Molecular mechanism of statin-mediated LOX-1 inhibition

Abstract

Statins are largely used in clinics in the treatment of patients with cardiovascular diseases for their effect on lowering circulating cholesterol. Lectin-like oxidized low-density lipoprotein (LOX-1), the primary receptor for ox-LDL, plays a central role in the pathogenesis of atherosclerosis and cardiovascular disorders. We have recently shown that chronic exposure of cells to lovastatin disrupts LOX-1 receptor cluster distribution in plasma membranes, leading to a marked loss of LOX-1 function. Here we investigated the molecular mechanism of statin-mediated LOX-1 inhibition and we demonstrate that all tested statins are able to displace the binding of fluorescent ox-LDL to LOX-1 by a direct interaction with LOX-1 receptors in a cell-based binding assay. Molecular docking simulations confirm the interaction and indicate that statins completely fill the hydrophobic tunnel that crosses the C-type lectin-like (CTLD) recognition domain of LOX-1. Classical molecular dynamics simulation technique applied to the LOX-1 CTLD, considered in the entire receptor structure with or without a statin ligand inside the tunnel, indicates that the presence of a ligand largely increases the dimer stability. Electrophoretic separation and western blot confirm that different statins binding stabilize the dimer assembly of LOX-1 receptors in vivo. The simulative and experimental results allow us to propose a CTLD clamp motion, which enables the receptor-substrate coupling. These findings reveal a novel and significant functional effect of statins.

Cholesterol level regulates lectin-like oxidized low-density lipoprotein receptor-1 function

“…Single aminoacid mutations on Ile149, which points to the empty space in the center of the tunnel, leads to a marked inhibition of the binding of ox-LDL and a series of oxidized phospholipids [19,21]. Similarly to these compounds, our experimental and simulative investigation confirms that statins completely fill the hydrophobic tunnel using a common mechanism and that residue Ile149 is always involved in stabilizing contacts with these drugs [7].…”

“…a direct interaction with LOX-1. All tested statins (lovastatin, atorvastatin, fluvastatin and pravastatin) are able to displace the binding of fluorescent ox-LDL to LOX-1 inhibiting LOX-1 activity [7]. In detail, short exposure with different statins at 4°C and for a time length not sufficient to induce any cholesterol synthesis reduction (60 min), results in a very marked decrease of ox-LDL bound to LOX-1 (Fig.…”

“…Molecular docking simulations indicate that statins completely fill the hydrophobic tunnel that crosses the C-type lectin-like (CTLD) recognition domain of LOX-1, blocking the binding to natural substrates, including ox-LDL [7]. As mentioned above the hydrophobic tunnel plays a crucial role in the recognition of ox-LDL and other ligands.…”

“…(ii) by extracting bound DiI-ox-LDL from positive stained cells with isopropanol and spectrofluorometric analysis [7]. The assay allows quantifying the inhibitory effect, being 100% of binding the amount of fluorescence in not-treated cells.…”

Multiple molecular dynamics simulations of human LOX-1 and Trp150Ala mutant reveal the structural determinants causing the full deactivation of the receptor

“…Three 200 ns MD simulations of the mutant Trp150Ala receptor including the CTLD, the NECK and the transmembrane domain (Table ), arranged as above described for the wild‐type receptor system. We have chosen to include the NECK domain in both the MD systems since previous simulations carried out in presence and absence of this domain and experimental data indicate that the NECK influences the CTLD behavior and function and is considered essential for the ligand binding activity of this receptor.…”