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. 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.

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. 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.

Electronic Polarizability Tunes the Function of the Human Bestrophin 1 Cl⁻ Channel.

Mechanisms of anion permeation within ion channels and nanopores remain poorly understood. Recent cryo-electron microscopy structures of the human bestrophin 1 chloride channel (hBest1) provide an opportunity to evaluate ion interactions predicted by molecular dynamics (MD) simulations against experimental observations. We implement the fully polarizable forcefield AMOEBA in MD simulations of open and partially-open states of the hBest1. The AMOEBA forcefield models multipole moments up to the quadrupole; therefore, it captures induced dipole and anion-π interactions. By including polarization we demonstrate the key role that aromatic residues play in ion permeation and the functional advantages of pore asymmetry within the highly conserved hydrophobic neck of the pore. We establish that these only arise when electronic polarization is included in the molecular models. We also show that Cl⁻ permeation in this region can be achieved through hydrophobic solvation concomitant with partial ion dehydration, which is compensated for by the formation of contacts with the edge of the phenylalanine ring. Furthermore, we demonstrate how polarizable simulations can help determine the identity of ion-like densities within high-resolution cryo-EM structures. Crucially, neglecting polarization in simulation of these systems results in the localization of Cl⁻ at positions that do not correspond with their experimentally resolved location. Overall, our results demonstrate the importance of including electronic polarization in realistic and physically accurate models of biological systems.

Structure of the native chemotaxis core signaling unit from phage E-protein lysed E. coli cells.

IMPORTANCE: Bacterial chemotaxis is a ubiquitous behavior that enables cell movement toward or away from specific chemicals. It serves as an important model for understanding cell sensory signal transduction and motility. Characterization of the molecular mechanisms underlying chemotaxis is of fundamental interest and requires a high-resolution structural picture of the sensing machinery, the chemosensory array. In this study, we combine cryo-electron tomography and molecular simulation to present the complete structure of the core signaling unit, the basic building block of chemosensory arrays, from Escherichia coli. Our results provide new insight into previously poorly-resolved regions of the complex and offer a structural basis for designing new experiments to test mechanistic hypotheses.

Heterogeneous biological membranes regulate protein partitioning via fluctuating diffusivity.

Cell membranes phase separate into ordered Lo and disordered Ld domains depending on their compositions. This membrane compartmentalization is heterogeneous and regulates the localization of specific proteins related to cell signaling and trafficking. However, it is unclear how the heterogeneity of the membranes affects the diffusion and localization of proteins in Lo and Ld domains. Here, using Langevin dynamics simulations coupled with the phase-field (LDPF) method, we investigate several tens of milliseconds-scale diffusion and localization of proteins in heterogeneous biological membrane models showing phase separation into Lo and Ld domains. The diffusivity of proteins exhibits temporal fluctuations depending on the field composition. Increases in molecular concentrations and domain preference of the molecule induce subdiffusive behavior due to molecular collisions by crowding and confinement effects, respectively. Moreover, we quantitatively demonstrate that the protein partitioning into the Lo domain is determined by the difference in molecular diffusivity between domains, molecular preference of domain, and molecular concentration. These results pave the way for understanding how biological reactions caused by molecular partitioning may be controlled in heterogeneous media. Moreover, the methodology proposed here is applicable not only to biological membrane systems but also to the study of diffusion and localization phenomena of molecules in various heterogeneous systems.

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. 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.

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. 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.

The Role of C2 Domains in Two Different Phosphatases: PTEN and SHIP2.

Phosphatase and tensin homologue (PTEN) and SH2-containing inositol 5'-phosphatase 2 (SHIP2) are structurally and functionally similar. They both consist of a phosphatase (Ptase) domain and an adjacent C2 domain, and both proteins dephosphorylate phosphoinositol-tri(3,4,5)phosphate, PI(3,4,5)P3; PTEN at the 3-phophate and SHIP2 at the 5-phosphate. Therefore, they play pivotal roles in the PI3K/Akt pathway. Here, we investigate the role of the C2 domain in membrane interactions of PTEN and SHIP2, using molecular dynamics simulations and free energy calculations. It is generally accepted that for PTEN, the C2 domain interacts strongly with anionic lipids and therefore significantly contributes to membrane recruitment. In contrast, for the C2 domain in SHIP2, we previously found much weaker binding affinity for anionic membranes. Our simulations confirm the membrane anchor role of the C2 domain in PTEN, as well as its necessity for the Ptase domain in gaining its productive membrane-binding conformation. In contrast, we identified that the C2 domain in SHIP2 undertakes neither of these roles, which are generally proposed for C2 domains. Our data support a model in which the main role of the C2 domain in SHIP2 is to introduce allosteric interdomain changes that enhance catalytic activity of the Ptase domain.

Influence of electronic polarization on the binding of anions to a chloride-pumping rhodopsin.

The functional properties of some biological ion channels and membrane transport proteins are proposed to exploit anion-hydrophobic interactions. Here, we investigate a chloride-pumping rhodopsin as an example of a membrane protein known to contain a defined anion binding site composed predominantly of hydrophobic residues. Using molecular dynamics simulations, we explore Cl- binding to this hydrophobic site and compare the dynamics arising when electronic polarization is neglected (CHARMM36 [c36] fixed-charge force field), included implicitly (via the prosECCo force field), or included explicitly (through the polarizable force field, AMOEBA). Free energy landscapes of Cl- moving out of the binding site and into bulk solution demonstrate that the inclusion of polarization results in stronger ion binding and a second metastable binding site in chloride-pumping rhodopsin. Simulations focused on this hydrophobic binding site also indicate longer binding durations and closer ion proximity when polarization is included. Furthermore, simulations reveal that Cl- within this binding site interacts with an adjacent loop to facilitate rebinding events that are not observed when polarization is neglected. These results demonstrate how the inclusion of polarization can influence the behavior of anions within protein binding sites and can yield results comparable with more accurate and computationally demanding methods.

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 -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.

Cardiolipin, and not monolysocardiolipin, preferentially binds to the interface of complexes III and IV.

The mitochondrial electron transport chain comprises a series of protein complexes embedded in the inner mitochondrial membrane that generate a proton motive force via oxidative phosphorylation, ultimately generating ATP. These protein complexes can oligomerize to form larger structures called supercomplexes. Cardiolipin (CL), a conical lipid, unique within eukaryotes to the inner mitochondrial membrane, has proven essential in maintaining the stability and function of supercomplexes. Monolysocardiolipin (MLCL) is a CL variant that accumulates in people with Barth syndrome (BTHS). BTHS is caused by defects in CL biosynthesis and characterised by abnormal mitochondrial bioenergetics and destabilised supercomplexes. However, the mechanisms by which MLCL causes pathogenesis remain unclear. Here, multiscale molecular dynamics characterise the interactions of CL and MLCL with yeast and mammalian mitochondrial supercomplexes containing complex III (CIII) and complex IV (CIV). Coarse-grained simulations reveal that both CL and MLCL bind to sites at the interface between CIII and CIV of the supercomplex. Free energy perturbation calculations show that MLCL interaction is weaker than that of CL and suggest that interaction with CIV drives this difference. Atomistic contact analyses show that, although interaction with CIII is similar for CL and MLCL, CIV makes more contacts with CL than MLCL, demonstrating that CL is a more successful "glue" between the two complexes. Simulations of the human CIII2CIV supercomplex show that this interface site is maintained between species. Our study suggests that MLCL accumulation in people with BTHS disrupts supercomplex stability by formation of relatively weak interactions at the interface lipid binding site.

Martini 3 Coarse-Grained Force Field for Carbohydrates.

The Martini 3 force field is a full reparametrization of the Martini coarse-grained model for biomolecular simulations. Due to the improved interaction balance, it allows for a more accurate description of condensed phase systems. In the present work, we develop a consistent strategy to parametrize carbohydrate molecules accurately within the framework of Martini 3. In particular, we develop a canonical mapping scheme which decomposes arbitrarily large carbohydrates into a limited number of fragments. Bead types for these fragments have been assigned by matching physicochemical properties of mono- and disaccharides. In addition, guidelines for assigning bonds, angles, and dihedrals were developed. These guidelines enable a more accurate description of carbohydrate conformations than in the Martini 2 force field. We show that models obtained with this approach are able to accurately reproduce osmotic pressures of carbohydrate water solutions. Furthermore, we provide evidence that the model differentiates correctly the solubility of the polyglucoses dextran (water-soluble) and cellulose (water insoluble but soluble in ionic liquids). Finally, we demonstrate that the new building blocks can be applied to glycolipids. We show they are able to reproduce membrane properties and induce binding of peripheral membrane proteins. These test cases demonstrate the validity and transferability of our approach.

Mass spectrometry captures biased signalling and allosteric modulation of a G-protein-coupled receptor.

G-protein-coupled receptors signal through cognate G proteins. Despite the widespread importance of these receptors, their regulatory mechanisms for G-protein selectivity are not fully understood. Here we present a native mass spectrometry-based approach to interrogate both biased signalling and allosteric modulation of the ß1-adrenergic receptor in response to various ligands. By simultaneously capturing the effects of ligand binding and receptor coupling to different G proteins, we probed the relative importance of specific interactions with the receptor through systematic changes in 14 ligands, including isoprenaline derivatives, full and partial agonists, and antagonists. We observed enhanced dynamics of the intracellular loop 3 in the presence of isoprenaline, which is capable of acting as a biased agonist. We also show here that endogenous zinc ions augment the binding in receptor-Gs complexes and propose a zinc ion-binding hotspot at the TM5/TM6 intracellular interface of the receptor-Gs complex. Further interrogation led us to propose a mechanism in which zinc ions facilitate a structural transition of the intermediate complex towards the stable state.

An N-glycan on the C2 domain of JAGGED1 is important for Notch activation.

The canonical members of the Jagged/Serrate and Delta families of transmembrane ligands have an extracellular, amino-terminal C2 domain that binds to phospholipids and is required for optimal activation of the Notch receptor. Somatic mutations that cause amino substitutions in the C2 domain in human JAGGED1 (JAG1) have been identified in tumors. We found in reporter cell assays that mutations affecting an N-glycosylation site reduced the ligand's ability to activate Notch. This N-glycosylation site located in the C2 domain is conserved in the Jagged/Serrate family but is lacking in the Delta family. Site-specific glycan analysis of the JAG1 amino terminus demonstrated that occupancy of this site by either a complex-type or high-mannose N-glycan was required for full Notch activation in reporter cell assays. Similarly to JAG1 variants with defects in Notch binding, N-glycan removal, either by mutagenesis of the glycosylation site or by endoglycosidase treatment, reduced receptor activation. The N-glycan variants also reduced receptor activation in a Notch signaling-dependent vascular smooth muscle cell differentiation assay. Loss of the C2 N-glycan reduced JAG1 binding to liposomes to a similar extent as the loss of the entire C2 domain. Molecular dynamics simulations suggested that the presence of the N-glycan limits the orientation of JAG1 relative to the membrane, thus facilitating Notch binding. These data are consistent with a critical role for the N-glycan in promoting a lipid-binding conformation that is required to orient Jagged at the cell membrane for full Notch activation.

Structural basis for cannabinoid-induced potentiation of alpha1-glycine receptors in lipid nanodiscs.

Nociception and motor coordination are critically governed by glycine receptor (GlyR) function at inhibitory synapses. Consequentially, GlyRs are attractive targets in the management of chronic pain and in the treatment of several neurological disorders. High-resolution mechanistic details of GlyR function and its modulation are just emerging. While it has been known that cannabinoids such as Δ9-tetrahydrocannabinol (THC), the principal psychoactive constituent in marijuana, potentiate GlyR in the therapeutically relevant concentration range, the molecular mechanism underlying this effect is still not understood. Here, we present Cryo-EM structures of full-length GlyR reconstituted into lipid nanodisc in complex with THC under varying concentrations of glycine. The GlyR-THC complexes are captured in multiple conformational states that reveal the basis for THC-mediated potentiation, manifested as different extents of opening at the level of the channel pore. Taken together, these structural findings, combined with molecular dynamics simulations and functional analysis, provide insights into the potential THC binding site and the allosteric coupling to the channel pore.

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. 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.

The guidance and adhesion protein FLRT2 dimerizes in cis via dual small-X3-small transmembrane motifs.

Fibronectin Leucine-rich Repeat Transmembrane (FLRT 1-3) proteins are a family of broadly expressed single-spanning transmembrane receptors that play key roles in development. Their extracellular domains mediate homotypic cell-cell adhesion and heterotypic protein interactions with other receptors to regulate cell adhesion and guidance. These in trans FLRT interactions determine the formation of signaling complexes of varying complexity and function. Whether FLRTs also interact at the surface of the same cell, in cis, remains unknown. Here, molecular dynamics simulations reveal two dimerization motifs in the FLRT2 transmembrane helix. Single particle tracking experiments show that these Small-X3-Small motifs synergize with a third dimerization motif encoded in the extracellular domain to permit the cis association and co-diffusion patterns of FLRT2 receptors on cells. These results may point to a competitive switching mechanism between in cis and in trans interactions, which suggests that homotypic FLRT interaction mirrors the functionalities of classic adhesion molecules.

Hepatocyte cholesterol content modulates glucagon receptor signalling.

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.

Influence of effective polarization on ion and water interactions within a biomimetic nanopore.

Interactions between ions and water at hydrophobic interfaces within ion channels and nanopores are suggested to play a key role in the movement of ions across biological membranes. Previous molecular-dynamics simulations have shown that anion affinity for aqueous/hydrophobic interfaces can be markedly influenced by including polarization effects through an electronic continuum correction. Here, we designed a model biomimetic nanopore to imitate the polar pore openings and hydrophobic gating regions found in pentameric ligand-gated ion channels. Molecular-dynamics simulations were then performed using both a non-polarizable force field and the electronic-continuum-correction method to investigate the behavior of water, Na+, and Cl- ions confined within the hydrophobic region of the nanopore. Number-density distributions revealed preferential Cl- adsorption to the hydrophobic pore walls, with this interfacial layer largely devoid of Na+. Free-energy profiles for Na+ and Cl- permeating the pore also display an energy-barrier reduction associated with the localization of Cl- to this hydrophobic interface, and the hydration-number profiles reflect a corresponding reduction in the first hydration shell of Cl-. Crucially, these ion effects were only observed through inclusion of effective polarization, which therefore suggests that polarizability may be essential for an accurate description for the behavior of ions and water within hydrophobic nanoscale pores, especially those that conduct Cl-.

Specific interactions of peripheral membrane proteins with lipids: what can molecular simulations show us?

Peripheral membrane proteins (PMPs) can reversibly and specifically bind to biological membranes to carry out functions such as cell signalling, enzymatic activity, or membrane remodelling. Structures of these proteins and of their lipid-binding domains are typically solved in a soluble form, sometimes with a lipid or lipid headgroup at the binding site. To provide a detailed molecular view of PMP interactions with the membrane, computational methods such as molecular dynamics (MD) simulations can be applied. Here, we outline recent attempts to characterise these binding interactions, focusing on both intracellular proteins, such as phosphatidylinositol phosphate (PIP)-binding domains, and extracellular proteins such as glycolipid-binding bacterial exotoxins. We compare methods used to identify and analyse lipid-binding sites from simulation data and highlight recent work characterising the energetics of these interactions using free energy calculations. We describe how improvements in methodologies and computing power will help MD simulations to continue to contribute to this field in the future.