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1.
Elife ; 82019 02 25.
Article in English | MEDLINE | ID: mdl-30802206

ABSTRACT

UNC-13 proteins play an essential role in synaptic transmission by recruiting synaptic vesicles (SVs) to become available for release, which is termed SV priming. Here we show that the C2A domain of UNC-13L, like the corresponding domain in mammalian Munc13-1, displays two conserved binding modes: forming C2A/C2A homodimers, or forming a heterodimer with the zinc finger domain of UNC-10/RIM (C2A/RIM). Functional analysis revealed that UNC-13L's C2A promotes synaptic transmission by regulating a post-priming process. Stimulus-evoked release but not SV priming, was impaired in unc-10 mutants deficient for C2A/RIM heterodimerization, leading to decreased release probability. Disrupting C2A/C2A homodimerization in UNC-13L-rescued animals had no effect on synaptic transmission, but fully restored the evoked release and the release probability of unc-10/RIM mutants deficient for C2A/RIM heterodimerization. Thus, our results support the model that RIM binding C2A releases UNC-13L from an autoinhibitory homodimeric complex to become fusion-competent by functioning as a switch only.


Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , Carrier Proteins/metabolism , Membrane Proteins/metabolism , Nerve Tissue Proteins/metabolism , Protein Multimerization , Synaptic Transmission , Synaptic Vesicles/metabolism , Animals , Protein Binding , Protein Domains
3.
Cell ; 174(2): 312-324.e16, 2018 07 12.
Article in English | MEDLINE | ID: mdl-29804838

ABSTRACT

The seven-transmembrane-spanning protein Smoothened is the central transducer in Hedgehog signaling, a pathway fundamental in development and in cancer. Smoothened is activated by cholesterol binding to its extracellular cysteine-rich domain (CRD). How this interaction leads to changes in the transmembrane domain and Smoothened activation is unknown. Here, we report crystal structures of sterol-activated Smoothened. The CRD undergoes a dramatic reorientation, allosterically causing the transmembrane domain to adopt a conformation similar to active G-protein-coupled receptors. We show that Smoothened contains a unique inhibitory π-cation lock, which is broken on activation and is disrupted in constitutively active oncogenic mutants. Smoothened activation opens a hydrophobic tunnel, suggesting a pathway for cholesterol movement from the inner membrane leaflet to the CRD. All Smoothened antagonists bind the transmembrane domain and block tunnel opening, but cyclopamine also binds the CRD, inducing the active transmembrane conformation. Together, these results define the mechanisms of Smoothened activation and inhibition.


Subject(s)
Hedgehog Proteins/metabolism , Smoothened Receptor/chemistry , Xenopus Proteins/chemistry , Allosteric Regulation , Animals , Binding Sites , Cell Line , Cholesterol/chemistry , Cholesterol/metabolism , Crystallography, X-Ray , Flow Cytometry , Hedgehog Proteins/genetics , Humans , Mice , Molecular Dynamics Simulation , Protein Binding , Protein Domains , Protein Structure, Tertiary , Signal Transduction , Small Molecule Libraries/chemical synthesis , Small Molecule Libraries/chemistry , Small Molecule Libraries/metabolism , Smoothened Receptor/antagonists & inhibitors , Smoothened Receptor/metabolism , Veratrum Alkaloids/chemistry , Veratrum Alkaloids/metabolism , Xenopus Proteins/antagonists & inhibitors , Xenopus Proteins/metabolism , Xenopus laevis/metabolism
4.
Neuron ; 95(2): 326-340.e5, 2017 Jul 19.
Article in English | MEDLINE | ID: mdl-28669545

ABSTRACT

The synaptic adhesion molecules Neurexin and Neuroligin alter the development and function of synapses and are linked to autism in humans. In C. elegans, post-synaptic Neurexin (NRX-1) and pre-synaptic Neuroligin (NLG-1) mediate a retrograde synaptic signal that inhibits acetylcholine (ACh) release at neuromuscular junctions. Here, we show that the retrograde signal decreases ACh release by inhibiting the function of pre-synaptic UNC-2/CaV2 calcium channels. Post-synaptic NRX-1 binds to an auxiliary subunit of pre-synaptic UNC-2/CaV2 channels (UNC-36/α2δ), decreasing UNC-36 abundance at pre-synaptic elements. Retrograde inhibition is mediated by a soluble form of NRX-1's ectodomain, which is released from the post-synaptic membrane by the SUP-17/ADAM10 protease. Mammalian Neurexin-1α binds α2δ-3 and decreases CaV2.2 current in transfected cells, whereas Neurexin-1α has no effect on CaV2.2 reconstituted with α2δ-1 and α2δ-2. Collectively, these results suggest that α-Neurexin binding to α2δ is a conserved mechanism for regulating synaptic transmission.


Subject(s)
Biophysical Phenomena/physiology , Calcium Channels, N-Type/metabolism , Glycoproteins/metabolism , Neuropeptides/metabolism , Synapses/metabolism , Synaptic Transmission/physiology , Acetylcholine/metabolism , Animals , Caenorhabditis elegans , Caenorhabditis elegans Proteins/metabolism , Cell Adhesion Molecules, Neuronal/metabolism , Humans , Nerve Tissue Proteins/metabolism , Neuromuscular Junction/metabolism , Protein Subunits/metabolism
5.
Cell ; 166(5): 1176-1187.e14, 2016 Aug 25.
Article in English | MEDLINE | ID: mdl-27545348

ABSTRACT

In vertebrates, sterols are necessary for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Sterols activate the membrane protein Smoothened by binding its extracellular, cysteine-rich domain (CRD). Major unanswered questions concern the nature of the endogenous, activating sterol and the mechanism by which it regulates Smoothened. We report crystal structures of CRD complexed with sterols and alone, revealing that sterols induce a dramatic conformational change of the binding site, which is sufficient for Smoothened activation and is unique among CRD-containing receptors. We demonstrate that Hedgehog signaling requires sterol binding to Smoothened and define key residues for sterol recognition and activity. We also show that cholesterol itself binds and activates Smoothened. Furthermore, the effect of oxysterols is abolished in Smoothened mutants that retain activation by cholesterol and Hedgehog. We propose that the endogenous Smoothened activator is cholesterol, not oxysterols, and that vertebrate Hedgehog signaling controls Smoothened by regulating its access to cholesterol.


Subject(s)
Cholesterol/metabolism , Hedgehog Proteins/metabolism , Smoothened Receptor/agonists , Animals , Cholesterol/chemistry , Crystallography, X-Ray , Mice , NIH 3T3 Cells , Oxysterols/chemistry , Oxysterols/metabolism , Protein Binding , Protein Conformation , Signal Transduction , Smoothened Receptor/chemistry , Smoothened Receptor/metabolism , Xenopus laevis
6.
Chembiochem ; 16(4): 611-7, 2015 Mar 02.
Article in English | MEDLINE | ID: mdl-25663046

ABSTRACT

Cholesterol is a fundamental lipid component of eukaryotic membranes and a precursor of potent signaling molecules, such as oxysterols and steroid hormones. Cholesterol and oxysterols are also essential for Hedgehog signaling, a pathway critical in embryogenesis and cancer. Despite their importance, the use of imaging sterols in cells is currently very limited. We introduce a robust and versatile method for sterol microscopy based on C19 alkyne cholesterol and oxysterol analogues. These sterol analogues are fully functional; they rescue growth of cholesterol auxotrophic cells and faithfully recapitulate the multiple roles that sterols play in Hedgehog signal transduction. Alkyne sterol analogues incorporate efficiently into cellular membranes and can be imaged with high resolution after copper(I)-catalyzed azide-alkyne cycloaddition reaction with fluorescent azides. We demonstrate the use of alkyne sterol probes for visualizing the subcellular distribution of cholesterol and for two-color imaging of sterols and choline phospholipids. Our imaging strategy should be broadly applicable to studying the role of sterols in normal physiology and disease.


Subject(s)
Hedgehog Proteins/metabolism , Optical Imaging , Signal Transduction , Sterols/analysis , Alkynes/chemistry , Animals , Azides/chemistry , Cholesterol/analogs & derivatives , Click Chemistry , Copper/chemistry , Cycloaddition Reaction , Humans , Mice , Microscopy/methods , Molecular Probes/chemistry , NIH 3T3 Cells , Optical Imaging/methods , Sterols/metabolism
7.
Nat Chem Biol ; 9(9): 557-64, 2013 Sep.
Article in English | MEDLINE | ID: mdl-23831757

ABSTRACT

Oxysterols bind the seven-transmembrane protein Smo (Smo) and potently activate vertebrate Hedgehog (Hh) signaling, a pathway essential in embryonic development, adult stem cell maintenance and cancer. It is unknown, however, whether oxysterols are important for normal vertebrate Hh signaling and whether antagonizing oxysterols can inhibit the Hh pathway. We developed azasterols that block Hh signaling by binding the oxysterol-binding site of Smo. We show that the binding site for oxysterols and azasterols maps to the extracellular, cysteine-rich domain of Smo and is completely separable from the site bound by other small-molecule modulators, located within the heptahelical bundle of Smo. Smo mutants in which oxysterol binding is abolished no longer respond to oxysterols and cannot be maximally activated by the Hh ligand. Our results show that oxysterol binding to vertebrate Smo is required for normal Hh signaling and that targeting the oxysterol-binding site is an effective strategy to inhibit Smo.


Subject(s)
Hedgehog Proteins/metabolism , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/metabolism , Signal Transduction , Sterols/metabolism , Amino Acid Sequence , Animals , Binding Sites/drug effects , Drosophila melanogaster , Evolution, Molecular , Mice , Molecular Sequence Data , Protein Structure, Tertiary , Receptors, G-Protein-Coupled/antagonists & inhibitors , Receptors, G-Protein-Coupled/genetics , Signal Transduction/drug effects , Smoothened Receptor , Sterols/chemistry , Sterols/pharmacology , Structure-Activity Relationship , Xenopus laevis
8.
Proc Natl Acad Sci U S A ; 104(1): 48-53, 2007 Jan 02.
Article in English | MEDLINE | ID: mdl-17179210

ABSTRACT

To determine which of seven library design algorithms best introduces new protein function without destroying it altogether, seven combinatorial libraries of green fluorescent protein variants were designed and synthesized. Each was evaluated by distributions of emission intensity and color compiled from measurements made in vivo. Additional comparisons were made with a library constructed by error-prone PCR. Among the designed libraries, fluorescent function was preserved for the greatest fraction of samples in a library designed by using a structure-based computational method developed and described here. A trend was observed toward greater diversity of color in designed libraries that better preserved fluorescence. Contrary to trends observed among libraries constructed by error-prone PCR, preservation of function was observed to increase with a library's average mutation level among the four libraries designed with structure-based computational methods.


Subject(s)
Combinatorial Chemistry Techniques/methods , Green Fluorescent Proteins/chemistry , Protein Engineering/methods , Drug Design , Green Fluorescent Proteins/physiology , Peptide Library
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