Structure and function of the ROR2 cysteine-rich domain in vertebrate noncanonical WNT5A signaling.

The receptor tyrosine kinase ROR2 mediates noncanonical WNT5A signaling to orchestrate tissue morphogenetic processes, and dysfunction of the pathway causes Robinow syndrome, brachydactyly B, and metastatic diseases. The domain(s) and mechanisms required for ROR2 function, however, remain unclear. We solved the crystal structure of the extracellular cysteine-rich (CRD) and Kringle (Kr) domains of ROR2 and found that, unlike other CRDs, the ROR2 CRD lacks the signature hydrophobic pocket that binds lipids/lipid-modified proteins, such as WNTs, suggesting a novel mechanism of ligand reception.

Mapping structural and dynamic divergence across the MBOAT family.

Membrane-bound O-acyltransferases (MBOATs) are membrane-embedded enzymes that catalyze acyl chain transfer to a diverse group of substrates, including lipids, small molecules, and proteins. MBOATs share a conserved structural core, despite wide-ranging functional specificity across both prokaryotes and eukaryotes. The structural basis of catalytic specificity, regulation and interactions with the surrounding environment remain uncertain.

Diverse genetic causes of amenorrhea in an ethnically homogeneous cohort and an evolving approach to diagnosis.

Research Question: Premature ovarian insufficiency (POI) is characterised by amenorrhea associated with elevated follicle stimulating hormone (FSH) under the age of 40 years and affects 1-3.7% women. Genetic factors explain 20-30% of POI cases, but most causes remain unknown despite genomic advancements.

Design, Synthesis, and Evaluation of Inhibitors of Hedgehog Acyltransferase.

Hedgehog signaling is involved in embryonic development and cancer growth. Functional activity of secreted Hedgehog signaling proteins is dependent on -terminal palmitoylation, making the palmitoyl transferase Hedgehog acyltransferase (HHAT), a potential drug target and a series of 4,5,6,7-tetrahydrothieno[3,2-]pyridines have been identified as HHAT inhibitors. Based on structural data, we designed and synthesized 37 new analogues which we profiled alongside 13 previously reported analogues in enzymatic and cellular assays.

LipIDens: simulation assisted interpretation of lipid densities in cryo-EM structures of membrane proteins.

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.

The energetics and ion coupling of cholesterol transport through Patched1.

Patched1 (PTCH1) is a tumor suppressor protein of the mammalian Hedgehog (HH) signaling pathway, implicated in embryogenesis and tissue homeostasis. PTCH1 inhibits the G protein-coupled receptor Smoothened (SMO) via a debated mechanism involving modulating ciliary cholesterol accessibility. Using extensive molecular dynamics simulations and free energy calculations to evaluate cholesterol transport through PTCH1, we find an energetic barrier of ~15 to 20 kilojoule per mole for cholesterol export.

Rocking the MBOAT: Structural insights into the membrane bound O-acyltransferase family.

The membrane-bound O-acyltransferase (MBOAT) superfamily catalyses the transfer of acyl chains to substrates implicated in essential cellular functions. Aberrant function of MBOATs is associated with various diseases and MBOATs are promising drug targets. There has been recent progress in structural characterisation of MBOATs, advancing our understanding of their functional mechanism.

The Inseparable Relationship Between Cholesterol and Hedgehog Signaling.

Ligands of the Hedgehog (HH) pathway are paracrine signaling molecules that coordinate tissue development in metazoans. A remarkable feature of HH signaling is the repeated use of cholesterol in steps spanning ligand biogenesis, secretion, dispersal, and reception on target cells. A cholesterol molecule covalently attached to HH ligands is used as a molecular baton by transfer proteins to guide their secretion, spread, and reception.

The Energetics and Ion Coupling of Cholesterol Transport Through Patched1.

Patched1 (PTCH1) is the principal tumour suppressor protein of the mammalian Hedgehog (HH) signalling pathway, implicated in embryogenesis and tissue homeostasis. PTCH1 inhibits the Class F G protein-coupled receptor Smoothened (SMO) via a debated mechanism involving modulating accessible cholesterol levels within ciliary membranes. Using extensive molecular dynamics (MD) simulations and free energy calculations to evaluate cholesterol transport through PTCH1, we find an energetic barrier of ~15-20 kJ mol for cholesterol export.

Patched 1 regulates Smoothened by controlling sterol binding to its extracellular cysteine-rich domain.

Smoothened (SMO) transduces the Hedgehog (Hh) signal across the plasma membrane in response to accessible cholesterol. Cholesterol binds SMO at two sites: one in the extracellular cysteine-rich domain (CRD) and a second in the transmembrane domain (TMD). How these two sterol-binding sites mediate SMO activation in response to the ligand Sonic Hedgehog (SHH) remains unknown.

Structure, mechanism, and inhibition of Hedgehog acyltransferase.

The Sonic Hedgehog (SHH) morphogen pathway is fundamental for embryonic development and stem cell maintenance and is implicated in various cancers. A key step in signaling is transfer of a palmitate group to the SHH N terminus, catalyzed by the multi-pass transmembrane enzyme Hedgehog acyltransferase (HHAT). We present the high-resolution cryo-EM structure of HHAT bound to substrate analog palmityl-coenzyme A and a SHH-mimetic megabody, revealing a heme group bound to HHAT that is essential for HHAT function.

Hedgehog-Interacting Protein is a multimodal antagonist of Hedgehog signalling.

Hedgehog (HH) morphogen signalling, crucial for cell growth and tissue patterning in animals, is initiated by the binding of dually lipidated HH ligands to cell surface receptors. Hedgehog-Interacting Protein (HHIP), the only reported secreted inhibitor of Sonic Hedgehog (SHH) signalling, binds directly to SHH with high nanomolar affinity, sequestering SHH. Here, we report the structure of the HHIP N-terminal domain (HHIP-N) in complex with a glycosaminoglycan (GAG).

Patched 1 reduces the accessibility of cholesterol in the outer leaflet of membranes.

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.

Relative Affinities of Protein-Cholesterol Interactions from Equilibrium Molecular Dynamics Simulations.

Specific interactions of lipids with membrane proteins contribute to protein stability and function. Multiple lipid interactions surrounding a membrane protein are often identified in molecular dynamics (MD) simulations and are, increasingly, resolved in cryo-electron microscopy (cryo-EM) densities. Determining the relative importance of specific interaction sites is aided by determination of lipid binding affinities using experimental or simulation methods.

Photochemical Probe Identification of a Small-Molecule Inhibitor Binding Site in Hedgehog Acyltransferase (HHAT).

The mammalian membrane-bound -acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports rational development of a photochemical probe to interrogate a novel small-molecule inhibitor binding site in the human MBOAT Hedgehog acyltransferase (HHAT).

Photochemical Probe Identification of a Small-Molecule Inhibitor Binding Site in Hedgehog Acyltransferase (HHAT)*.

The mammalian membrane-bound O-acyltransferase (MBOAT) superfamily is involved in biological processes including growth, development and appetite sensing. MBOATs are attractive drug targets in cancer and obesity; however, information on the binding site and molecular mechanisms underlying small-molecule inhibition is elusive. This study reports rational development of a photochemical probe to interrogate a novel small-molecule inhibitor binding site in the human MBOAT Hedgehog acyltransferase (HHAT).

Simultaneous binding of Guidance Cues NET1 and RGM blocks extracellular NEO1 signaling.

During cell migration or differentiation, cell surface receptors are simultaneously exposed to different ligands. However, it is often unclear how these extracellular signals are integrated. Neogenin (NEO1) acts as an attractive guidance receptor when the Netrin-1 (NET1) ligand binds, but it mediates repulsion via repulsive guidance molecule (RGM) ligands.

Cholesterol access in cellular membranes controls Hedgehog signaling.

The Hedgehog (Hh) signaling pathway coordinates cell-cell communication in development and regeneration. Defects in this pathway underlie diseases ranging from birth defects to cancer. Hh signals are transmitted across the plasma membrane by two proteins, Patched 1 (PTCH1) and Smoothened (SMO).

Glypicans shield the Wnt lipid moiety to enable signalling at a distance.

A relatively small number of proteins have been suggested to act as morphogens-signalling molecules that spread within tissues to organize tissue repair and the specification of cell fate during development. Among them are Wnt proteins, which carry a palmitoleate moiety that is essential for signalling activity. How a hydrophobic lipoprotein can spread in the aqueous extracellular space is unknown.

Repulsive guidance molecules lock growth differentiation factor 5 in an inhibitory complex.

Repulsive guidance molecules (RGMs) are cell surface proteins that regulate the development and homeostasis of many tissues and organs, including the nervous, skeletal, and immune systems. They control fundamental biological processes, such as migration and differentiation by direct interaction with the Neogenin (NEO1) receptor and function as coreceptors for the bone morphogenetic protein (BMP)/growth differentiation factor (GDF) family. We determined crystal structures of all three human RGM family members in complex with GDF5, as well as the ternary NEO1-RGMB-GDF5 assembly.

R-spondins engage heparan sulfate proteoglycans to potentiate WNT signaling.

R-spondins (RSPOs) amplify WNT signaling during development and regenerative responses. We previously demonstrated that RSPOs 2 and 3 potentiate WNT/β-catenin signaling in cells lacking leucine-rich repeat-containing G-protein coupled receptors (LGRs) 4, 5 and 6 (Lebensohn and Rohatgi, 2018). We now show that heparan sulfate proteoglycans (HSPGs) act as alternative co-receptors for RSPO3 using a combination of ligand mutagenesis and ligand engineering.

Acylation-coupled lipophilic induction of polarisation (Acyl-cLIP): a universal assay for lipid transferase and hydrolase enzymes.

Posttranslational attachment of lipids to proteins is important for many cellular functions, and the enzymes responsible for these modifications are implicated in many diseases, from cancer to neurodegeneration. Lipid transferases and hydrolases are increasingly tractable therapeutic targets, but present unique challenges for high-throughput biochemical enzyme assays which hinder development of new inhibitors. We present Acylation-coupled Lipophilic Induction of Polarisation (Acyl-cLIP) as the first universally applicable biochemical lipidation assay, exploiting the hydrophobic nature of lipidated peptides to drive a polarised fluorescence readout.

Cholesterol accessibility at the ciliary membrane controls hedgehog signaling.

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.

The morphogen Sonic hedgehog inhibits its receptor Patched by a pincer grasp mechanism.

Hedgehog (HH) ligands, classical morphogens that pattern embryonic tissues in all animals, are covalently coupled to two lipids-a palmitoyl group at the N terminus and a cholesteroyl group at the C terminus. While the palmitoyl group binds and inactivates Patched 1 (PTCH1), the main receptor for HH ligands, the function of the cholesterol modification has remained mysterious. Using structural and biochemical studies, along with reassessment of previous cryo-electron microscopy structures, we find that the C-terminal cholesterol attached to Sonic hedgehog (Shh) binds the first extracellular domain of PTCH1 and promotes its inactivation, thus triggering HH signaling.