RESUMO
Aquaporins are transmembrane proteins that facilitate the flow of water through cellular membranes. An unusual characteristic of yeast aquaporins is that they frequently contain an extended N terminus of unknown function. Here we present the X-ray structure of the yeast aquaporin Aqy1 from Pichia pastoris at 1.15 A resolution. Our crystal structure reveals that the water channel is closed by the N terminus, which arranges as a tightly wound helical bundle, with Tyr31 forming H-bond interactions to a water molecule within the pore and thereby occluding the channel entrance. Nevertheless, functional assays show that Aqy1 has appreciable water transport activity that aids survival during rapid freezing of P. pastoris. These findings establish that Aqy1 is a gated water channel. Mutational studies in combination with molecular dynamics simulations imply that gating may be regulated by a combination of phosphorylation and mechanosensitivity.
Assuntos
Aquaporinas/química , Aquaporinas/metabolismo , Ativação do Canal Iônico , Pichia/química , Transporte Biológico , Simulação por Computador , Cristalografia por Raios X , Congelamento , Viabilidade Microbiana , Modelos Moleculares , Fosforilação , Estrutura Secundária de Proteína , Spinacia oleracea/química , Homologia Estrutural de Proteína , Tirosina/metabolismo , ÁguaRESUMO
This work presents a comparison of the crystal packing of three eukaryotic membrane proteins: human aquaporin 1, human aquaporin 5 and a spinach plasma membrane aquaporin. All were purified from expression constructs both with and without affinity tags. With the exception of tagged aquaporin 1, all constructs yielded crystals. Two significant effects of the affinity tags were observed: crystals containing a tag typically diffracted to lower resolution than those from constructs encoding the protein sequence alone and constructs without a tag frequently produced crystals that suffered from merohedral twinning. Twinning is a challenging crystallographic problem that can seriously hinder solution of the structure. Thus, for integral membrane proteins, the addition of an affinity tag may help to disrupt the approximate symmetry of the protein and thereby reduce or avoid merohedral twinning.
Assuntos
Marcadores de Afinidade/química , Aquaporinas/química , Pichia , Proteínas de Plantas/química , Proteínas Recombinantes/química , Marcadores de Afinidade/metabolismo , Aquaporinas/genética , Aquaporinas/metabolismo , Cromatografia de Afinidade , Clonagem Molecular , Cristalização , Cristalografia por Raios X/métodos , Humanos , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estrutura Secundária de Proteína/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spinacia oleraceaRESUMO
Accumulating evidence suggests that dysregulated glycerol metabolism contributes to the pathophysiology of obesity and type 2 diabetes. Glycerol efflux from adipocytes is regulated by the aquaglyceroporin AQP7, which is translocated upon hormone stimulation. Here, we propose a molecular mechanism where the AQP7 mobility in adipocytes is dependent on perilipin 1 and protein kinase A. Biochemical analyses combined with ex vivo studies in human primary adipocytes, demonstrate that perilipin 1 binds to AQP7, and that catecholamine activated protein kinase A phosphorylates the N-terminus of AQP7, thereby reducing complex formation. Together, these findings are indicative of how glycerol release is controlled in adipocytes, and may pave the way for the future design of drugs against human metabolic pathologies.