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1.
mSystems ; 9(6): e0036924, 2024 Jun 18.
Article in English | MEDLINE | ID: mdl-38717159

ABSTRACT

Most of Earth's trees rely on critical soil nutrients that ectomycorrhizal fungi (EcMF) liberate and provide, and all of Earth's land plants associate with bacteria that help them survive in nature. Yet, our understanding of how the presence of EcMF modifies soil bacterial communities, soil food webs, and root chemistry requires direct experimental evidence to comprehend the effects that EcMF may generate in the belowground plant microbiome. To this end, we grew Pinus muricata plants in soils that were either inoculated with EcMF and native forest bacterial communities or only native bacterial communities. We then profiled the soil bacterial communities, applied metabolomics and lipidomics, and linked omics data sets to understand how the presence of EcMF modifies belowground biogeochemistry, bacterial community structure, and their functional potential. We found that the presence of EcMF (i) enriches soil bacteria linked to enhanced plant growth in nature, (ii) alters the quantity and composition of lipid and non-lipid soil metabolites, and (iii) modifies plant root chemistry toward pathogen suppression, enzymatic conservation, and reactive oxygen species scavenging. Using this multi-omic approach, we therefore show that this widespread fungal symbiosis may be a common factor for structuring soil food webs.IMPORTANCEUnderstanding how soil microbes interact with one another and their host plant will help us combat the negative effects that climate change has on terrestrial ecosystems. Unfortunately, we lack a clear understanding of how the presence of ectomycorrhizal fungi (EcMF)-one of the most dominant soil microbial groups on Earth-shapes belowground organic resources and the composition of bacterial communities. To address this knowledge gap, we profiled lipid and non-lipid metabolites in soils and plant roots, characterized soil bacterial communities, and compared soils amended either with or without EcMF. Our results show that the presence of EcMF changes soil organic resource availability, impacts the proliferation of different bacterial communities (in terms of both type and potential function), and primes plant root chemistry for pathogen suppression and energy conservation. Our findings therefore provide much-needed insight into how two of the most dominant soil microbial groups interact with one another and with their host plant.


Subject(s)
Bacteria , Food Chain , Microbiota , Mycorrhizae , Pinus , Plant Roots , Soil Microbiology , Mycorrhizae/physiology , Pinus/microbiology , Bacteria/metabolism , Bacteria/genetics , Plant Roots/microbiology , Plant Roots/metabolism , Microbiota/physiology , Symbiosis , Soil/chemistry
2.
Structure ; 2024 Apr 05.
Article in English | MEDLINE | ID: mdl-38636523

ABSTRACT

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. Here, we combine comparative molecular dynamics (MD) simulations with bioinformatics to assess molecular and interactional divergence across the family. In simulations, MBOATs differentially distort the bilayer depending on their substrate type. Additionally, we identify lipid binding sites surrounding reactant gates in the surrounding membrane. Complementary bioinformatic analyses reveal a conserved role for re-entrant loop-2 in MBOAT fold stabilization and a key hydrogen bond bridging DGAT1 dimerization. Finally, we predict differences in MBOAT solvation and water gating properties. These data are pertinent to the design of MBOAT-specific inhibitors that encompass dynamic information within cellular mimetic environments.

3.
Nat Commun ; 14(1): 7774, 2023 Nov 27.
Article in English | MEDLINE | ID: mdl-38012131

ABSTRACT

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.


Subject(s)
Membrane Proteins , Molecular Dynamics Simulation , Membrane Proteins/chemistry , Cryoelectron Microscopy , Membrane Lipids , Protein Conformation
4.
Sci Adv ; 9(34): eadh1609, 2023 08 25.
Article in English | MEDLINE | ID: mdl-37611095

ABSTRACT

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. In silico data are coupled to in vivo biochemical assays of PTCH1 mutants to probe coupling between cation binding sites, transmembrane motions, and PTCH1 activity. Using complementary simulations of Dispatched1, we find that transition between "inward-open" and solvent "occluded" states is accompanied by Na+-induced pinching of intracellular helical segments. Thus, our findings illuminate the energetics and ion coupling stoichiometries of PTCH1 transport mechanisms, whereby one to three Na+ or two to three K+ couple to cholesterol export, and provide the first molecular description of transitions between distinct transport states.


Subject(s)
Biological Assay , Hedgehog Proteins , Animals , Biological Transport , Binding Sites , Embryonic Development , Mammals
5.
Curr Opin Struct Biol ; 80: 102589, 2023 06.
Article in English | MEDLINE | ID: mdl-37040671

ABSTRACT

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. Integrating information across the MBOAT family, we characterise a common MBOAT fold and provide a blueprint for substrate and inhibitor engagement. This work provides context for the diverse substrates, mechanisms, and evolutionary relationships of protein and small-molecule MBOATs. Further work should aim to characterise MBOATs, as inherently lipid-associated proteins, within their membrane environment.


Subject(s)
Acyltransferases , Acyltransferases/chemistry
6.
bioRxiv ; 2023 Feb 15.
Article in English | MEDLINE | ID: mdl-36824746

ABSTRACT

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 -1 for cholesterol export. In simulations we identify cation binding sites within the PTCH1 transmembrane domain (TMD) which may provide the energetic impetus for cholesterol transport. In silico data are coupled to in vivo biochemical assays of PTCH1 mutants to probe coupling between transmembrane motions and PTCH1 activity. Using complementary simulations of Dispatched1 (DISP1) we find that transition between 'inward-open' and solvent 'occluded' states is accompanied by Na + induced pinching of intracellular helical segments. Thus, our findings illuminate the energetics and ion-coupling stoichiometries of PTCH1 transport mechanisms, whereby 1-3 Na + or 2-3 K + couple to cholesterol export, and provide the first molecular description of transitions between distinct transport states.

7.
Sci Adv ; 8(22): eabm5563, 2022 06 03.
Article in English | MEDLINE | ID: mdl-35658032

ABSTRACT

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. We find that mutations in the CRD (but not the TMD) reduce the fold increase in SMO activity triggered by SHH. SHH also promotes the photocrosslinking of a sterol analog to the CRD in intact cells. In contrast, sterol binding to the TMD site boosts SMO activity regardless of SHH exposure. Mutational and computational analyses show that these sites are in allosteric communication despite being 45 angstroms apart. Hence, sterols function as both SHH-regulated orthosteric ligands at the CRD and allosteric ligands at the TMD to regulate SMO activity and Hh signaling.


Subject(s)
Cysteine , Hedgehog Proteins , Cholesterol/metabolism , Hedgehog Proteins/chemistry , Ligands , Sterols/chemistry
8.
Mol Metab ; 63: 101530, 2022 09.
Article in English | MEDLINE | ID: mdl-35718339

ABSTRACT

OBJECTIVE: To determine whether glucagon receptor (GCGR) actions are modulated by cellular cholesterol levels. METHODS: We determined the effects of experimental cholesterol depletion and loading on glucagon-mediated cAMP production, ligand internalisation and glucose production in human hepatoma cells, mouse and human hepatocytes. GCGR interactions with lipid bilayers were explored using coarse-grained molecular dynamic simulations. Glucagon responsiveness was measured in mice fed a high cholesterol diet with or without simvastatin to modulate hepatocyte cholesterol content. RESULTS: GCGR cAMP signalling was reduced by higher cholesterol levels across different cellular models. Ex vivo glucagon-induced glucose output from mouse hepatocytes was enhanced by simvastatin treatment. Mice fed a high cholesterol diet had increased hepatic cholesterol and a blunted hyperglycaemic response to glucagon, both of which were partially reversed by simvastatin. Simulations identified likely membrane-exposed cholesterol binding sites on the GCGR, including a site where cholesterol is a putative negative allosteric modulator. CONCLUSIONS: Our results indicate that cellular cholesterol content influences glucagon sensitivity and indicate a potential molecular basis for this phenomenon. This could be relevant to the pathogenesis of non-alcoholic fatty liver disease, which is associated with both hepatic cholesterol accumulation and glucagon resistance.


Subject(s)
Cholesterol , Glucagon , Glucose , Hepatocytes , Receptors, Glucagon , Animals , Cholesterol/analysis , Cholesterol/metabolism , Glucagon/metabolism , Glucose/metabolism , Hepatocytes/chemistry , Hepatocytes/metabolism , Humans , Mice , Receptors, Glucagon/metabolism , Simvastatin/metabolism , Simvastatin/pharmacology
9.
J Chem Theory Comput ; 18(2): 1188-1201, 2022 Feb 08.
Article in English | MEDLINE | ID: mdl-35020380

ABSTRACT

Lipids play important modulatory and structural roles for membrane proteins. Molecular dynamics simulations are frequently used to provide insights into the nature of these protein-lipid interactions. Systematic comparative analysis requires tools that provide algorithms for objective assessment of such interactions. We introduce PyLipID, a Python package for the identification and characterization of specific lipid interactions and binding sites on membrane proteins from molecular dynamics simulations. PyLipID uses a community analysis approach for binding site detection, calculating lipid residence times for both the individual protein residues and the detected binding sites. To assist structural analysis, PyLipID produces representative bound lipid poses from simulation data, using a density-based scoring function. To estimate residue contacts robustly, PyLipID uses a dual-cutoff scheme to differentiate between lipid conformational rearrangements while bound from full dissociation events. In addition to the characterization of protein-lipid interactions, PyLipID is applicable to analysis of the interactions of membrane proteins with other ligands. By combining automated analysis, efficient algorithms, and open-source distribution, PyLipID facilitates the systematic analysis of lipid interactions from large simulation data sets of multiple species of membrane proteins.


Subject(s)
Membrane Proteins , Molecular Dynamics Simulation , Binding Sites , Ligands , Lipid Bilayers/chemistry , Lipids , Membrane Proteins/chemistry
10.
Mol Cell ; 81(24): 5025-5038.e10, 2021 12 16.
Article in English | MEDLINE | ID: mdl-34890564

ABSTRACT

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. A structure of HHAT bound to potent small-molecule inhibitor IMP-1575 revealed conformational changes in the active site that occlude substrate binding. Our multidisciplinary analysis provides a detailed view of the mechanism by which HHAT adapts the membrane environment to transfer an acyl chain across the endoplasmic reticulum membrane. This structure of a membrane-bound O-acyltransferase (MBOAT) superfamily member provides a blueprint for other protein-substrate MBOATs and a template for future drug discovery.


Subject(s)
Acyltransferases/antagonists & inhibitors , Acyltransferases/metabolism , Enzyme Inhibitors/pharmacology , Hedgehog Proteins/metabolism , Membrane Proteins/metabolism , Acylation , Acyltransferases/genetics , Acyltransferases/ultrastructure , Allosteric Regulation , Animals , COS Cells , Catalytic Domain , Chlorocebus aethiops , Cryoelectron Microscopy , HEK293 Cells , Heme/metabolism , Humans , Membrane Proteins/antagonists & inhibitors , Membrane Proteins/genetics , Membrane Proteins/ultrastructure , Molecular Dynamics Simulation , Palmitoyl Coenzyme A/metabolism , Protein Conformation , Signal Transduction , Structure-Activity Relationship
11.
J Chem Theory Comput ; 17(10): 6548-6558, 2021 Oct 12.
Article in English | MEDLINE | ID: mdl-34523933

ABSTRACT

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. Here, we develop a method for determining protein-lipid binding affinities from equilibrium coarse-grained MD simulations using binding saturation curves, designed to mimic experimental protocols. We apply this method to directly obtain affinities for cholesterol binding to multiple sites on a range of membrane proteins and compare our results with free energies obtained from density-based equilibrium methods and with potential of mean force calculations, getting good agreement with respect to the ranking of affinities for different sites. Thus, our binding saturation method provides a robust, high-throughput alternative for determining the relative consequence of individual sites seen in, e.g., cryo-EM derived membrane protein structures surrounded by an array of ancillary lipid densities.


Subject(s)
Cholesterol , Lipids/chemistry , Membrane Proteins/chemistry , Molecular Dynamics Simulation , Cryoelectron Microscopy
12.
Sci Adv ; 7(34)2021 08.
Article in English | MEDLINE | ID: mdl-34417182

ABSTRACT

Integral membrane proteins are localized and/or regulated by lipids present in the surrounding bilayer. While bacteria have relatively simple membranes, there is ample evidence that many bacterial proteins bind to specific lipids, especially the anionic lipid cardiolipin. Here, we apply molecular dynamics simulations to assess lipid binding to 42 different Escherichia coli inner membrane proteins. Our data reveal an asymmetry between the membrane leaflets, with increased anionic lipid binding to the inner leaflet regions of the proteins, particularly for cardiolipin. From our simulations, we identify >700 independent cardiolipin binding sites, allowing us to identify the molecular basis of a prototypical cardiolipin binding site, which we validate against structures of bacterial proteins bound to cardiolipin. This allows us to construct a set of metrics for defining a high-affinity cardiolipin binding site on bacterial membrane proteins, paving the way for a heuristic approach to defining other protein-lipid interactions.


Subject(s)
Cardiolipins , Escherichia coli , Bacterial Proteins/metabolism , Cardiolipins/chemistry , Escherichia coli/metabolism , Lipid Bilayers/chemistry , Membrane Proteins/metabolism , Molecular Dynamics Simulation
13.
Biophys J ; 119(2): 300-313, 2020 07 21.
Article in English | MEDLINE | ID: mdl-32610088

ABSTRACT

The extracellular domain (ECD) of class B1 G-protein-coupled receptors (GPCRs) plays a central role in signal transduction and is uniquely positioned to sense both the extracellular and membrane environments. Although recent studies suggest a role for membrane lipids in the modulation of class A and class F GPCR signaling properties, little is known about the effect of lipids on class B1 receptors. In this study, we employed multiscale molecular dynamics simulations to access the dynamics of the glucagon receptor (GCGR) ECD in the presence of native-like membrane bilayers. Simulations showed that the ECD could move about a hinge region formed by residues Q122-E126 to adopt both closed and open conformations relative to the transmembrane domain. ECD movements were modulated by binding of the glycosphingolipid GM3. These large-scale fluctuations in ECD conformation may affect the ligand binding and receptor activation properties. We also identify a unique phosphatidylinositol (4,5)-bisphosphate (PIP2) interaction profile near intracellular loop (ICL) 2/TM3 at the G-protein-coupling interface, suggesting a mechanism of engaging G-proteins that may have a distinct dependence on PIP2 compared with class A GPCRs. Given the structural conservation of class B1 GPCRs, the modulatory effects of GM3 and PIP2 on GCGR may be conserved across these receptors, offering new insights into potential therapeutic targeting.


Subject(s)
Glycosphingolipids , Receptors, Glucagon , Molecular Dynamics Simulation , Protein Binding , Receptors, G-Protein-Coupled/metabolism , Receptors, Glucagon/metabolism
14.
Nat Chem Biol ; 15(10): 975-982, 2019 10.
Article in English | MEDLINE | ID: mdl-31548691

ABSTRACT

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. Molecular dynamics simulations show that this interaction leads to the closure of a tunnel through PTCH1 that serves as the putative conduit for sterol transport. Thus, Shh inactivates PTCH1 by grasping its extracellular domain with two lipidic pincers, the N-terminal palmitate and the C-terminal cholesterol, which are both inserted into the PTCH1 protein core.


Subject(s)
Hedgehog Proteins/metabolism , Patched-1 Receptor/metabolism , Animals , Cholesterol/chemistry , Gene Expression Regulation , HEK293 Cells , Hedgehog Proteins/chemistry , Hedgehog Proteins/genetics , Humans , Mice , Models, Molecular , NIH 3T3 Cells , Patched-1 Receptor/chemistry , Protein Binding , Protein Conformation , Single-Domain Antibodies
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