Molecular dynamics simulation of human LOX-1 provides an explanation for the lack of OxLDL binding to the Trp150Ala mutant
© Falconi et al; licensee BioMed Central Ltd. 2007
Received: 21 May 2007
Accepted: 07 November 2007
Published: 07 November 2007
Dimeric lectin-like oxidized low-density lipoprotein receptor-1 LOX-1 is the target receptor for oxidized low density lipoprotein in endothelial cells. In vivo assays revealed that in LOX-1 the basic spine arginine residues are important for binding, which is lost upon mutation of Trp150 with alanine. Molecular dynamics simulations of the wild-type LOX-1 and of the Trp150Ala mutant C-type lectin-like domains, have been carried out to gain insight into the severe inactivating effect.
The mutation does not alter the dimer stability, but a different dynamical behaviour differentiates the two proteins. As described by the residues fluctuation, the dynamic cross correlation map and the principal component analysis in the wild-type the two monomers display a symmetrical motion that is not observed in the mutant.
The symmetrical motion of monomers is completely damped by the structural rearrangement caused by the Trp150Ala mutation. An improper dynamical coupling of the monomers and different fluctuations of the basic spine residues are observed, with a consequent altered binding affinity.
Low density lipoprotein (LDL) is oxidized in vascular endothelial cells to OxLDL, a highly detrimental product that results in endothelial cell injury and is implicated in the development of atherosclerosis. Vascular endothelial cells also internalize and degrade external OxLDL though the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) [1–3]. OxLDL causes vascular endothelial cell activation and dysfunction, resulting in pro-inflammatory responses, pro-oxidative conditions, and apoptosis, all of which are pro-atherogenic. LOX-1 has been characterized as the primary receptor for OxLDL on the surface of vascular endothelial cells and is up-regulated in atherosclerotic lesions [2, 3]. Upon recognition of OxLDL, LOX-1 is observed to initiate OxLDL internalization and degradation as well as the induction of a variety of pro-atherogenic cellular responses, including reduction of nitric oxide (NO) release , secretion of monocyte chemoattractant protein-1 (MCP-1) , production of reactive oxygen species , expression of matrix metalloproteinase-1 and -3 , monocyte adhesion , and apoptosis .
LOX-1 is a member of the scavenger receptor family, a structurally diverse group of cell surface receptors of the innate immune system that recognize modified lipoproteins. It is a disulfide-linked homodimeric type II transmembrane protein with a short 34-residue cytoplasmic region, a single transmembrane region, and an extracellular region consisting of an 80-residue domain, predicted to be a coiled coil called "neck domain", followed by a 130-residue C-terminal C-type lectin-like domain (CTLD) [2, 9].
Several positively charged CTLD LOX-1 residues are known to play a role in the recognition of OxLDL [13–15], and a detailed understanding of this interaction could be of significant medical interest because specific antagonists potentially could mitigate the progression of atherosclerosis. In vivo functional assays with LOX-1 mutants revealed that linearly aligned basic residues at the dimer surface, that has been referred as the basic spine (i.e. arginines 208, 229, 231 and 248), are responsible for ligand binding . In fact single elimination of each arginine reduces the binding activity. This effect is even more evident upon mutation of Trp150, a residue located at the dimer interface, into alanine, suggesting that an altered inter-subunit interaction strongly affect the OxLDL binding region . OxLDL has been suggested to have amphipathic α-helices on its surface , and the basic spine structure of LOX-1 has been proposed to provide an appropriate platform for the interaction with these α-helices .
In this work we have investigated the not naturally occurring LOX-1 Trp150Ala mutation through molecular dynamics (MD) simulation to study its structural and dynamical properties in comparison to the wild-type protein . Our results show that both the native and mutated proteins have a stable dimeric structure, but they display different overall motion. In the native protein a collective motion generates a symmetrical rotation of each monomer one against the other, while in the mutant this coordinated inter-subunit movement is absent. As a consequence an altered dynamical coupling of the monomers and different fluctuations of the basic spine residues are observed, providing an explanation for the drastic reduction of the OxLDL binding affinity of the mutant protein.
Results and discussion
Root Mean Square Deviations and Fluctuations
RMSF values of the basic spine residues calculated from the simulation of the wild-type and mutant proteins compared to the RMSF values converted from the experimental B-factors.
Wild-type RMSF (Å)
Trp150Ala RMSF (Å)
Converted X-ray B-factors (Å)
For the native protein the residue RMSF values reproduce well the crystallographic B-factors  (Fig. 3A). This is strictly true for the helices and the β-strands, while the loops between regular secondary structures segments have fluctuations larger than the corresponding converted B-factors, likely due to the higher degree of hydration of the simulations when compared to the crystal . The B-factor values of basic spine arginines, extracted from the PDB file 1YPQ and converted to RMSF values for comparison (see Methods), are very close to the residue RMSF values detected in the wild-type simulation (see Table 1).
Secondary structures and cavities
Two large cavities are present in the LOX-1 CTLD. The first cavity is represented by the "hydrophobic tunnel", which is a 20 Å, mostly non-polar, tunnel localized at the center of the dimer interface . This tunnel is 7–8 Å in diameter except for a constriction that narrows the middle of the tunnel to a diameter of 4 Å . The second cavity, located below the first one and above the inter-chain Cys140.A-Cys140.B disulfide bridge, is smaller and shaped by hydrophobic residues including Pro143, Cys144, Pro145, Trp148, Ile149 and Trp150 . In the mutant protein the amino acid substitution Trp150Ala generates a volume increase of the second cavity (not shown). The volume of the two cavities, monitored along the trajectories of the two proteins by using the program Surfnet , is preserved in both simulations.
Hydrogen bond analysis
The LOX-1 dimer structure shows that Trp150 contributes not only to dimer formation but also to the maintenance of the proper CTLD fold through inter and intra-chain hydrogen bonds . In the wild-type simulation, the maintenance of the short β 0-β 1 antiparallel β-ribbon is ensured by hydrogen bond network between Trp150.Nε 1-Gly152.O, Asp147.N-Trp150.O and His151.N-Asn154.O.
In the mutant protein the introduction of an alanine in position 150 disrupts the hydrogen bond between the indole group and Gly152 in both subunits and prevents, in the B subunit, the hydrogen bond between His151 and Asn154, thereby generating the asymmetric unfolding of the β 0 segment (see Fig. 4). However, new inter-subunit hydrogen bonds arise between Gln146.Nε 2-Ala150.O and Ala150.N-Trp148.O enforcing the dimeric interactions.
Cross-correlations and principal component analysis
The principal component analysis (PCA), or essential dynamics [20, 21], has been also applied to highlight the correlation differences between the native and mutated protein. This analysis is based on the diagonalisation of the covariance matrix built from the atomic fluctuations after the removal of the translational and rotational movement, and permits the identification of the main 3N directions along which the majority of the protein motion is defined. The analysis, carried out on the 268 Cα atoms of the two proteins, indicates that although the motion is dispersed over 804 eigenvectors, about 80% of the motion depends on the first 30 eigenvectors having the largest eigenvalues (see additional file 2) as generally found in many different systems [22, 23].
In mutant LOX-1 (Fig. 6B) the coupling of the inter-subunit motion is cancelled by the mutation that generates a rigid subunit interface and strongly restrains the synchronized motion observed in the wild-type protein. The unique regions having a relative high mobility are now represented by the loops L1, L2 and L3 and the amino and carboxy terminal tails that release the motion gathered by the dimeric structure in the absence of a bendable interface hinge (see also additional file 4). The different motion induces a different behaviour of the residues belonging to the four basic spine arginines of the two subunits, represented by van der Waals spheres in Fig. 6. The arginines, in fact, move in an opposite direction in the two proteins, as indicated by the reverse position of their blue and red colours.
Our analyses indicate that the mutant displays a different dynamical coupling of the monomers, when compared to the native protein, and a different fluctuations of the basic spine arginines, two factors that may prevent the molecular recognition of OxLDL.
The results obtained from molecular dynamics simulations indicate that both the native and the Trp150Ala mutated protein display a stable dimeric structure that is fully maintained over the entire simulation time. In fact, elimination of the Trp150, located at the inter-subunit interface, mainly induces a dynamical perturbation and only in part a structural rearrangement.
The first important dynamical effect is the occurrence of a different flexibility of two of the four arginine residues (Arg229 and Arg248), which belong to the basic spine (see Fig. 3 and Table 1). These display high flexibility only in one of the two subunits of the Trp150Ala mutant. This asymmetric dynamical behaviour is coupled to the asymmetric destructuration of the β 0 strand that occurs only in a single subunit of the mutant (see Fig. 4). This is due to the alteration of the hydrogen bond network that, instead, is fully maintained in the native protein. The loss of this short β-strand, located at the dimer interface, damps the dimer symmetric motion present in the wild-type as detected through the PCA analysis (Fig. 6A and 6B and additional files 3 and 4). The two monomers in the wild-type undergo a symmetric rotation that pushes the monomers one against the other, using the inter-subunit surface as a flexible hinge (Fig. 6A and additional file 3). On the other hand, in the mutant the inter-subunit surface becomes rigid and the two monomers do not move anymore in a symmetric way (Fig. 6B and additional file 4). This alteration of basic spine dynamical properties disengages the molecular recognition, indicating that the OxLDL needs a regular motion of the monomers for its efficient binding on the receptor surface.
Because the LOX-1 receptor plays a crucial role in atherosclerosis plaque formation, unravelling the molecular mechanism of OxLDL-LOX-1 interaction is of clinical interest. To understand the dynamical aspects of the recognition site could very well be the first step towards the development and therapeutical application of OxLDL antagonists.
Size, box dimensions and number of damped configurations of the two simulated systems.
Box side X (Å)
Box side Y (Å)
Box side Z (Å)
Thermalization scheme of the two simulated systems.
Steps number and Δt
Position restraint (kcal/mol·Å)
25000 – 0.5 fs
25000 – 1.0 fs
10000 – 2.0 fs
20000 – 2.0 fs
50000 – 2.0 fs
where Δri is the displacement from the mean position of the ith atom and the symbol ⟨⟩ represent the time average over the whole trajectory.
This work was in part supported by Italian Ministry of University and Research (MUR) and through R.E.D.D. s.r.l., a spin-off of the Tor Vergata University of Rome.
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