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Molecular dynamics simulations of monomeric apolipoprotein A-I from a recent X-ray structure
No full textWe have examined the X-ray crystal structure recently refined by Ajees and colleagues (Ajees et al. 2006) for monomeric apolipoprotein A-I (apoA-I). Because the structure, which has been crystallized together with chromium organic compounds, possesses a substantially higher percentage of alpha helicity than is generally estimated experimentally for the lipid-free monomeric apoA-I in solution (~80% vs ~50%), we have performed molecular dynamics (MD) simulations for ~10 ns of the model in order to explore the dynamic behavior of the single apoA-I monomer at a physiological salt concentration and a temperature range of 310-410 K.
While 10 ns simulation is only a starting point, a few important observations have been made: i) the percentage of alpha helicity decreased substantially to below 70% (i.e., towards a lower experimental estimate); ii) the structure became more globular in overall appearance; iii) the flexible N-terminal domain (amino acid residues 1 to 43) has lost most of its alpha helicity; iv) the hydrophobic core of the 4-helix bundle is defined by stacking of a cluster of aromatic amino acid residues, outlined by a shell of aliphatic hydrophobic residues; v) The four helix bundle portion of the simulated structure is clustering around a pronounced stacking of aromatic residues derived from all four helixes and the aromatic cluster is overlaid by a shell of aliphatic hydrophobic residues. We conjecture that this aromatic cluster and its surrounding hydrophobic residues are the driving force for creation of a dynamic (molten globular) four helix bundle arrangement in lipid-free monomeric apoA-I in solution. This work was supported by NIH grant
Molecular dynamics simulations of normal and Milano rotamers of lipid-bound apolipoprotein A-I particles
No full textDynamics of Activation of Lecithin: Cholesterol Acyltransferase by Apolipoprotein A-I
No full textThe 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 I-CAT activation. Both the all-atom and CG simulations provide support for a model in which the helix 5/5 domains or 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
Validation of previous computer models and MD simulations of discoidal HDL by a recent crystal structure of apoA-I.
No full textHDL is a population of apoA-I-containing particles inversely correlated with heart disease. Because HDL is a soft form of matter deformable by thermal fluctuations, structure determination has been difficult. Here, we compare the recently published crystal structure of lipid-free (Δ185-243)apoA-I with apoA-I structure from models and molecular dynamics (MD) simulations of discoidal HDL. These analyses validate four of our previous structural findings for apoA-I: i) a baseline double belt diameter of 105 Å ii) central α helixes with an 11/3 pitch; iii) a "presentation tunnel" gap between pairwise helix 5 repeats hypothesized to move acyl chains and unesterified cholesterol from the lipid bilayer to the active sites of LCAT; and iv) interchain salt bridges hypothesized to stabilize the LL5/5 chain registry. These analyses are also consistent with our finding that multiple salt bridge-forming residues in the N-terminus of apoA-I render that conserved domain "sticky." Additionally, our crystal MD comparisons led to two new hypotheses: i) the interchain leucine-zippers previously reported between the pair-wise helix 5 repeats drive lipid-free apoA-I registration; ii) lipidation induces rotations of helix 5 to allow formation of interchain salt bridges, creating the LCAT presentation tunnel and "zip-locking" apoA-I into its full LL5/5 registration
Molecular Dynamics Simulated Annealing of Phospholipid- rich HDL
No full textAll-atom MD simulations by us of an atomistic double belt model for discoidal HDL produced particles where the apoA-I belt underwent conformational changes coordinated with a distortion of the bilayer disc into a minimal surface patch. Due to short simulations, these particles might represent kinetically trapped intermediates. Here we report all-atom MD simulated annealing as a more robust
approach to particle structure. Temperature jumps, in explicit water without constraints, were performed on the published 100:2 particle (molar ratio = POPC:D40apoA-I). While 525K produced rapid vaporization, three 500K simulations for 20ns produced no vaporization. Analyses of average changes in structural parameters during simulation showed: i) SASA of acyl chains rapidly increased 5-fold, then plateaued, a result of expansion of an intact bilayer. ii) Total
apoA-I helicity decreased from 95% to 72%. iii) SASA of the lipid-associating hydrophobic residues of apoA-I increased 60%. Using
stability at 500K as one measure of helix stability, the figure above shows changes in helicity of individual residues within tandem helical repeats averaged over the three simulations. We conclude
that much of apoA-I in PL-rich HDL has no “fixed” conformation, end domains being more labile than central ones
From Discoidal to Spheroidal HDL particles through Coarse Grained and All Atom Molecular Dynamics Simulations
No full textIn vivo high density lipoproteins (HDL) originate as discoidal complexes of apolipoprotein (apo) A-I, phospholipids (PL) and
cholesterol. These nascent HDL complexes are remodeled by the enzyme Lecithin Cholesterol Acyl Transferase (LCAT), that catalyses the transition from discoidal (PL-rich) to spherical HDL (the form of circulating HDL) by generating cholesteryl esters (CE) and lyso-PC. The phase separation of neutral lipids, CE and triglycerides
(TG), creates a hydrophobic core encapsulated by the protein and amphipathic lipid molecules. To investigate the conformational change of apoA-I in the transition from PL-rich to CE-rich HDL particles, we initially performed all atom (AA) and coarse grained (CG) molecular dynamics (MD) simulations at 310 K on two starting model discoidal HDL particles containing palmitoyloleoylphosphatidylcholine (POPC), cholesterol (UC) and full length apoA-I molecules with molar ratios of 160:24:2 and 160:64:2,
respectively. In the 100 ns coarse grained structures a fraction of the cholesterol molecules was mutated to cholesteryl oleate (CO)
molecules and an equivalent number of POPC molecules were
removed. The main goal was to mimic the LCAT activity in silico by simulating model CE-rich HDL particles representing small
HDL2 particles with a cholesterol concentration similar to that of circulating HDL. Then, the two mutated structures containing POPC:CO:UC:apoA-I molar ratios of 142:18:6:2 and 105:55:9:2, respectively, were subjected to a 100 ns CG MD simulation at 310K.
In both CG MD simulations CO molecules form a hydrophobic core in the 100 ns time scale, indicating that hydrophobic interactions
play an active role in the stabilization of spheroidal HDL particles. It is also interesting to note the separation of UC molecules, as
observed experimentally, into two distinct environments: free and bound to the protein.
This work was supported by the National Institute of Health
Molecular Dynamics Simulations of Protein-Encapsulated Lipid Bilayers Converge Rapidly to form Novel Minimal Surfaces
No full textAssessment of the Validity of the Double Superhelix Model for Reconstituted High Density Lipoproteins A COMBINED COMPUTATIONAL-EXPERIMENTAL APPROACH
No full textFor several decades, the standard model for high density lipoprotein (HDL) particles reconstituted from apolipoprotein A-I (apoA-I) and phospholipid (apoA-I/HDL) has been a discoidal particle similar to 100 angstrom in diameter and the thickness of a phospholipid bilayer. Recently, Wu et al. (Wu, Z., Gogonea, V., Lee, X., Wagner, M. A., Li, X. M., Huang, Y., Undurti, A., May, R. P., Haertlein, M., Moulin, M., Gutsche, I., Zaccai, G., Didonato, J. A., and Hazen, S. L. (2009) J. Biol. Chem. 284, 3660536619) used small angle neutron scattering to develop a new model they termed double superhelix (DSH) apoA-I that is dramatically different from the standard model. Their model possesses an open helical shape that wraps around a prolate ellipsoidal type I hexagonal lyotropic liquid crystalline phase. Here, we used three independent approaches, molecular dynamics, EM tomography, and fluorescence resonance energy transfer spectroscopy (FRET) to assess the validity of the DSH model. (i) By using molecular dynamics, two different approaches, all-atom simulated annealing and coarse-grained simulation, show that initial ellipsoidal DSH particles rapidly collapse to discoidal bilayer structures. These results suggest that, compatible with current knowledge of lipid phase diagrams, apoA-I cannot stabilize hexagonal I phase particles of phospholipid. (ii) By using EM, two different approaches, negative stain and cryo-EM tomography, show that reconstituted apoA-I/HDL particles are discoidal in shape. (iii) By using FRET, reconstituted apoA-I/HDL particles show a 28 -34-angstrom intermolecular separation between terminal domain residues 40 and 240, a distance that is incompatible with the dimensions of the DSH model. Therefore, we suggest that, although novel, the DSH model is energetically unfavorable and not likely to be correct. Rather, we conclude that all evidence supports the likelihood that reconstituted apoA-I/HDL particles, in general, are discoidal in shape
From Discoidal to Spheroidal HDL particles through All Atom and Coarse Grained Molecular Dynamics simulations
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