Allosteric modulation of the adenosine A2A receptor by cholesterol

Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A2A receptor (A2AR) in reconstituted phospholipid nanodiscs. This model system conveniently excludes possible effects arising from cholesterol-induced phase separation or receptor oligomerization and focuses on the question of allostery. GTP hydrolysis assays show that cholesterol weakly enhances the basal signaling of A2AR while decreasing the agonist EC50. Fluorine nuclear magnetic resonance (19F NMR) spectroscopy shows that this enhancement arises from an increase in the receptor’s active state population and a G-protein-bound precoupled state. 19F NMR of fluorinated cholesterol analogs reveals transient interactions with A2AR, indicating a lack of high-affinity binding or direct allosteric modulation. The combined results suggest that the observed allosteric effects are largely indirect and originate from cholesterol-mediated changes in membrane properties, as shown by membrane fluidity measurements and high-pressure NMR.

Mechanisms underlying drug-mediated regulation of membrane protein function

The hydrophobic coupling between membrane proteins and their host lipid bilayer provides a mechanism by which bilayer-modifying drugs may alter protein function. Drug regulation of membrane protein function thus may be mediated by both direct interactions with the protein and drug-induced alterations of bilayer properties, in which the latter will alter the energetics of protein conformational changes. To tease apart these mechanisms, we examine how the prototypical, proton-gated bacterial potassium channel KcsA is regulated by bilayer-modifying drugs using a fluorescence-based approach to quantify changes in both KcsA function and lipid bilayer properties (using gramicidin channels as probes). All tested drugs inhibited KcsA activity, and the changes in the different gating steps varied with bilayer thickness, suggesting a coupling to the bilayer. Examining the correlations between changes in KcsA gating steps and bilayer properties reveals that drug-induced regulation of membrane protein function indeed involves bilayer-mediated mechanisms. Both direct, either specific or nonspecific, binding and bilayer-mediated mechanisms therefore are likely to be important whenever there is overlap between the concentration ranges at which a drug alters membrane protein function and bilayer properties. Because changes in bilayer properties will impact many diverse membrane proteins, they may cause indiscriminate changes in protein function.

Lysine acetylation regulates the interaction between proteins and membranes

Lysine acetylation regulates the function of soluble proteins in vivo, yet it remains largely unexplored whether lysine acetylation regulates membrane protein function. Here, we use bioinformatics, biophysical analysis of recombinant proteins, live-cell fluorescent imaging and genetic manipulation of Drosophila to explore lysine acetylation in peripheral membrane proteins. Analysis of 50 peripheral membrane proteins harboring BAR, PX, C2, or EHD membrane-binding domains reveals that lysine acetylation predominates in membrane-interaction regions. Acetylation and acetylation-mimicking mutations in three test proteins, amphiphysin, EHD2, and synaptotagmin1, strongly reduce membrane binding affinity, attenuate membrane remodeling in vitro and alter subcellular localization. This effect is likely due to the loss of positive charge, which weakens interactions with negatively charged membranes. In Drosophila, acetylation-mimicking mutations of amphiphysin cause severe disruption of T-tubule organization and yield a flightless phenotype. Our data provide mechanistic insights into how lysine acetylation regulates membrane protein function, potentially impacting a plethora of membrane-related processes.

Allosteric modulation of the adenosine A2A receptor by cholesterol

Cholesterol is a major component of the cell membrane and commonly regulates membrane protein function. Here, we investigate how cholesterol modulates the conformational equilibria and signaling of the adenosine A2A receptor (A2AR) in reconstituted phospholipid bilayers. GTP hydrolysis assays show that cholesterol is a weak positive allosteric modulator of A2AR, as seen through enhanced basal signaling and a small decrease in agonist EC50. Fluorine nuclear magnetic resonance (19F NMR) spectroscopy suggests that this enhancement arises from an increase in the receptor’s active state populations and stronger G protein coupling. 19F NMR of fluorinated cholesterol analogs reveals transient and non-specific interactions with A2AR, indicating a lack of high-affinity binding sites or direct allosteric modulation. This is confirmed by computational analysis which suggests that cholesterol contacts confer a weak and possibly negative allosteric effect. The combined results suggest that the observed cholesterol allostery in A2AR is likely a result of indirect membrane effects through cholesterol-mediated changes in membrane properties, as shown by membrane fluidity measurements and high-pressure NMR.

Assessing the Role of Lipids in the Molecular Mechanism of Membrane Proteins

Membrane proteins have evolved to work optimally within the complex environment of the biological membrane. Consequently, interactions with surrounding lipids are part of their molecular mechanism. Yet, the identification of lipid–protein interactions and the assessment of their molecular role is an experimental challenge. Recently, biophysical approaches have emerged that are compatible with the study of membrane proteins in an environment closer to the biological membrane. These novel approaches revealed specific mechanisms of regulation of membrane protein function. Lipids have been shown to play a role in oligomerization, conformational transitions or allosteric coupling. In this review, we summarize the recent biophysical approaches, or combination thereof, that allow to decipher the role of lipid–protein interactions in the mechanism of membrane proteins.

Tracking Membrane Protein Dynamics in Real Time

Membrane proteins govern critical cellular processes and are central to human health and associated disease. Understanding of membrane protein function is obscured by the vast ranges of structural dynamics—both in the spatial and time regime—displayed in the protein and surrounding membrane. The membrane lipids have emerged as allosteric modulators of membrane protein function, which further adds to the complexity. In this review, we discuss several examples of membrane dependency. A particular focus is on how molecular dynamics (MD) simulation have aided to map membrane protein dynamics and how enhanced sampling methods can enable observing the otherwise inaccessible biological time scale. Also, time-resolved X-ray scattering in solution is highlighted as a powerful tool to track membrane protein dynamics, in particular when combined with MD simulation to identify transient intermediate states. Finally, we discuss future directions of how to further develop this promising approach to determine structural dynamics of both the protein and the surrounding lipids. Graphic Abstract

A DNA nanoassembly-based approach to map membrane protein nanoenvironments

Super-resolution imaging has revealed that most proteins at the plasma membrane are not uniformly distributed but localize to dynamic domains of nanoscale dimensions. To investigate their functional relevance, there is a need for methods that enable comprehensive mapping of the compositions and spatial organizations of membrane protein nanodomains in cell populations. However, current superresolution methods are limited to analysing small, preselected subsets of proteins, at very low sampling fractions. Here we describe the development of a non-microscopy based super-resolution method for unbiased ensemble analysis of membrane protein nanodomains. The method, termed NANOscale DEciphEring of membrane Protein nanodomains (NanoDeep), is based on the use of DNA nanoassemblies to translate membrane protein organization information into a DNA sequencing readout. Using NanoDeep, we characterized the nanoenvironments of Her2, a membrane receptor of critical relevance in cancer. We found that the occupancies of Her2, Her3 and EGFR in the nanoenvironments surrounding Her2 were similar in two cell lines with vastly different expression levels of Her2. Further, we found that adding Heregulin-β1 to cancer cells led to increased occupancy of Her2 and Her3, and to a lesser extent EGFR, in Her2 nanoenvironments. NanoDeep has the potential to provide new insights into the roles of the composition and spatial organization of protein nanoenvironments in the regulation of membrane protein function.