George Hedger

Structural basis of Smoothened regulation by its extracellular domains

Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzled-class 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. The CRD is stacked atop the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD–linker domain–TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains.

Lipid interaction sites on channels, transporters and receptors: Recent insights from molecular dynamics simulations.

Lipid molecules are able to selectively interact with specific sites on integral membrane proteins, and modulate their structure and function. Identification and characterization of these sites are of importance for our understanding of the molecular basis of membrane protein function and stability, and may facilitate the design of lipid-like drug molecules. Molecular dynamics simulations provide a powerful tool for the identification of these sites, complementing advances in membrane protein structural biology and biophysics. We describe recent notable biomolecular simulation studies which have identified lipid interaction sites on a range of different membrane proteins. The sites identified in these simulation studies agree well with those identified by complementary experimental techniques. This demonstrates the power of the molecular dynamics approach in the prediction and characterization of lipid interaction sites on integral membrane proteins. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

The juxtamembrane regions of human receptor tyrosine kinases exhibit conserved interaction sites with anionic lipids

Receptor tyrosine kinases (RTKs) play a critical role in diverse cellular processes and their activity is regulated by lipids in the surrounding membrane, including PIP2 (phosphatidylinositol-4,5-bisphosphate) in the inner leaflet, and GM3 (monosialodihexosylganglioside) in the outer leaflet. However, the precise details of the interactions at the molecular level remain to be fully characterised. Using a multiscale molecular dynamics simulation approach, we comprehensively characterise anionic lipid interactions with all 58 known human RTKs. Our results demonstrate that the juxtamembrane (JM) regions of RTKs are critical for inducing clustering of anionic lipids, including PIP2, both in simple asymmetric bilayers, and in more complex mixed membranes. Clustering is predominantly driven by interactions between a conserved cluster of basic residues within the first five positions of the JM region, and negatively charged lipid headgroups. This highlights a conserved interaction pattern shared across the human RTK family. In particular predominantly the N-terminal residues of the JM region are involved in the interactions with PIP2, whilst residues within the distal JM region exhibit comparatively less lipid specificity. Our results suggest that JM–lipid interactions play a key role in RTK structure and function, and more generally in the nanoscale organisation of receptor-containing cell membranes.

Convergence and Sampling in Determining Free Energy Landscapes for Membrane Protein Association

Potential of mean force (PMF) calculations are used to characterize the free energy landscape of protein–lipid and protein–protein association within membranes. Coarse-grained simulations allow binding free energies to be determined with reasonable statistical error. This accuracy relies on defining a good collective variable to describe the binding and unbinding transitions, and upon criteria for assessing the convergence of the simulation toward representative equilibrium sampling. As examples, we calculate protein–lipid binding PMFs for ANT/cardiolipin and Kir2.2/PIP2, using umbrella sampling on a distance coordinate. These highlight the importance of replica exchange between windows for convergence. The use of two independent sets of simulations, initiated from bound and unbound states, provide strong evidence for simulation convergence. For a model protein–protein interaction within a membrane, center-of-mass distance is shown to be a poor collective variable for describing transmembrane helix–helix dimerization. Instead, we employ an alternative intermolecular distance matrix RMS (DRMS) coordinate to obtain converged PMFs for the association of the glycophorin transmembrane domain. While the coarse-grained force field gives a reasonable Kd for dimerization, the majority of the bound population is revealed to be in a near-native conformation. Thus, the combination of a refined reaction coordinate with improved sampling reveals previously unnoticed complexities of the dimerization free energy landscape. We propose the use of replica-exchange umbrella sampling starting from different initial conditions as a robust approach for calculation of the binding energies in membrane simulations.

Free Energy Landscape of Lipid Interactions with Regulatory Binding Sites on the Transmembrane Domain of the EGF Receptor.

Lipid molecules can bind to specific sites on integral membrane proteins, modulating their structure and function. We have undertaken coarse-grained simulations to calculate free energy profiles for glycolipids and phospholipids interacting with modulatory sites on the transmembrane helix dimer of the EGF receptor within a lipid bilayer environment. We identify lipid interaction sites at each end of the transmembrane domain and compute interaction free energy profiles for lipids with these sites. Interaction free energies ranged from ca. −40 to −4 kJ/mol for different lipid species. Those lipids (glycolipid GM3 and phosphoinositide PIP2) known to modulate EGFR function exhibit the strongest binding to interaction sites on the EGFR, and we are able to reproduce the preference for interaction with GM3 over other glycolipids suggested by experiment. Mutation of amino acid residues essential for EGFR function reduce the binding free energy of these key lipid species. The residues interacting with the lipids in the simulations are in agreement with those suggested by experimental (mutational) studies. This approach provides a generalizable tool for characterizing the interactions of lipids that bind to specific sites on integral membrane proteins.

Molecular simulations of glycolipids: Towards mammalian cell membrane models

Glycolipids are key components of mammalian cell membranes, influencing a diverse range of cellular functions. For example, a number of receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR), are allosterically regulated by the glycolipid monosialodihexosylganglioside (GM3). Recent advances in molecular dynamics methods, especially the development of coarse-grained models, have enabled simulations of increasingly complex models of cell membranes. We demonstrate these methodological developments via a case study of a coarse-grained model for the ganglioside GM3. This glycolipid is included in simulations of a mixed lipid bilayer model reflecting the compositional complexity of a mammalian cell membrane. The resultant membrane model is used to simulate the interactions of GM3 with the transmembrane domain of the EGFR.

PtdIns(4,5)P2 stabilizes active states of GPCRs and enhances selectivity of G-protein coupling.

G-protein-coupled receptors (GPCRs) are involved in many physiological processes and are therefore key drug targets1. Although detailed structural information is available for GPCRs, the effects of lipids on the receptors, and on downstream coupling of GPCRs to G proteins are largely unknown. Here we use native mass spectrometry to identify endogenous lipids bound to three class A GPCRs. We observed preferential binding of phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) over related lipids and confirm that the intracellular surface of the receptors contain hotspots for PtdIns(4,5)P2 binding. Endogenous lipids were also observed bound directly to the trimeric Gαsβγ protein complex of the adenosine A2A receptor (A2AR) in the gas phase. Using engineered Gα subunits (mini-Gαs, mini-Gαi and mini-Gα12)2, we demonstrate that the complex of mini-Gαs with the β1 adrenergic receptor (β1AR) is stabilized by the binding of two PtdIns(4,5)P2 molecules. By contrast, PtdIns(4,5)P2 does not stabilize coupling between β1AR and other Gα subunits (mini-Gαi or mini-Gα12) or a high-affinity nanobody. Other endogenous lipids that bind to these receptors have no effect on coupling, highlighting the specificity of PtdIns(4,5)P2. Calculations of potential of mean force and increased GTP turnover by the activated neurotensin receptor when coupled to trimeric Gαiβγ complex in the presence of PtdIns(4,5)P2 provide further evidence for a specific effect of PtdIns(4,5)P2 on coupling. We identify key residues on cognate Gα subunits through which PtdIns(4,5)P2 forms bridging interactions with basic residues on class A GPCRs. These modulating effects of lipids on receptors suggest consequences for understanding function, G-protein selectivity and drug targeting of class A GPCRs.Mass spectrometry-based assays are used to reveal specificity and structural determinants of lipid binding to class A G-protein-coupled receptors, and the effects of specific lipids on receptor coupling to G proteins.

Cholesterol interaction sites on the transmembrane domain of the hedgehog signal transducer and Class F G protein-coupled receptor Smoothened

Transduction of hedgehog signals across the plasma membrane is a key process during animal development. This is facilitated by the Class F G-protein-coupled-receptor (GPCR) Smoothened (SMO), a major drug target in the treatment of basal cell carcinomas. Recent studies have suggested that SMO is modulated via interactions of its transmembrane (TM) domain with cholesterol. Long time scale (>0.35 ms of simulation time) molecular dynamics simulations of SMO embedded in two different cholesterol containing lipid bilayers reveal direct interactions of cholesterol with the transmembrane domain at regions distinct from those observed in Class A GPCRs. In particular the extracellular tips of helices TM2 and TM3 form a well-defined cholesterol interaction site, robust to changes in membrane composition and in force field parameters. Potential of mean force calculations for cholesterol interactions yield a free energy landscape for cholesterol binding. Combined with analysis of equilibrium cholesterol occupancy these results reveal the existence of a dynamic ‘greasy patch’ interaction with the TM domain of SMO, which may be compared to previously identified lipid interaction sites on other membrane proteins. These predictions provide molecular level insights into cholesterol interactions with a biomedically relevant Class F GPCR, suggesting potential druggable sites.

Interactions of the EphA2 Kinase Domain with PIPs in Membranes: Implications for Receptor Function.

Summary EphA2 is a member of the receptor tyrosine kinase family. Interactions of the cytoplasmic region of EphA2 with the cell membrane are functionally important and yet remain incompletely characterized. Molecular dynamics simulations combined with biochemical studies reveal the interactions of the transmembrane, juxtamembrane (JM), and kinase domains with the membrane. We describe how the kinase domain is oriented relative to the membrane and how the JM region can modulate this interaction. We highlight the role of phosphatidylinositol phosphates (PIPs) in mediating the interaction of the kinase domain with the membrane and, conversely, how positively charged patches at the kinase surface and in the JM region induce the formation of nanoclusters of PIP molecules in the membrane. Integration of these results with those from previous studies enable computational reconstitution of a near complete EphA2 receptor within a membrane, suggesting a role for receptor-lipid interactions in modulation of EphA2.

GPCRs: What Can We Learn from Molecular Dynamics Simulations?

Advances in the structural biology of G-protein Coupled Receptors have resulted in a significant step forward in our understanding of how this important class of drug targets function at the molecular level. However, it has also become apparent that they are very dynamic molecules, and moreover, that the underlying dynamics is crucial in shaping the response to different ligands. Molecular dynamics simulations can provide unique insight into the dynamic properties of GPCRs in a way that is complementary to many experimental approaches. In this chapter, we describe progress in three distinct areas that are particularly difficult to study with other techniques: atomic level investigation of the conformational changes that occur when moving between the various states that GPCRs can exist in, the pathways that ligands adopt during binding/unbinding events and finally, the influence of lipids on the conformational dynamics of GPCRs.