Long Timescale Dynamics of Cholesterol in Lipid Bilayers

Proposal to the Biomedical Applications Group at the Pittsburgh Supercomputing Center, funded by the National Institute of General Medical Science June 23, 2016

Summary

Cholesterol, the ubiquitous sterol present in mammalian cells, is a key player in maintaining and modifying characteristics of cell membranes, modulating the function of membrane-embedded and peripheral proteins. Cholesterol concentrations are cell type dependent, dynamic, and tightly regulated. Despite decades of experimental and computational studies, due to the fluidity and heterogeneity of the membrane environment, fundamental aspects of cholesterol function and dynamics are not yet understood. We propose to combine long timescale molecular dynamics with state-of-the-art magic-angle spinning solid-state NMR (SSNMR) experiments to yield an atomistically-resolved understanding of cholesterol. We have developed isotopic labeling and customized SSNMR measurements to provide site-resolved chemical shifts, order parameters and relaxation rates, which are experimental parameters dependent upon the hierarchical dynamics of the sterol in true lipid bilayers. These highly precise experimental data sets, when synergistically combined with computational analysis leveraging the unprecedented computational power of Anton 2, will enable a major breakthrough in understanding the molecular interactions of cholesterol in membranes. Our major goals are to use the long timescale MD simulations to discern for the first time the molecular interactions that lead to the hierarchical cholesterol-lipid and cholesterol-cholesterol dynamics.

PI: Chad M. Rienstra Professor of Chemistry University of Illinois at Urbana-Champaign 600 South Mathews Avenue, Urbana, IL 61801 Telephone: (217) 244-4655 FAX: (217) 244-3186 Email: [email protected]

Co-PI: Taras V. Pogorelov Research Assistant Professor of Chemistry Beckman Institute for Advanced Science and Technology National Center for Supercomputing Applications School of Chemical Sciences University of Illinois at Urbana-Champaign 600 South Mathews Avenue, Urbana, IL 61801 Telephone: (217) 244-4655 FAX: (217) 244-3186 Email: [email protected]

1 Background Sterols are vitally important in numerous biochemical and biophysical processes in the cell. Cholesterol, the major sterol present in mammalian cells, is a key regulator of membrane order, permeability, thickness and lateral organization1-3 and protein function1,4,5. Cholesterol also acts as a precursor to steroid hormones and bile acids6. The regulatory roles of cholesterol are postulated to depend directly upon interactions with other sterols and phospholipid molecules7-9. Recently it was shown that the orientation and dynamics of cholesterol are essential to its regulatory role10,11, though molecular details of the mechanism are still lacking. The importance of cholesterol to human health is appreciated by the fact that cholesterol-lowering drugs are a multibillion dollar per year global market, and almost 100 million Americans have high blood cholesterol (American Heart Association, 2013). Our research team is uniquely positioned to provide insights into this intense area of research by combining state-of-art solid-state NMR (SSNMR), biosynthesis and isotopic labeling, and molecular dynamics (MD) simulations to investigate the atomistically resolved hierarchical dynamics of cholesterol in lipid bilayers. Extensive prior studies of cholesterol have reported order parameters12 and restraints on the orientation in the bilayer from 2H SSNMR13,14. Additionally, there have been numerous MD simulations of cholesterol in the bilayer to ascertain the orientation and fast timescale dynamics (<100 ns)10,15. The limitations of these studies were the relatively small numbers of site- resolved experimental parameters and inadequate simulation timescales to examine biologically relevant events. We propose to rectify this situation in the current study and elucidate two fundamental aspects of cholesterol function: (1) a quantitative description of hierarchical dynamics and (2) cholesterol cluster formation and its influence on membrane. In both cases we plan to make extensive comparisons and analyses using both long timescale MD trajectories from Anton 2 and high-resolution 3D SSNMR data sets acquired at the University of Illinois. In our preliminary studies, we have generated foundational experimental data using 3D separated local field spectroscopy SSNMR methods in combination with highly 13C-enriched cholesterol samples prepared with an engineered yeast strain17. We thereby have determined site-specific chemical shifts and dipolar order parameters for cholesterol in multiple lipid bilayer environments above and below the gel-to-liquid-crystal phase transition, and we are in the process of measuring cross-correlated dipolar fields and relaxation parameters. These observables serve as high-resolution benchmarks that will be directly computed from the long timescale simulations to investigate hierarchical dynamics. The timescales of previous computational studies were too short to compute the important microsecond timescale motions involving clusters of cholesterol molecules. The unmatched computational power of Anton 2 (Ref. 18) will enable us to achieve an atomic-resolution description of cholesterol in phosphatidylcholine lipids with varying tail lengths to investigate the orientation, which depends upon lipid bilayer thickness10. The two lipid systems we propose to investigate are 10:3 POPC:Cholesterol and 10:3 DLPC:Cholesterol. These two lipid types, while having the same head-group, have a large difference in lipid tail length and membrane thickness. With the opportunity to capture long timescale simulations, we will be able to investigate the hierarchy of cholesterol’s diverse motions19: uniaxial molecular rotation, molecular axis wobble, chain reorientation and association to form cholesterol clusters (sometimes referred to as patches or rafts). Our data will lend insight into the distribution of size of these clusters, as well as their intermolecular association energetics and dynamics. Performing Anton 2 simulations at two different temperatures, corresponding to the liquid and gel phases, will distinguish between possible dynamic modes of cholesterol, that we are now able to capture with SSNMR. For example, the combination of experimental and computational data will distinguish among cholesterol rotating about its axis, rotating on the surface of a cone, and rotating and exploring the region within a cone.

2 Cholesterol is a vital part of the cell membrane and Anton 2 simulations proposed here when used in tandem with the unique SSNMR data will enable next level of understanding of the molecular mechanisms involved in regulation of the cell wall environment. Additionally, the direct comparison of SSNMR data with the long scale MD simulations will provide news insights into possible improvements of force fields.

Scientific Objectives

The Rienstra group has collected SSNMR data for most of the sites of cholesterol and now can connect experimental data directly with long timescale MD simulations. We have investigated cholesterol-lipid systems at two temperatures as these are conditions in which cholesterol is in environments both above and below the liquid-gel phase transition. Our SSNMR data indicate that substantial molecular motions remain even below the bulk phase transition temperature. In addition to the major fast (<100 ns) motional modes of uniaxial rotation and tail libration, slower motions (>microsecond) include translational diffusion, wobble on the surface of a cone, large amplitude tail reorientations and association events to form dimers and higher-order oligomers. Populations, rates, and Arrhenius activation energies for each of these processes can be extracted from the temperature-dependent SSNMR data for comparison with simulation. Our experimental results will complement the dynamic atomic-level description of how cholesterol interacts with the lipids. Anton 2 provides unique opportunities to model these systems on a long timescale to investigate both cholesterol-lipid and cholesterol-cholesterol interactions with experimental validation. We propose to simulate cholesterol-lipid systems (~120,000 atoms, Fig. 1) to fulfill our objectives. Effects of lipid tail length and temperature on cholesterol dynamics. To investigate how motions of cholesterol are influenced by the lipid tail length and hence membrane thickness, we will perform equilibrium simulations of the following cholesterol-lipid systems to match the SSNMR samples: 1) 10:3 POPC:Cholesterol and 2) 10:3 DLPC:Cholesterol. The former lipid tail has 16 and 18 carbon atoms while the latter has 12, and therefore the bilayer thickness differs and the orientation of cholesterol is altered dramatically. We anticipate fundamental changes in the interactions of cholesterol in each bilayer due to the altered

Figure 1. (A) 13C-13C 2D Spectrum of cholesterol in POPC (B) Line-shape examples for C8 and C12, black is experimental and red is simulated compared the rigid CA of N-acetyl valine (NAV) (C) Cholesterol molecule with line-shape carbon sites highlighted and cholesterol depicted (purple) in POPC bilayers.

3 orientation and differences in available surface area for interaction with phospholipids. To match the conditions under which SSNMR data was collected and various dynamics modes were detected, we will perform simulations of each system at 37 °C and -25 °C. The systems will have approximately 120,000 atoms with 400 lipids and 120 cholesterol molecules per system. The systems will be assembled built using CHARMM-GUI Membrane builder20 with cholesterol molecules initially randomly placed. To capture the microsecond hierarchical motions of cholesterol, we plan to simulate each of two systems at higher and lower temperatures for 40 μs. We expect that these long time simulations will capture the axial molecular rotation, molecular axis wobble, and chain reorientation of cholesterol, as well as address our second objective that focuses on cholesterol-cholesterol interactions. The motional timescales of lipid rafts and cholesterol- cholesterol interactions are on the order of microseconds to milliseconds21,22.The size of the proposed systems, including the concentration of cholesterol (there will be approximately 60 cholesterol molecules per leaflet) will allow for sufficient interaction between cholesterol molecules. This system composition is physiologically relevant, as plasma membranes can contain up to 50 mole% cholesterol, relative to the lipid23. To our knowledge these will be the first MD simulations at the timescale sufficient to match the cholesterol dynamics measured and will enable characterization of cholesterol influence on lipids, which are currently not labeled. These studies will lead the way to future SSNMR/MD studies of membrane-cholesterol system interaction with membrane proteins and provide a high-resolution picture of cholesterol interaction with putative binding sites of proteins or molecules of interest. Specifically, current efforts in the PI labs are focused on interactions with the powerful yet toxic antifungal drug Amphotericin B (AmB)24. Our team is studying the interactions with AmB and sterols, in an attempt to elucidate the mechanism of binding, with the goal of creating an AmB analogue with greater therapeutic index and lower toxicity. The initial cholesterol experiments described here will pave the way towards future experiments investigating the molecular mechanism of how cholesterol interacts with AmB. The combination of biosynthesis, high-resolution SSNMR and unmatched MD capabilities will allow us to elucidate the specific molecular mechanism behind cholesterol in lipids at the atomic level.

Project Feasibility

The proposed studies are ideal for Anton 2 as newly accessible timescales (40 μs) will enable to match the hierarchical dynamics of cholesterol measured with SSNMR.

1. MD force fields. The lipids with be described with the CHARMM36 lipid force field25. While cholesterol molecules will be described with the specifically updated CHARMM36c force field known for a better prediction of deuterium order parameters26. The TIP3P water model will be used27.

2. Simulation cell. Cubic box of water with 400 lipid and 120 cholesterol molecules in each system will be prepared resulting in cell size of 1.2x1.2x1.2 nm.

3. Simulation system. The simulated lipid-cholesterol systems will have ~120,000 atoms including only lipid, cholesterol, and water molecules.

4. MD simulation. The simulation performed on Anton 2 will be standard equilibrium unbiased molecular dynamics simulations in the NPT (constant number of molecules, pressure, and temperature) ensemble. No restraints will be used.

4 5. PI expertise. The investigators have studied membrane-associated phenomena by means of SSNMR and MD simulations for many years. The PI (C.M.R.) has a long history of SSNMR methods development and cutting-edge diverse applications24,28-42. The co-PI (T.V.P.) has been developing novel methods of membrane modeling45,46, that now are extensively used47 and in collaboration implemented in freely available resources48. The PIs have an established history of collaboration in combining computational and experimental methods to study membrane- based systems49,50. Please note, that the co-PI is also submitting an independent Anton 2 proposal on an unrelated project. This was confirmed as allowed with the PSC staff via email.

Requested resources

To address the proposed studies of cholesterol domain formation and to match the experimental systems we will need to accommodate a sufficient number of lipid and cholesterol, which will lead to ~120,000 atoms. Based on the published Anton 2 benchmark we estimate performance of ~20 μs/machine-day. To capture the hierarchical dynamics of cholesterol-lipid and cholesterol-cholesterol interactions will propose four simulations 40 μs/each, thus we request 8 machine-days.

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