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.

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.

Bile acid biosynthesis in Smith-Lemli-Opitz syndrome bypassing cholesterol: Potential importance of pathway intermediates.

Bile acids are the end products of cholesterol metabolism secreted into bile. They are essential for the absorption of lipids and lipid soluble compounds from the intestine. Here we have identified a series of unusual Δ-unsaturated bile acids in plasma and urine of patients with Smith-Lemli-Opitz syndrome (SLOS), a defect in cholesterol biosynthesis resulting in elevated levels of 7-dehydrocholesterol (7-DHC), an immediate precursor of cholesterol.

G protein-coupled receptors control the sensitivity of cells to the morphogen Sonic Hedgehog.

The morphogen Sonic Hedgehog (SHH) patterns tissues during development by directing cell fates in a concentration-dependent manner. The SHH signal is transmitted across the membrane of target cells by the heptahelical transmembrane protein Smoothened (SMO), which activates the GLI family of transcription factors through a mechanism that is undefined in vertebrates. Using CRISPR-edited null alleles and small-molecule inhibitors, we systematically analyzed the epistatic interactions between SMO and three proteins implicated in SMO signaling: the heterotrimeric G protein subunit Gα, the G protein-coupled receptor kinase 2 (GRK2), and the Gα-coupled receptor GPR161.

Production, characterization, and assessment of a stable analog of the response regulator CheY-phosphate from Thermotoga maritima.

Phosphorylation of CheY promotes association with the flagellar motor and ultimately controls the directional bias of the motor. However, biochemical studies of activated CheY-phosphate have been challenging due to the rapid hydrolysis of the aspartyl-phosphate in vitro. An inert analog of Tm CheY-phosphate, phosphono-CheY, was synthesized by chemical modification and purified by cation-exchange chromatography.

Co-Folding of a FliF-FliG Split Domain Forms the Basis of the MS:C Ring Interface within the Bacterial Flagellar Motor.

The interface between the membrane (MS) and cytoplasmic (C) rings of the bacterial flagellar motor couples torque generation to rotation within the membrane. The structure of the C-terminal helices of the integral membrane protein FliF (FliF) bound to the N terminal domain of the switch complex protein FliG (FliG) reveals that FliG folds around FliF to produce a topology that closely resembles both the middle and C-terminal domains of FliG. The interface is consistent with solution-state nuclear magnetic resonance, small-angle X-ray scattering, in vivo interaction studies, and cellular motility assays.

Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling.

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.

Structural basis of Smoothened regulation by its extracellular domains.

Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzledclass 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.

Assembly states of FliM and FliG within the flagellar switch complex.

At the base of the bacterial flagella, a cytoplasmic rotor (the C-ring) generates torque and reverses rotation sense in response to stimuli. The bulk of the C-ring forms from many copies of the proteins FliG, FliM, and FliN, which together constitute the switch complex. To help resolve outstanding issues regarding C-ring architecture, we have investigated interactions between FliM and FliG from Thermotoga maritima with X-ray crystallography and pulsed dipolar ESR spectroscopy (PDS).

Structure and activity of the flagellar rotor protein FliY: a member of the CheC phosphatase family.

Background: FliY is a flagellar rotor protein of the CheC phosphatase family.

Results: The FliY structure resembles that of the rotor protein FliM but contains two active centers for CheY dephosphorylation.

Conclusion: FliY incorporates properties of the FliM/FliN rotor proteins and the CheC/CheX phosphatases to serve multiple functions in the flagellar switch.

Architecture of the soluble receptor Aer2 indicates an in-line mechanism for PAS and HAMP domain signaling.

Bacterial receptors typically contain modular architectures with distinct functional domains that combine to send signals in response to stimuli. Although the properties of individual components have been investigated in many contexts, there is little information about how diverse sets of modules work together in full-length receptors. Here, we investigate the architecture of Aer2, a soluble gas-sensing receptor that has emerged as a model for PAS (Per-Arnt-Sim) and poly-HAMP (histidine kinase-adenylyl cyclase-methyl-accepting chemotaxis protein-phosphatase) domain signaling.

Self-association of the histidine kinase CheA as studied by pulsed dipolar ESR spectroscopy.

Biologically important protein complexes often involve molecular interactions that are low affinity or transient. We apply pulsed dipolar electron spin resonance spectroscopy and site-directed spin labeling in what to our knowledge is a new approach to study aggregation and to identify regions on protein surfaces that participate in weak, but specific molecular interactions. As a test case, we have probed the self-association of the chemotaxis kinase CheA, which forms signaling clusters with chemoreceptors and the coupling protein CheW at the poles of bacterial cells.