Dynamics of activation of lecithin:cholesterol acyltransferase by apolipoprotein A-I

Abstract

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 all-atom 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.

FIGURE 1. Analysis of changes in particles during the 30 ns of the MDSA protocol

A. Plot of MDSA protocol (temperature in K versus time in ns). RMSD calculated for protein α-carbon atoms as a function of time of MDSA for: B. MDSA-1. C. MDSA-2. D. MDSA-3. E. MDSA-4.

FIGURE 2. Structural features of the amphipathic presentation tunnel in the four MDSA particles

A. Full spacefilling models of final particle structures viewed from the solvent side of the helix 5/5 domain. Protein: Pro (yellow), helix 5 (residues 122–142, green), helix 1 (residues 44–65, blue), helix 8 (residues 187–208, cyan), helix 10 (residues 221–241, red), and the remainder of the protein (light blue). POPC: Acyl chains (black), phosphate groups P (gold) and O (red), and choline groups (skyblue). UC: CPK colors. Models of the helix 5/5 domain of the final structures showing POPC molecules whose terminal methyls are inserted in the amphipathic presentation tunnel and the UC nearest the amphipathic presentation tunnel shown from: B. Solvent view, C. Lipid view. Protein: Spacefilling, K133 and K140 (blue), E125 and E136 (red), G129 (magenta), L130 (white), Leu (gold), Pro (yellow) and the remainder of helix 5 (green). POPC: Stick representation, acyl chains (black), phosphate groups P (gold) and O (red), choline groups (skyblue) and terminal methyls (cyan spheres). UC: Spacefilling, CPK colors.

FIGURE 3. Dynamics of the helix 5/5 gap in the four MDSA particles

A–D. Area of the gap measured by SASA of all POPC or UC atoms within 7 Å of G129 plotted as a function of time for: (A) MDSA-1, (B) MDSA-2, (C) MDSA-3 and (D) MDSA-4.

FIGURE 4. Analyses of the MDSA protocol of the dynamics of eight POPC molecules that insert into the amphipathic presentation tunnel at least 5% of the time

A. MDSA-1: POPC-1 (thick black line), POPC-2 (thick gray line). B. MDSA-2: POPC-1 (thick black line), POPC-2 (thick gray line), POPC-3 (thin black line. C. MDSA-4. The Y-axis plots the distance of each terminal methyl from any G129.

FIGURE 5. Four views of one snapshot of the CGMD simulation of CGMD-1 illustrating insertion of a terminal methyl pseudoatom into the amphipathic presentation tunnel

All lipid molecules are shown in spacefilling mode. Protein: Helix 5 (green), Pro (yellow), helix 1 (blue), helix 10 (red). POPC: Headgroups (cyan), terminal methyl pseudoatoms (gold), other acyl pseudoatoms (white); UC: Nonpolar portion (brown), hydroxyl (cream). A. Full particle with α carbons of protein shown in tube mode except solvent inaccessible salt bridges plus K133 and G129 that are shown in spacefilling mode. B. Particle as shown in A rotated approximately 60° clockwise along the vertical axis. C. Full particle as shown in A with protein in spacefilling mode. D. Cross-eyed stereo view of the center of the helix 5 domain of particle representation as shown in C.

FIGURE 6. Cumulative plots of the degree of terminal methyl insertion into the amphipathic presentation tunnels for all-atom versus CGMD simulations

The distance of every terminal methyl group from the nearest G129 α carbon was counted over every frame of the selected portion of each MD simulation. A. Last 10 ns of all-atom MDSA-1, MDSA-2 and MDSA-4 simulations. B. Full 20 µsec of CGMD simulations CGMD-1, CGMD-2 and CGMD-3. The resulting tables were used to create cumulative plots of the fraction of trajectory frames that had ≥ 1, ≥ 2, ≥ 3, ≥ 4, or ≥ 5 terminal methyls (Me) within a given distance (Å) of the nearest G129 residue.

FIGURE 7. Four views of one snapshot of the CGMD simulation of CGMD-2 illustrating insertion of hydroxyl pseudoatoms of UC into the amphipathic presentation tunnel and between the solvent inaccessible salt bridges of the helix7/3 domain

All lipid molecules are shown in spacefilling mode. Protein: Helix (green), Pro (yellow), helix 1 (blue), helix 1 ( red); POPC: Headgroups (cyan), terminal methyl pseudoatoms (gold), other acyl pseudoatoms (white); UC: Nonpolar portion (brown), hydroxyl (cream), UC_c (central UC in helix 5/5 domain), UC_L (left hand UC in helix 3/7 domain), UC_R (right hand UC in helix 7/3 domain). A. Full particle with α carbons of protein shown in tube mode except solvent inaccessible salt bridges plus K133 and G129 that are shown in spacefilling mode. B. Particle as shown in A rotated approximately 80 ° counterclockwise along vertical axis. C. Full particle as shown in A with protein in spacefilling mode. D. Cross-eyed stereo view of the center of the helix 5 domain of particle representation as shown in C.

FIGURE 8. Cumulative plots of the degree of UC hydroxyl insertion into the amphipathic presentation tunnel over the full 20 µsec of CGMD simulation of CGMD-1, CGMD-2 and CGMD-3

The distance of every UC hydroxyl group pseudoatom from the nearest charged pseudoatom for K133, R177 and H155 was counted over every frame of each MD simulation. The resulting tables were used to create cumulative plots of the fraction of trajectory frames that had ≥ 1, ≥ 2, or ≥ 3 UC hydroxyl (UC-OH) pseudoatoms within a given distance (Å) of the nearest charged pseudoatom for: A. K133, B. R177 and C. H155.

FIGURE 9. Analysis of interactions of POPC and UC with the apoA-I double belt

ADF plots of: A. Center of mass (COM) of POPC from protein; B. Center of mass (COM) of UC from protein; C. P1 of POPC from protein. The number of UC hydroxyl oxygens ≤ 10 Å from each residue were counted over the last 20% of the MDSA frames and the average number was plotted for: D. MDSA-1, MDSA-2 and MDSA-4. E. MDSA-3. The lower arrowheads show the location of the six solvent inaccessible salt bridges (acidic, gray; basic, black) and K133 (black. Helical repeats (indicated at top) are denoted with alternating white and gray rectangles. The upper open arrowheads indicate location of UC clusters associated with the single helix 5/5 and duplicate helix 7/3 domains.

FIGURE 10. Structural representations of the helix 5/5 domain of the four MDSA simulations of the 57:16:6:2 CE-rich spheroidal HDL particles

All lipid molecules are represented in spacefilling mode. The protein is represented in ribbons mode. Protein: Helix 5 (green), Pro (yellow spacefilling), remainder of protein (light blue); POPC: Acyl chains (black), phosphate groups P (gold) and O (red), and choline groups (skyblue); UC (CPK colors); CE (magenta). A. CGMD-1. B. CGMD-2. C. CGMD-3. D. CGMD-4.

FIGURE 11. Cross-eyed stereo images of interactions of terminal methyl, UC hydroxyl and CE pseudoatoms with the amphipathic presentation tunnel during CGMD simulations of 57:16:6:2 CE-rich spheroidal HDL particles

All lipid molecules are represented in spacefilling mode while α carbons of the protein are in tube mode except α carbons of certain residues denoted below that are in spacefilling mode. Protein: Helix 5 (green), Pro (yellow spacefilling), helix 1 (blue), helix 10 (red); residues 1–43 (magenta), remainder of protein (gray), K133 (blue spacefilling), G129 (magenta spacefilling), E125 and E136 (red spacefilling); POPC (white); UC: nonpolar portion (brown), hydroxyl (cream); CE (blue-green). A. Insertion of terminal methyl pseudoatom. B. Insertion of UC-OH. C. Blockage by CE.

FIGURE 12. Cumulative plot of the degree of blockage of the amphipathic presentation tunnel by CE over the full 20 µsec of CGMD simulations of 57:16:6:2 CE-rich spheroidal HDL particles

The distance of every CE pseudoatom from the nearest G129 pseudoatom was counted over every frame of every simulation. The resulting table was used to create a cumulative plot of the fraction of trajectory frames that had at least one pseudoatom of ≥ 1, ≥ 2, or ≥ 3 CE within a given distance (Å) of the nearest G129 pseudoatom.