RESUMEN
Sorting nexins (SNXs) or phox homology (PX) domain containing proteins are central regulators of cell trafficking and signaling. A subfamily of PX domain proteins possesses two unique PX-associated domains, as well as a regulator of G protein-coupled receptor signaling (RGS) domain that attenuates Gαs-coupled G protein-coupled receptor signaling. Here we delineate the structural organization of these RGS-PX proteins, revealing a protein family with a modular architecture that is conserved in all eukaryotes. The one exception to this is mammalian SNX19, which lacks the typical RGS structure but preserves all other domains. The PX domain is a sensor of membrane phosphoinositide lipids and we find that specific sequence alterations in the PX domains of the mammalian RGS-PX proteins, SNX13, SNX14, SNX19, and SNX25, confer differential phosphoinositide binding preferences. Although SNX13 and SNX19 PX domains bind the early endosomal lipid phosphatidylinositol 3-phosphate, SNX14 shows no membrane binding at all. Crystal structures of the SNX19 and SNX14 PX domains reveal key differences, with alterations in SNX14 leading to closure of the binding pocket to prevent phosphoinositide association. Our findings suggest a role for alternative membrane interactions in spatial control of RGS-PX proteins in cell signaling and trafficking.
Asunto(s)
Proteínas de Unión al GTP/metabolismo , Fosfatos de Fosfatidilinositol/química , Proteínas Recombinantes de Fusión/química , Nexinas de Clasificación/química , Secuencia de Aminoácidos , Animales , Sitios de Unión , Movimiento Celular , Secuencia Conservada , Cristalografía por Rayos X , Endosomas/química , Endosomas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Unión al GTP/genética , Expresión Génica , Regulación de la Expresión Génica , Células HeLa , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Fosfatos de Fosfatidilinositol/metabolismo , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Transducción de Señal , Nexinas de Clasificación/genética , Nexinas de Clasificación/metabolismoRESUMEN
The remarkable potency and pharmacological diversity of animal venoms has made them an increasingly valuable source of lead molecules for drug and insecticide discovery. Nevertheless, most of the chemical diversity encoded within these venoms remains uncharacterized, despite decades of research, in part because of the small quantities of venom available. However, recent advances in the miniaturization of bioassays and improvements in the sensitivity of mass spectrometry and NMR spectroscopy have allowed unprecedented access to the molecular diversity of animal venoms. Here, we discuss these technological developments in the context of establishing a high-throughput pipeline for venoms-based drug discovery.