Xavier Prasanna

Cholesterol Modulates the Dimer Interface of the β2-Adrenergic Receptor via Cholesterol Occupancy Sites

The β2-adrenergic receptor is an important member of the G-protein-coupled receptor (GPCR) superfamily, whose stability and function are modulated by membrane cholesterol. The recent high-resolution crystal structure of the β2-adrenergic receptor revealed the presence of possible cholesterol-binding sites in the receptor. However, the functional relevance of cholesterol binding to the receptor remains unexplored. We used MARTINI coarse-grained molecular-dynamics simulations to explore dimerization of the β2-adrenergic receptor in lipid bilayers containing cholesterol. A novel (to our knowledge) aspect of our results is that receptor dimerization is modulated by membrane cholesterol. We show that cholesterol binds to transmembrane helix IV, and cholesterol occupancy at this site restricts its involvement at the dimer interface. With increasing cholesterol concentration, an increased presence of transmembrane helices I and II, but a reduced presence of transmembrane helix IV, is observed at the dimer interface. To our knowledge, this study is one of the first to explore the correlation between cholesterol occupancy and GPCR organization. Our results indicate that dimer plasticity is relevant not just as an organizational principle but also as a subtle regulatory principle for GPCR function. We believe these results constitute an important step toward designing better drugs for GPCR dimer targets.

Cholesterol-dependent Conformational Plasticity in GPCR Dimers

The organization and function of the serotonin1A receptor, an important member of the GPCR family, have been shown to be cholesterol-dependent, although the molecular mechanism is not clear. We performed a comprehensive structural and dynamic analysis of dimerization of the serotonin1A receptor by coarse-grain molecular dynamics simulations totaling 3.6 ms to explore the molecular details of its cholesterol-dependent association. A major finding is that the plasticity and flexibility of the receptor dimers increase with increased cholesterol concentration. In particular, a dimer interface formed by transmembrane helices I-I was found to be sensitive to cholesterol. The modulation of dimer interface appears to arise from a combination of direct cholesterol occupancy and indirect membrane effects. Interestingly, the presence of cholesterol at the dimer interface is correlated with increased dimer plasticity and flexibility. These results represent an important step in characterizing the molecular interactions in GPCR organization with potential relevance to therapeutic interventions.

Role of Lipid-Mediated Effects in β2-Adrenergic Receptor Dimerization

G protein-coupled receptors (GPCRs) are an important family of mammalian membrane proteins whose function has been shown to be modulated by membrane lipid composition. The β2-adrenergic receptor is one of the most well characterized GPCRs. Structural characterization of the β2-adrenergic receptor and other related receptors has revealed putative lipid binding sites. In addition, indirect lipid effects, such as hydrophobic mismatch, have also been implicated in receptor function and organization. Despite these advances in understanding the receptor in the membrane environment, our understanding of the protein-lipid interactions remains limited. Here, we have used MARTINI coarse-grain molecular dynamics simulations to explore receptor-lipid interactions of the β2-adrenergic receptor. We analyze the indirect membrane effects such as hydrophobic mismatch and correlate its role in driving receptor association. We also study direct receptor-lipid interactions and identify a novel lipid binding site. The sites of increased receptor-lipid interactions, could play an important role in modulating receptor association. Our results provide novel insights into the correlation between direct and indirect lipid effects with GPCR organization. We believe these results constitute an important step in understanding GPCR organization and dynamics in the cell membrane.

Differential dynamics of the serotonin1A receptor in membrane bilayers of varying cholesterol content revealed by all atom molecular dynamics simulation

Abstract The serotonin1A receptor belongs to the superfamily of G protein-coupled receptors (GPCRs) and is a potential drug target in neuropsychiatric disorders. The receptor has been shown to require membrane cholesterol for its organization, dynamics and function. Although recent work suggests a close interaction of cholesterol with the receptor, the structural integrity of the serotonin1A receptor in the presence of cholesterol has not been explored. In this work, we have carried out all atom molecular dynamics simulations, totaling to 3 μs, to analyze the effect of cholesterol on the structure and dynamics of the serotonin1A receptor. Our results show that the presence of physiologically relevant concentration of membrane cholesterol alters conformational dynamics of the serotonin1A receptor and, on an average lowers conformational fluctuations. Our results show that, in general, transmembrane helix VII is most affected by the absence of membrane cholesterol. These results are in overall agreement with experimental data showing enhancement of GPCR stability in the presence of membrane cholesterol. Our results constitute a molecular level understanding of GPCR-cholesterol interaction, and represent an important step in our overall understanding of GPCR function in health and disease.

Protein-dependent membrane interaction of a partially disordered protein complex with oleic acid : Implications for cancer lipidomics

Bovine α-lactalbumin (BLA) forms cytotoxic complexes with oleic acid (OA) that perturbs tumor cell membranes, but molecular determinants of these membrane-interactions remain poorly understood. Here, we aim to obtain molecular insights into the interaction of BLA/BLA-OA complex with model membranes. We characterized the folding state of BLA-OA complex using tryptophan fluorescence and resolved residue-specific interactions of BLA with OA using molecular dynamics simulation. We integrated membrane-binding data using a voltage-sensitive probe and molecular dynamics (MD) to demonstrate the preferential interaction of the BLA-OA complex with negatively charged membranes. We identified amino acid residues of BLA and BLA-OA complex as determinants of these membrane interactions using MD, functionally corroborated by uptake of the corresponding α-LA peptides across tumor cell membranes. The results suggest that the α-LA component of these cytotoxic complexes confers specificity for tumor cell membranes through protein interactions that are maintained even in the lipid complex, in the presence of OA.

Exploring GPCR–Lipid Interactions by Molecular Dynamics Simulations: Excitements, Challenges, and the Way Forward

Gprotein-coupled receptors (GPCRs) are seven transmembrane receptors that mediate a large number of cellular responses and are important drug targets. One of the current challenges in GPCR biology is to analyze the molecular signatures of receptor-lipid interactions and their subsequent effects on GPCR structure, organization, and function. Molecular dynamics simulation studies have been successful in predicting molecular determinants of receptor-lipid interactions. In particular, predicted cholesterol interaction sites appear to correspond well with experimentally determined binding sites and estimated time scales of association. In spite of several success stories, the methodologies in molecular dynamics simulations are still emerging. In this Feature Article, we provide a comprehensive overview of coarse-grain and atomistic molecular dynamics simulations of GPCR-lipid interaction in the context of experimental observations. In addition, we discuss the effect of secondary and tertiary structural constraints in coarse-grain simulations in the context of functional dynamics and structural plasticity of GPCRs. We envision that this comprehensive overview will help resolve differences in computational studies and provide a way forward.

Chapter 5:Experimental and Computational Approaches to Study Membranes and Lipid–Protein Interactions

Biological membranes are complex two-dimensional, non-covalent assemblies of a diverse variety of lipids and proteins. A hallmark of membrane organization is varying degrees of spatiotemporal heterogeneity spanning a wide range. Membrane proteins are implicated in a wide variety of cellular functions, and comprise ∼30% of the human proteome and ∼50% of the current drug targets. Their interactions with membrane lipids are recognized as crucial elements in their function. In this article, we provide an overview of experimental and theoretical approaches to analyze membrane organization, dynamics, and lipid–protein interactions. In this context, we highlight the wide range of time scales that membrane events span, and approaches that are suitable for a given time scale. We discuss representative fluorescence-based approaches (FRET and FRAP) that help to address questions on lipid–protein and protein–cytoskeleton interactions in membranes. In a complimentary fashion, we discuss computational methods, atomistic and coarse-grain, that are required to address a given membrane problem at an appropriate scale. We believe that the synthesis of knowledge gained from experimental and computational approaches will enable us to probe membrane organization, dynamics, and interactions at increasing spatiotemporal resolution, thereby providing a robust model for the membrane in health and disease.