Despite the vast amount of experimental and theoretical studies on the binding affinity of cations -especially the biologically relevant Na + and Ca 2+ -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 Ca 2+ 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 Ca 2+ binding in all the tested models, no model had sufficient quantitative accuracy to interpret the Ca 2+ :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.

ApoA-I is a uniquely flexible lipid-scavenging protein capable of incorporating phospholipids into stable particles. Here we report molecular dynamics simulations on a series of progressively smaller discoidal high density lipoprotein particles produced by incremental removal of palmitoyloleoylphosphatidylcholine via four different pathways. The starting model contained 160 palmitoyloleoylphosphatidylcholines and a belt of two antiparallel amphipathic helical lipid-associating domains of apolipoprotein (apo) A-I. The results are particularly compelling. After a few nanoseconds of molecular dynamics simulation, independent of the starting particle and method of size reduction, all simulated double belts of the four lipidated apoA-I particles have helical domains that impressively approximate the x-ray crystal structure of lipid-free apoA-I, particularly between residues 88 and 186. These results provide atomic resolution models for two of the particles produced by in vitro reconstitution of nascent high density lipoprotein particles. These particles, measuring 95 angstroms and 78 angstroms by nondenaturing gradient gel electrophoresis, correspond in composition and in size/shape (by negative stain electron microscopy) to the simulated particles with molar ratios of 100:2 and 50:2, respectively. The lipids of the 100:2 particle family form minimal surfaces at their monolayer-monolayer interface, whereas the 50:2 particle family displays a lipid pocket capable of binding a dynamic range of phospholipid molecules.

Spheroidal high-density lipoprotein (HDL) particles circulating in the blood are formed through an enzymatic process activated by apoA-I, leading to the esterification of cholesterol, which creates a hydrophobic core of cholesteryl ester molecules in the middle of the discoidal phospholipid bilayer. In this study, we investigated the conformation of apoA-I in model spheroidal HDL (ms-HDL) particles using both atomistic and coarse-grained molecular dynamics simulations, which are found to provide consistent results for all HDL properties we studied. The observed small contribution of cholesteryl oleate molecules to the solvent-accessible surface area of the entire ms-HDL particle indicates that palmitoyloleoylphosphatidylcholines and apoA-I molecules cover the hydrophobic core comprised of cholesteryl esters particularly well. The ms-HDL particles are found to form a prolate ellipsoidal shape, with sizes consistent with experimental results. Large rigid domains and low mobility of the protein are seen in all the simulations. Additionally, the average number of contacts of cholesteryl ester molecules with apoA-I residues indicates that cholesteryl esters interact with protein residues mainly through their cholesterol moiety. We propose that the interaction of annular cholesteryl oleate molecules contributes to apoA-I rigidity stabilizing and regulating the structure and function of the ms-HDL particle.

We study the structure and dynamics of spherical high density lipoprotein (HDL) particles through coarse-grained multi-microsecond molecular dynamics simulations. We simulate both a lipid droplet without the apolipoprotein A-I (apoA-I) and the full HDL particle including two apoA-I molecules surrounding the lipid compartment. The present models are the first ones among computational studies where the size and lipid composition of HDL are realistic, corresponding to human serum HDL. We focus on the role of lipids in HDL structure and dynamics. Particular attention is paid to the assembly of lipids and the influence of lipid-protein interactions on HDL properties. We find that the properties of lipids depend significantly on their location in the particle (core, intermediate region, surface). Unlike the hydrophobic core, the intermediate and surface regions are characterized by prominent conformational lipid order. Yet, not only the conformations but also the dynamics of lipids are found to be distinctly different in the different regions of HDL, highlighting the importance of dynamics in considering the functionalization of HDL. The structure of the lipid droplet close to the HDL-water interface is altered by the presence of apoA-Is, with most prominent changes being observed for cholesterol and polar lipids. For cholesterol, slow trafficking between the surface layer and the regimes underneath is observed. The lipid-protein interactions are strongest for cholesterol, in particular its interaction with hydrophobic residues of apoA-I. Our results reveal that not only hydrophobicity but also conformational entropy of the molecules are the driving forces in the formation of HDL structure. The results provide the first detailed structural model for HDL and its dynamics with and without apoA-I, and indicate how the interplay and competition between entropy and detailed interactions may be used in nanoparticle and drug design through self-assembly.

High density lipoproteins (HDL) represent a heterogeneous population of particles with apoA-I as the major protein (1). Whether apoA-I/HDL 2 plays a direct role in cardiovascular disease prevention (e.g. removal of cholesterol from clogged arteries) or an indirect one (e.g. acts as a platform for the clustering of protective molecules, such as anti-inflammatory or antioxidant proteins), detailed knowledge of HDL structure is a key to understanding the molecular mechanism underlying these processes. Because the conformation of apoA-I on HDL is highly plastic (1), understanding apoA-I/HDL structure and dynamics is not straightforward.Since the early 1970s, the standard model for HDL particles reconstituted from apolipoprotein A-I (apoA-I) and phospholipid (apoA-I/HDL) has been that of a discoidal particle on the order of 100 Å in diameter and the thickness of a phospholipid bilayer. The initial observation of discoidal HDL particles was made by Forte et al. (2) when they examined HDL from patients with familial lecithin:cholesterol acyltransferase deficiency by negative stain EM. Reconstituted apoA-I/HDL particles (3) and nascent HDL from lymph (4) were then observed by negative stain EM to also have a discoidal shape. X-ray and neutron scattering studies (5, 6) provided further support for the standard discoidal model. The standard discoidal model has been used to interpret the results of a large number of experimental studies of the structure of reconstituted apoA-I/HDL particles. For review, see however, Wu et al. (11) used small angle neutron scattering to develop a model for apoA-I/HDL particles that is dramatically different from the standard model. In their model, termed a double superhelix (DSH), apoA-I possesses an open helical shape that twists around a central prolate ellipsoidal particle resembling a partial type I hexagonal lyotropic liquid crystalline phase.The DSH model is interesting. It is similar to MD simulation-based models (12) in that the proposed double superhelix forms a left-handed spiral as it wraps around the prolate ellip-

The product of transesterification of phospholipid acyl chains and unesterified cholesterol (UC) by the enzyme lecithin: cholesterol acyltransferase (LCAT) is cholesteryl ester (CE). Activation of LCAT by apolipoprotein (apo) A-I on nascent (discoidal) high density lipoproteins (HDL) is essential for formation of mature (spheroidal) HDL during the antiatherogenic process of reverse cholesterol transport. Here we report all-atom and coarse grained (CG) molecular dynamics (MD) simulations of HDL particles that have major implications for mechanisms of LCAT activation. Both the allatom and CG simulations provide support for a model in which the helix 5/5 domains of apoA-I create an amphipathic "presentation tunnel" that exposes methyl ends of acyl chains at the bilayer center to solvent. Further, CG simulations show that UC also becomes inserted with high efficiency into the amphipathic presentation tunnel with its hydroxyl moiety (UC-OH) exposed to solvent; these results are consistent with trajectory analyses of the all-atom simulations showing that UC is being concentrated in the vicinity of the presentation tunnel. Finally, consistent with known product inhibition of CE-rich HDL by CE, CG simulations of CE-rich spheroidal HDL indicate partial blockage of the amphipathic presentation tunnel by CE. These results lead us to propose the following working hypothesis: After attachment of LCAT to discoidal HDL, the helix 5/5 domains in apoA-I form amphipathic presentation tunnels for migration of hydrophobic acyl chains and amphipathic UC from the bilayer to the phospholipase A2-like and esterification active sites of LCAT, respectively. This hypothesis is currently being tested by site-directed mutagenesis.Of the possible mechanisms suggested to explain the atheroprotective role of apolipoprotein (apo) A-I, a process called reverse cholesterol transport is most completely understood at the molecular level. Activation of the plasma enzyme lecithin:cholesterol acyltransferase (LCAT) (1) by apoA-I is necessary for esterification of the unesterified cholesterol (UC) molecules of high density lipoproteins (HDL) to cholesteryl ester (CE) and leads to the conversion of discoidal phospholipid-rich HDL to spheroidal, CE-rich (circulating) HDL, a central step of reverse cholesterol transport (2). A fuller understanding of reverse cholesterol transport demands knowledge of the structure and dynamics of the various HDL particles and intermediates in their assembly. † This work was supported in part by the National Institutes of Health Grant P01 HL-34343 (to JPS).* Author to whom correspondence should be addressed: Jere P. Segrest, 1808 7 th Avenue South, Boshell Diabetes Building 630, Departments of Medicine and Biochemistry and Molecular Genetics, and Center for Computational and Structural Dynamics, Birmingham, AL 35294, Phone 205 934 4420, Fax 205 975 8070, segrest@uab.edu . SUPPORTING INFORMATION AVAILABLEFigs. S1-S5 detail additional dynamics of the amphipathic presentation tunnel including a movie (S3). Fig. S6 il...

Apolipoprotein (apo) A-I is an unusually flexible protein whose lipid-associated structure is poorly understood. Thermal denaturation, which is used to measure the global helix stability of high-density lipoprotein (HDL)-associated apoA-I, provides no information about local helix stability. Here we report the use of temperature jump molecular dynamics (MD) simulations to scan the per-residue helix stability of apoA-I in phospholipid-rich HDL. When three 20 ns MD simulations were performed at 500 K on each of two particles created by MD simulations at 310 K, bilayers remained intact but expanded by 40%, and total apoA-I helicity decreased from 95% to 72%. Of significance, the conformations of the overlapping N- and C-terminal domains of apoA-I in the particles were unusually mobile, exposing hydrocarbon regions of the phospholipid to solvent; a lack of buried interhelical salt bridges in the terminal domains correlated with increased mobility. Nondenaturing gradient gels show that 40% expansion of the phospholipid surface of 100:2 particles by addition of palmitoyloleoylphosphatidylcholine exceeds the threshold of particle stability. As a unifying hypothesis, we propose that the terminal domains of apoA-I are phospholipid concentration-sensitive molecular triggers for fusion/remodeling of HDL particles. Since HDL remodeling is necessary for cholesterol transport, our model for remodeling has substantial biomedical implications.

Conversion of discoidal phospholipid (PL)-rich high density lipoprotein (HDL) to spheroidal cholesteryl ester-rich HDL is a central step in reverse cholesterol transport. A detailed understanding of this process and the atheroprotective role of apolipoprotein A-I (apoA-I) requires knowledge of the structure and dynamics of these various particles. This study, combining computation with experimentation, illuminates structural features of apoA-I allowing it to incorporate varying amounts of PL. Molecular dynamics simulated annealing of PL-rich HDL models containing unesterified cholesterol results in double belt structures with the same general saddle-shaped conformation of both our previous molecular dynamics simulations at 310 K and the x-ray structure of lipid-free apoA-I. Conversion from a discoidal to a saddle-shaped particle involves loss of helicity and formation of loops in opposing antiparallel parts of the double belt. During surface expansion caused by the temperature-jump step, the curved palmitoyloleoylphosphatidylcholine bilayer surfaces approach planarity. Relaxation back into saddle-shaped structures after cool down and equilibration further supports the saddle-shaped particle model. Our kinetic analyses of reconstituted particles demonstrate that PL-rich particles exist in discrete sizes corresponding to local energetic minima. Agreement of experimental and computational determinations of particle size/shape and apoA-I helicity provide additional support for the saddle-shaped particle model. Truncation experiments combined with simulations suggest that the N-terminal prolinerich domain of apoA-I influences the stability of PL-rich HDL particles. We propose that apoA-I incorporates increasing PL in the form of minimal surface bilayers through the incremental unwinding of an initially twisted saddle-shaped apoA-I double belt structure.