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Structural studies show that the activity of the G-protein-coupled receptor Smoothened is modulated by ligand-regulated interactions between its extracellular and transmembrane domains. Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzled-class G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked atop the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD–linker domain–TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains. Smoothened structure — with added cholesterol Smoothened (SMO) is a G-protein-coupled receptor that transduces Hedgehog (Hh) signals across the membrane in all animals. Despite being a key developmental regulator, oncoprotein and drug target in oncology, the mechanism by which SMO is activated has remained unknown. These authors solve the 3.2 Å crystal structure of SMO containing its extracellular cysteine-rich (CRD), linker and heptahelical G-protein-coupled receptor (TMD) domains. Surprisingly, a cholesterol molecule was bound to SMO in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of SMO to transmit native Hh signals. Binding of the potent antagonist and anti-cancer drug vismodegib leads to a number of conformational changes and the loss of cholesterol from the CRD–linker domain–TMD interface.

Structural, biochemical and biophysical studies of eukaryotic soluble and membrane proteins require their production in milligram quantities. Although large-scale protein expression strategies based on transient or stable transfection of mammalian cells are well established, they are associated with high consumable costs, limited transfection efficiency or long and tedious selection of clonal cell lines. Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines. In this protocol, we describe the design and step-by-step application of a lentiviral plasmid suite, termed pHR-CMV-TetO2, for the constitutive or inducible large-scale production of soluble and membrane proteins in HEK293 cell lines. Optional features include bicistronic co-expression of fluorescent marker proteins for enrichment of co-transduced cells using cell sorting and of biotin ligase for in vivo biotinylation. We demonstrate the efficacy of the method for a set of soluble proteins and for the G-protein-coupled receptor (GPCR) Smoothened (SMO). We further compare this method with baculovirus transduction of mammalian cells (BacMam), using the type-A γ-aminobutyric acid receptor (GABAAR) β3 homopentamer as a test case. The protocols described here are optimized for simplicity, speed and affordability; lead to a stable polyclonal cell line and milligram-scale amounts of protein in 3–4 weeks; and routinely achieve an approximately three- to tenfold improvement in protein production yield per cell as compared to transient transduction or transfection.

Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility. Cells must communicate with each other to coordinate the development of most tissues and organs. Damage to these communication systems is often seen in degenerative disorders and in cancer. The Hedgehog signaling pathway is one of a handful of these critical systems. Reduced Hedgehog signals can lead to birth defects, while excessive Hedgehog signals can lead to skin and brain cancers. Cells transmit the Hedgehog signal by releasing a protein into their surroundings, where it can influence neighboring cells. Despite years of study, it is not understood how the Hedgehog signal is transmitted from the outside to the inside of a receiving cell. Studies first done in flies and subsequently confirmed in humans have shown that a protein called Smoothened is needed to transmit the Hedgehog signal across the membrane of receiving cells. But it was not known how Smoothened carries out this critical signaling step to influence gene activation inside the cell and consequently to change cell behavior. Now, Luchetti, Sircar et al. find that cholesterol, an important component of the cell membrane, directly binds to Smoothened and changes its shape so that it can activate Hedgehog signaling components inside cells. The experiments made use of mouse cells, and the discovery shows that cholesterol may play a previously underappreciated role in cell-to-cell communication. This newly discovered role for cholesterol has implications for diseases, including a unique set of developmental disorders caused by abnormalities in pathways that produce cholesterol in human cells. Furthermore, this unexpected insight into Smoothened’s activity may be clinically important, because Smoothened can cause cancer when mutated and is the target of anti-cancer drugs that are being used in the clinic. Following on from these findings, a major step will be to uncover if and how Hedgehog signals regulate cholesterol to allow Smoothened to transmit these signals across the cell membrane.

Previously we proposed that transmission of the hedgehog signal across the plasma membrane by Smoothened is triggered by its interaction with cholesterol (Luchetti et al., 2016). But how is cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct pools of cholesterol, we find that Smoothened activation and hedgehog signaling are driven by a biochemically-defined, small fraction of membrane cholesterol, termed accessible cholesterol. Increasing cholesterol accessibility by depletion of sphingomyelin, which sequesters cholesterol in complexes, amplifies hedgehog signaling. Hedgehog ligands increase cholesterol accessibility in the membrane of the primary cilium by inactivating the transporter-like protein Patched 1. Trapping this accessible cholesterol blocks hedgehog signal transmission across the membrane. Our work shows that the organization of cholesterol in the ciliary membrane can be modified by extracellular ligands to control the activity of cilia-localized signaling proteins.

Ionotropic glutamate receptor (iGIuR) family members are integrated into supramolecular complexes that modulate their location and function at excitatory synapses. However, a lack of structural information beyond isolated receptors or fragments thereof currently limits the mechanistic understanding of physiological iGIuR signaling. Here, we report structural and functional analyses of the prototypical molecular bridge linking postsynaptic iGIuR δ2 (GIuD2) and presynaptic β-neurexin 1 (β-NRX1) via CbIn1, a C1q-like synaptic organizer. We show how CbIn1 hexamers \"anchor\" GIuD2 amino-terminal domain dimers to monomeric β-NRX1. This arrangement promotes synaptogenesis and is essential for D-serine–dependent GIuD2 signaling in vivo, which underlies long-term depression of cerebellar parallel fiber–Purkinje cell (PF-PC) synapses and motor coordination in developing mice. These results lead to a model where protein and small-molecule ligands synergistically control synaptic iGIuR function.

Cryo-electron microscopy (cryo-EM) enables the determination of membrane protein structures in native-like environments. Characterising how membrane proteins interact with the surrounding membrane lipid environment is assisted by resolution of lipid-like densities visible in cryo-EM maps. Nevertheless, establishing the molecular identity of putative lipid and/or detergent densities remains challenging. Here we present LipIDens, a pipeline for molecular dynamics (MD) simulation-assisted interpretation of lipid and lipid-like densities in cryo-EM structures. The pipeline integrates the implementation and analysis of multi-scale MD simulations for identification, ranking and refinement of lipid binding poses which superpose onto cryo-EM map densities. Thus, LipIDens enables direct integration of experimental and computational structural approaches to facilitate the interpretation of lipid-like cryo-EM densities and to reveal the molecular identities of protein-lipid interactions within a bilayer environment. We demonstrate this by application of our open-source LipIDens code to ten diverse membrane protein structures which exhibit lipid-like densities. Interpretation of lipid-like densities in cryo-EM structures of membrane proteins is challenging. Here authors present LipIDens, enabling molecular dynamics analysis of protein-lipid interactions.

A long-standing mystery in vertebrate Hedgehog signaling is how Patched 1 (PTCH1), the receptor for Hedgehog ligands, inhibits the activity of Smoothened, the protein that transmits the signal across the membrane. We previously proposed (Kinnebrew et al., 2019) that PTCH1 inhibits Smoothened by depleting accessible cholesterol from the ciliary membrane. Using a new imaging-based assay to directly measure the transport activity of PTCH1, we find that PTCH1 depletes accessible cholesterol from the outer leaflet of the plasma membrane. This transport activity is terminated by binding of Hedgehog ligands to PTCH1 or by dissipation of the transmembrane potassium gradient. These results point to the unexpected model that PTCH1 moves cholesterol from the outer to the inner leaflet of the membrane in exchange for potassium ion export in the opposite direction. Our study provides a plausible solution for how PTCH1 inhibits SMO by changing the organization of cholesterol in membranes and establishes a general framework for studying how proteins change cholesterol accessibility to regulate membrane-dependent processes in cells.

Semaphorin-plexin cell-cell signaling is important in tissue development, with roles in axon guidance, immunity and cancer. The structure of the complex formed between semaphorin-3, plexin-A and their co-receptor neuropilin, combined with mutagenesis, reveals how neuropilin contributes to stabilizing the signaling complex. Co-receptors add complexity to cell-cell signaling systems. The secreted semaphorin 3s (Sema3s) require a co-receptor, neuropilin (Nrp), to signal through plexin As (PlxnAs) in functions ranging from axon guidance to bone homeostasis, but the role of the co-receptor is obscure. Here we present the low-resolution crystal structure of a mouse semaphorin–plexin–Nrp complex alongside unliganded component structures. Dimeric semaphorin, two copies of plexin and two copies of Nrp are arranged as a dimer of heterotrimers. In each heterotrimer subcomplex, semaphorin contacts plexin, similar to in co-receptor–independent signaling complexes. The Nrp1s cross brace the assembly, bridging between sema domains of the Sema3A and PlxnA2 subunits from the two heterotrimers. Biophysical and cellular analyses confirm that this Nrp binding mode stabilizes a canonical, but weakened, Sema3–PlxnA interaction, adding co-receptor control over the mechanism by which receptor dimerization and/or oligomerization triggers signaling.

Key Points The strength of the genetic association between specific MHC class II alleles and an individual's susceptibility to particular chronic inflammatory diseases renders these alleles the main known risk factor for many such diseases. The peptide-binding grooves of MHC class II molecules can be described in terms of pockets that must accommodate the side chains of residues at positions P1, P4, P6 and P9 of the peptide. Analyses of the characteristics of these pockets, as revealed by the crystal structures of MHC class II molecules, provide insights into how sequence polymorphisms determine the population of peptides a particular MHC class II molecule can bind, and indicate molecular mechanisms that could determine disease susceptibility. Structure-based analysis indicates that differential peptide binding between two closely related HLA-DQ6 molecules is central to their positive and negative association with the chronic neurological disorder narcolepsy, an observation that is consistent with narcolepsy being an autoimmune disease. Coeliac disease is an autoimmune-like disorder that is caused by an immune response to antigens present in wheat gluten. HLA-DQ2, and to a lesser extent HLA-DQ8, have peptide-binding-groove characteristics that strongly favour the binding of gluten-derived peptides, consistent with the association of these MHC class II molecules with coeliac disease. Crystal structures for the type-1-diabetes-associated MHC class II molecules HLA-DQ8, HLA-DQ2 and mouse H2-IA g7 reveal a distinctive P9 pocket, which might indicate similar pathophysiological pathways for developing type 1 diabetes in humans and non-obese diabetic mice. A comparison of the structures of disease-associated versus protective MHC class II molecules reveals a second characteristic; the P6 pocket shows a consistent trend in volume size that correlates from positive to negative association with type 1 diabetes. T cells are thought to play an important role in the development of rheumatoid arthritis and an immunodominant T-cell epitope from type II collagen is a candidate autoantigen. The structures of the disease-associated HLA-DR4.1 and HLA-DR1 molecules reveal P4 pockets that have in common an ability to bind acidic residues, plus shallow P6 and P9 pockets that are particularly well suited to binding the glycine-rich sequences typical of type-II-collagen-derived peptides. Distinctive structural characteristics of the multiple-sclerosis-associated MHC class II molecules HLA-DR2a and HLA-DR2b separately result in peptide residues P6–P9 assuming a raised position above the respective peptide-binding grooves. This differs considerably from the canonical mode of peptide presentation by other MHC class II molecules and might favour T-cell receptors that sample a reduced portion of the peptide, hence increasing the likelihood of a disease inducing crossreactivity. MHC class II molecules are important factors that contribute to the susceptibility of an individual to autoimmune disease. Jones and colleagues look for clues to their involvement in disease by analysing crystal structures of peptide–MHC-class II complexes. MHC class II molecules on the surface of antigen-presenting cells display a range of peptides for recognition by the T-cell receptors of CD4 + T helper cells. Therefore, MHC class II molecules are central to effective adaptive immune responses, but conversely, genetic and epidemiological data have implicated these molecules in the pathogenesis of autoimmune diseases. Indeed, the strength of the associations between particular MHC class II alleles and disease render them the main genetic risk factors for autoimmune disorders such as type 1 diabetes. Here, we discuss the insights that the crystal structures of MHC class II molecules provide into the molecular mechanisms by which sequence polymorphisms might contribute to disease susceptibility.

WIF-1 inhibits Wnt signaling by binding Wnt ligands. Structural and biochemical analysis of WIF-1 shows the EGF-like domains wrapping back to contact the ligand-binding WD domain, which also binds a phospholipid near the interaction site for Wnt ligands. The tail of EGF-like domains also harbors a proteoglycan binding site, indicating that all domains of WIF-1 contribute to the regulation of Wnt signaling in vivo . Wnt morphogens control embryonic development and homeostasis in adult tissues. In vertebrates the N-terminal WIF domain (WIF-1 WD ) of Wnt inhibitory factor 1 (WIF-1) binds Wnt ligands. Our crystal structure of WIF-1 WD reveals a previously unidentified binding site for phospholipid; two acyl chains extend deep into the domain, and the head group is exposed to the surface. Biophysical and cellular assays indicate that there is a WIF-1 WD Wnt-binding surface proximal to the lipid head group but also implicate the five epidermal growth factor (EGF)-like domains (EGFs I–V) in Wnt binding. The six-domain WIF-1 crystal structure shows that EGFs I–V are wrapped back, interfacing with WIF-1 WD at EGF III. EGFs II–V contain a heparan sulfate proteoglycan (HSPG)-binding site, consistent with conserved positively charged residues on EGF IV. This combination of HSPG- and Wnt-binding properties suggests a modular model for the localization of WIF-1 and for signal inhibition within morphogen gradients.