Mechanism of allosteric regulation of ß2-adrenergic receptor by cholesterol.

There is evidence that lipids can be allosteric regulators of membrane protein structure and activation. However, there are no data showing how exactly the regulation emerges from specific lipid-protein interactions. Here we show in atomistic detail how the human ß2-adrenergic receptor (ß2AR) - a prototypical G protein-coupled receptor - is modulated by cholesterol in an allosteric fashion. Extensive atomistic simulations show that cholesterol regulates ß2AR by limiting its conformational variability. The mechanism of action is based on the binding of cholesterol at specific high-affinity sites located near the transmembrane helices 5-7 of the receptor. The alternative mechanism, where the ß2AR conformation would be modulated by membrane-mediated interactions, plays only a minor role. Cholesterol analogues also bind to cholesterol binding sites and impede the structural flexibility of ß2AR, however cholesterol generates the strongest effect. The results highlight the capacity of lipids to regulate the conformation of membrane receptors through specific interactions.

Molecular electrometer and binding of cations to phospholipid bilayers.

Despite the vast amount of experimental and theoretical studies on the binding affinity of cations - especially the biologically relevant Na+ and Ca2+ - for phospholipid bilayers, there is no consensus in the literature. Here we show that by interpreting changes in the choline headgroup order parameters according to the 'molecular electrometer' concept [Seelig et al., Biochemistry, 1987, 26, 7535], one can directly compare the ion binding affinities between simulations and experiments. Our findings strongly support the view that in contrast to Ca2+ and other multivalent ions, Na+ and other monovalent ions (except Li+) do not specifically bind to phosphatidylcholine lipid bilayers at sub-molar concentrations. However, the Na+ binding affinity was overestimated by several molecular dynamics simulation models, resulting in artificially positively charged bilayers and exaggerated structural effects in the lipid headgroups. While qualitatively correct headgroup order parameter response was observed with Ca2+ binding in all the tested models, no model had sufficient quantitative accuracy to interpret the Ca2+:lipid stoichiometry or the induced atomistic resolution structural changes. All scientific contributions to this open collaboration work were made publicly, using nmrlipids.blogspot.fi as the main communication platform.

How To Minimize Artifacts in Atomistic Simulations of Membrane Proteins, Whose Crystal Structure Is Heavily Engineered: ß2-Adrenergic Receptor in the Spotlight.

Atomistic molecular dynamics (MD) simulations are used extensively to elucidate membrane protein properties. These simulations are based on three-dimensional protein structures that in turn are often based on crystallography. The protein structures resolved in crystallographic studies typically do not correspond to pristine proteins, however. Instead the crystallized proteins are commonly engineered, including structural modifications (mutations, replacement of protein sequences by antibodies, bound ligands, etc.) whose impact on protein structure and dynamics is largely unknown. Here we explore this issue through atomistic MD simulations (∼5 µs in total), focusing on the ß2-adrenergic receptor (ß2AR) that is one of the most studied members of the G-protein coupled receptor superfamily. Starting from an inactive-state crystal structure of ß2AR, we remove the many modifications in ß2AR systematically one at a time, in six consecutive steps. After each step, we equilibrate the system and simulate it quite extensively. The results of this step-by-step approach highlight that the structural modifications used in crystallization can affect ligand and G-protein binding sites, packing at the transmembrane-helix interface region, and the dynamics of connecting loops in ß2AR. When the results of the systematic step-by-step approach are compared to an all-at-once technique where all modifications done on ß2AR are removed instantaneously at the same time, it turns out that the step-by-step method provides results that are superior in terms of maintaining protein structural stability. The results provide compelling evidence that for membrane proteins whose 3D structure is based on structural engineering, the preparation of protein structure for atomistic MD simulations is a delicate and sensitive process. The results show that most valid results are found when the structural modifications are reverted slowly, one at a time.

Toward Atomistic Resolution Structure of Phosphatidylcholine Headgroup and Glycerol Backbone at Different Ambient Conditions.

Phospholipids are essential building blocks of biological membranes. Despite a vast amount of very accurate experimental data, the atomistic resolution structures sampled by the glycerol backbone and choline headgroup in phoshatidylcholine bilayers are not known. Atomistic resolution molecular dynamics simulations have the potential to resolve the structures, and to give an arrestingly intuitive interpretation of the experimental data, but only if the simulations reproduce the data within experimental accuracy. In the present work, we simulated phosphatidylcholine (PC) lipid bilayers with 13 different atomistic models, and compared simulations with NMR experiments in terms of the highly structurally sensitive C-H bond vector order parameters. Focusing on the glycerol backbone and choline headgroups, we showed that the order parameter comparison can be used to judge the atomistic resolution structural accuracy of the models. Accurate models, in turn, allow molecular dynamics simulations to be used as an interpretation tool that translates these NMR data into a dynamic three-dimensional representation of biomolecules in biologically relevant conditions. In addition to lipid bilayers in fully hydrated conditions, we reviewed previous experimental data for dehydrated bilayers and cholesterol-containing bilayers, and interpreted them with simulations. Although none of the existing models reached experimental accuracy, by critically comparing them we were able to distill relevant chemical information: (1) increase of choline order parameters indicates the P-N vector tilting more parallel to the membrane, and (2) cholesterol induces only minor changes to the PC (glycerol backbone) structure. This work has been done as a fully open collaboration, using nmrlipids.blogspot.fi as a communication platform; all the scientific contributions were made publicly on this blog. During the open research process, the repository holding our simulation trajectories and files ( https://zenodo.org/collection/user-nmrlipids ) has become the most extensive publicly available collection of molecular dynamics simulation trajectories of lipid bilayers.

Experimental determination and computational interpretation of biophysical properties of lipid bilayers enriched by cholesteryl hemisuccinate.

Cholesteryl hemisuccinate (CHS) is one of the cholesterol-mimicking detergents not observed in nature. It is, however, widely used in protein crystallography, in biochemical studies of proteins, and in pharmacology. Here, we performed an extensive experimental and theoretical study on the behavior of CHS in lipid membranes rich in unsaturated phospholipids. We found that the deprotonated form of CHS (that is the predominant form under physiological conditions) does not mimic cholesterol very well. The protonated form of CHS does better in this regard, but also its ability to mimic the physical effects of cholesterol on lipid membranes is limited. Overall, although ordering and condensing effects characteristic to cholesterol are present in systems containing any form of CHS, their strength is appreciably weaker compared to cholesterol. Based on the considerable amount of experimental and atomistic simulation data, we conclude that these differences originate from the fact that the ester group of CHS does not anchor it in an optimal position at the water-membrane interface. The implications of these findings for considerations of protein-cholesterol interactions are briefly discussed.

How well does cholesteryl hemisuccinate mimic cholesterol in saturated phospholipid bilayers?

Cholesteryl hemisuccinate is a detergent that is often used to replace cholesterol in crystallization of membrane proteins. Here we employ atomistic molecular dynamics simulations to characterize how well the properties of cholesteryl hemisuccinate actually match those of cholesterol in saturated protein-free lipid membranes. We show that the protonated form of cholesteryl hemisuccinate mimics many of the membrane properties of cholesterol quite well, while the deprotonated form of cholesteryl hemisuccinate is less convincing in this respect. Based on the results, we suggest that cholesteryl hemisuccinate in its protonated form is a quite faithful mimic of cholesterol for membrane protein crystallization, if specific cholesterol-protein interactions (not investigated here) are not playing a crucial role.