RESUMEN
JIP3 and JIP4, two highly related scaffolding proteins for MAP kinases, are binding partners for two molecular motors as well as for the small G protein ARF6. The leucine zipper II (LZII) region of JIP3/4 is the binding site for these three partners. Previously, the crystal structure of ARF6 bound to JIP4 revealed LZII in a parallel coiled-coil arrangement. Here, the crystal structure of an N-terminally truncated form of LZII of JIP3 alone shows an unexpected antiparallel arrangement. Using molecular dynamics and modelling, the stability of this antiparallel LZII arrangement, as well as its specificity for ARF6, were investigated. This study highlights that N-terminal truncation of LZII can change its coiled-coil orientation without affecting its overall stability. Further, a conserved buried asparagine residue was pinpointed as a possible structural determinant for this dramatic structural rearrangement. Thus, LZII of JIP3/4 is a versatile structural motif, modifications of which can impact partner recognition and thus biological function.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/química , Proteínas del Tejido Nervioso/química , Secuencia de Aminoácidos , Cristalización , Cristalografía por Rayos X , Humanos , Leucina Zippers , Simulación de Dinámica Molecular , Fragmentos de Péptidos/química , Dominios y Motivos de Interacción de Proteínas , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
RGK proteins are atypical small GTP-binding proteins that are involved in the regulation of voltage-dependent calcium channels and actin cytoskeleton remodelling. The structure of the Rem2 G domain bound to GDP is reported here in a monoclinic crystal form at 2.66 Å resolution. It is very similar to the structure determined previously from an orthorhombic crystal form. However, differences in the crystal-packing environment revealed that the switch I and switch II regions are flexible and not ordered as previously reported. Comparison of the available RGK protein structures along with those of other small GTP-binding proteins highlights two structural features characteristic of this atypical family and suggests that the conserved tryptophan residue in the DXWEX motif may be a structural determinant of the nucleotide-binding affinity.
Asunto(s)
Proteínas de Unión al GTP Monoméricas/química , Dominios y Motivos de Interacción de Proteínas , Secuencia de Aminoácidos , Animales , Guanosina Difosfato , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas de Unión al GTP Monoméricas/metabolismo , Estructura Terciaria de Proteína , Ratas , Alineación de SecuenciaRESUMEN
G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors and are of major therapeutic importance. Structure determination of G protein-coupled receptors and other applications require milligram quantities of purified receptor proteins on a regular basis. Recombinant GPCRs fused to a heterologous biotinylation domain were produced in the yeast Pichia pastoris. We describe an efficient method for their rapid purification that relies on the capture of these receptors with streptavidin immobilized on agarose beads, and their subsequent release by enzymatic digestion with TEV protease. This method has been applied to several GPCRs belonging to the class A rhodopsin subfamily, leading to high yields of purified proteins; it represents a method of choice for biochemical and biophysical studies when large quantities of purified GPCRs are needed.