Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser

Abstract

G protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signaling to numerous G proteinindependent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly, in which rhodopsin uses distinct structural elements, including TM7 and Helix 8 to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ~20° rotation between the Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms § Correspondence to H. Eric Xu: Eric.Xu@vai.org. * These authors contributed equally.Contributions: Y.K. initiated the project, developed the expression and purification methods for rhodopsin-arrestin complex, and bulk-purified expression constructs and proteins used in LCP crystallization for the SFX method; X.E.Z. collected the synchrotron data, helped with the SFX data collection, processed the data, and solved the structures; X.G. expressed and purified rhodopsinarrestin complexes, characterized their binding and thermal stability, discovered the initial crystallization conditions with 9.7 MAG, prepared most crystals for synchrotron data collection, prepared all crystals for the final data collection by SFX, helped with SFX data collection, and established the initial cross-linking method for the rhodopsin-arrestin complex; Y.H. designed and performed Tango assays and disulfide bond cross-linking experiments; C.Z. developed the mammalian expression methods; P.W.dW helped with XFEL data processing and performed computational experiments; J.K., M.H.E.T., K. M. S-P., K. P., J. M., Y.J., X.Y.Z., and Q.C. performed cell culture, mutagenesis, protein purification, rhodopsin-arrestin binding experiments; W.L. and A.I. grew crystals and collected synchrotron data at APS and SFX data at LCLS, G.W.H. and Q.X. determined and validated the structure. Z.Z. and V.K. constructed the full model, the phosphorylated rhodopsin-arrestin model, and help writing the paper; D.W., S.L., D.J., C.K., Sh.B., and N.A. Z. helped with XFEL data collection and initial data analysis; S.B., M.M., and G.J.W. set up the XFEL experiment, performed the data collection, and commented on the paper. A.B., T.W., C.G., O.Y., and H.C. helped with XFEL data collection and data analysis, processed the data and helped with structure validation. G.M. W., B.P., and P.G. performed HDX experiments and helped with manuscript writing. J.L. helped initiate this collaborative project and with writing the paper. M.W. collected the 7.7 Å dataset at Swiss Light Source. A.M.,...

β-arrestin–biased signaling through the β 2 -adrenergic receptor promotes cardiomyocyte contraction

“…Upon GRK-mediated phosphorylation, many GPCRs, including the β 2 AR, bind β-arrestins with high affinity (11). Recently, visualization of the GPCR-arrestin interface was achieved by serial femtosecond X-ray laser crystallography of a rhodopsin/arrestin complex (24). Additional insight was gained through cocrystallization studies of an arrestin finger loop peptide and rhodopsin (25), and by electron microscopy and deuterium exchange analysis of a β-arrestin1 complex with a β 2 AR-vasopressin 2 receptor C-terminal tail fusion (26) (25).…”

Distinct conformations of GPCR–β-arrestin complexes mediate desensitization, signaling, and endocytosis

“…Moreover, GPCRs tend to either interact with βarr transiently, termed "class A" GPCRs [e.g., β 2 -adrenergic receptor (β 2 AR)], or tightly, known as "class B" GPCRs [e.g., vasopressin type 2 receptor (V 2 R)]. For the current study, we use a previously described chimeric β 2 V 2 R construct, which comprises the β 2 AR with its C-terminal tail exchanged with the V 2 R C-terminal tail (12)(13)(14). The β 2 V 2 R construct provides an ideal system for studying a GPCR-βarr complex in vitro, because it maintains identical pharmacological properties to the WT β 2 AR and has a robustly increased class B affinity for βarr1, which allows stable β 2 V 2 R-βarr complexes to be formed and purified.…”

“…A recent structural study of a constitutively active rhodopsin-arrestin fusion protein revealed high-resolution information about a single conformation of the complex in which the arrestin engaged via the transmembrane core of the receptor (12). However, negative-stain electron microscopy (EM) analysis of an antigen-binding fragment 30 (Fab30)-stabilized β 2 V 2 R-βarr1-Fab30 complex demonstrated that the β 2 V 2 R-βarr1 complex assumes two unique conformations: one in which ∼63% of the βarr1 in the complex is bound only to the phosphorylated receptor C-terminal tail and appears to hang from the receptor ("tail" conformation) and a second more fully engaged conformation representing ∼37%, in which, in addition to the tail interaction, the Significance β-Arrestins (βarrs) interact with G protein-coupled receptors (GPCRs) to desensitize G protein signaling, initiate signaling on their own, and mediate receptor endocytosis.…”

Accurate determination of segmented X-ray detector geometry

“…Indexed after geometry refinement Gd:Lysozyme [22] (4N5R) 391,000 42% (164,000) 56% (218,000) Cathepsin B [24] (4HWY) 358,000 56% (201,000) 59% (210,000) DgkA [25] 180,000 60% (109,000) 78% (140,500) Rhodopsin-Arrestin [26] (4ZWJ) 11,000 20% (2,200) 80% (8,800) For data from two published data sets, Gd:Lysozyme [22] (Dataset 1, Ref. [27]) and Cathepsin B [24], the indexing rate improved noticeably after geometry refinement.…”