMAT2, and are essential for H(+)-coupled transport. We conclude that BbMAT provides an excellent experimental paradigm for the study of its mammalian counterparts and bacterial multidrug transporters.