RESUMEN
ABCB10 is one of the three ATP-binding cassette (ABC) transporters found in the inner membrane of mitochondria. In mammals ABCB10 is essential for erythropoiesis, and for protection of mitochondria against oxidative stress. ABCB10 is therefore a potential therapeutic target for diseases in which increased mitochondrial reactive oxygen species production and oxidative stress play a major role. The crystal structure of apo-ABCB10 shows a classic exporter fold ABC transporter structure, in an open-inwards conformation, ready to bind the substrate or nucleotide from the inner mitochondrial matrix or membrane. Unexpectedly, however, ABCB10 adopts an open-inwards conformation when complexed with nonhydrolysable ATP analogs, in contrast to other transporter structures which adopt an open-outwards conformation in complex with ATP. The three complexes of ABCB10/ATP analogs reported here showed varying degrees of opening of the transport substrate binding site, indicating that in this conformation there is some flexibility between the two halves of the protein. These structures suggest that the observed plasticity, together with a portal between two helices in the transmembrane region of ABCB10, assist transport substrate entry into the substrate binding cavity. These structures indicate that ABC transporters may exist in an open-inwards conformation when nucleotide is bound. We discuss ways in which this observation can be aligned with the current views on mechanisms of ABC transporters.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Conformación Molecular , Nucleótidos/química , Estructura Terciaria de Proteína , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Cristalografía por Rayos X , Humanos , Membrana Dobles de Lípidos/química , Membrana Dobles de Lípidos/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutación , Nucleótidos/metabolismo , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Unión Proteica , Homología de Secuencia de Aminoácido , Células Sf9RESUMEN
TREK-2 (KCNK10/K2P10), a two-pore domain potassium (K2P) channel, is gated by multiple stimuli such as stretch, fatty acids, and pH and by several drugs. However, the mechanisms that control channel gating are unclear. Here we present crystal structures of the human TREK-2 channel (up to 3.4 angstrom resolution) in two conformations and in complex with norfluoxetine, the active metabolite of fluoxetine (Prozac) and a state-dependent blocker of TREK channels. Norfluoxetine binds within intramembrane fenestrations found in only one of these two conformations. Channel activation by arachidonic acid and mechanical stretch involves conversion between these states through movement of the pore-lining helices. These results provide an explanation for TREK channel mechanosensitivity, regulation by diverse stimuli, and possible off-target effects of the serotonin reuptake inhibitor Prozac.