Computational Studies of the Cholesterol Transport between NPC2 and the N‑Terminal Domain of NPC1 (NPC1(NTD))

The transport of cholesterol from NPC2 to NPC1 is essential for the maintenance of cholesterol homeostasis in late endosomes. On the basis of a rigid docking model of the crystal structures of the N-terminal cholesterol binding domain of NPC1­(NTD) and the soluble NPC2 protein, models of the NPC1­(NTD)-NPC2-cholesterol complexes at the beginning and the end of the transport as well as the unligated NPC1­(NTD)-NPC2 complex were studied using 86 ns MD simulations. Significant differences in the cholesterol binding mode and the overall structure of the two proteins compared to the crystal structures of the cholesterol binding separate units were obtained. Relevant residues for the binding are identified using MM/GBSA calculations and the influence of the mutations analyzed by modeling them <i>in silico</i>, rationalizing the results of previous mutagenesis experiments. From the calculated energies and the NEB (nudged elastic band) evaluation of the cholesterol transfer mechanism, an atomistic model is proposed of the transfer of cholesterol from NPC2 to NPC1­(NTD) through the formation of an intermediate NPC1­(NTD)-NPC2 complex

Computational Studies of the Cholesterol Transport between NPC2 and the N‑Terminal Domain of NPC1 (NPC1(NTD))

The transport of cholesterol from NPC2 to NPC1 is essential for the maintenance of cholesterol homeostasis in late endosomes. On the basis of a rigid docking model of the crystal structures of the N-terminal cholesterol binding domain of NPC1­(NTD) and the soluble NPC2 protein, models of the NPC1­(NTD)-NPC2-cholesterol complexes at the beginning and the end of the transport as well as the unligated NPC1­(NTD)-NPC2 complex were studied using 86 ns MD simulations. Significant differences in the cholesterol binding mode and the overall structure of the two proteins compared to the crystal structures of the cholesterol binding separate units were obtained. Relevant residues for the binding are identified using MM/GBSA calculations and the influence of the mutations analyzed by modeling them <i>in silico</i>, rationalizing the results of previous mutagenesis experiments. From the calculated energies and the NEB (nudged elastic band) evaluation of the cholesterol transfer mechanism, an atomistic model is proposed of the transfer of cholesterol from NPC2 to NPC1­(NTD) through the formation of an intermediate NPC1­(NTD)-NPC2 complex

Computational Studies of the Cholesterol Transport between NPC2 and the N‑Terminal Domain of NPC1 (NPC1(NTD))

The transport of cholesterol from NPC2 to NPC1 is essential for the maintenance of cholesterol homeostasis in late endosomes. On the basis of a rigid docking model of the crystal structures of the N-terminal cholesterol binding domain of NPC1­(NTD) and the soluble NPC2 protein, models of the NPC1­(NTD)-NPC2-cholesterol complexes at the beginning and the end of the transport as well as the unligated NPC1­(NTD)-NPC2 complex were studied using 86 ns MD simulations. Significant differences in the cholesterol binding mode and the overall structure of the two proteins compared to the crystal structures of the cholesterol binding separate units were obtained. Relevant residues for the binding are identified using MM/GBSA calculations and the influence of the mutations analyzed by modeling them <i>in silico</i>, rationalizing the results of previous mutagenesis experiments. From the calculated energies and the NEB (nudged elastic band) evaluation of the cholesterol transfer mechanism, an atomistic model is proposed of the transfer of cholesterol from NPC2 to NPC1­(NTD) through the formation of an intermediate NPC1­(NTD)-NPC2 complex

Computational Studies of the Cholesterol Transport between NPC2 and the N‑Terminal Domain of NPC1 (NPC1(NTD))

The transport of cholesterol from NPC2 to NPC1 is essential for the maintenance of cholesterol homeostasis in late endosomes. On the basis of a rigid docking model of the crystal structures of the N-terminal cholesterol binding domain of NPC1­(NTD) and the soluble NPC2 protein, models of the NPC1­(NTD)-NPC2-cholesterol complexes at the beginning and the end of the transport as well as the unligated NPC1­(NTD)-NPC2 complex were studied using 86 ns MD simulations. Significant differences in the cholesterol binding mode and the overall structure of the two proteins compared to the crystal structures of the cholesterol binding separate units were obtained. Relevant residues for the binding are identified using MM/GBSA calculations and the influence of the mutations analyzed by modeling them <i>in silico</i>, rationalizing the results of previous mutagenesis experiments. From the calculated energies and the NEB (nudged elastic band) evaluation of the cholesterol transfer mechanism, an atomistic model is proposed of the transfer of cholesterol from NPC2 to NPC1­(NTD) through the formation of an intermediate NPC1­(NTD)-NPC2 complex

Computational Studies of the Cholesterol Transport between NPC2 and the N‑Terminal Domain of NPC1 (NPC1(NTD))

The transport of cholesterol from NPC2 to NPC1 is essential for the maintenance of cholesterol homeostasis in late endosomes. On the basis of a rigid docking model of the crystal structures of the N-terminal cholesterol binding domain of NPC1­(NTD) and the soluble NPC2 protein, models of the NPC1­(NTD)-NPC2-cholesterol complexes at the beginning and the end of the transport as well as the unligated NPC1­(NTD)-NPC2 complex were studied using 86 ns MD simulations. Significant differences in the cholesterol binding mode and the overall structure of the two proteins compared to the crystal structures of the cholesterol binding separate units were obtained. Relevant residues for the binding are identified using MM/GBSA calculations and the influence of the mutations analyzed by modeling them <i>in silico</i>, rationalizing the results of previous mutagenesis experiments. From the calculated energies and the NEB (nudged elastic band) evaluation of the cholesterol transfer mechanism, an atomistic model is proposed of the transfer of cholesterol from NPC2 to NPC1­(NTD) through the formation of an intermediate NPC1­(NTD)-NPC2 complex

Computational Studies of the Cholesterol Transport between NPC2 and the N‑Terminal Domain of NPC1 (NPC1(NTD))

The transport of cholesterol from NPC2 to NPC1 is essential for the maintenance of cholesterol homeostasis in late endosomes. On the basis of a rigid docking model of the crystal structures of the N-terminal cholesterol binding domain of NPC1­(NTD) and the soluble NPC2 protein, models of the NPC1­(NTD)-NPC2-cholesterol complexes at the beginning and the end of the transport as well as the unligated NPC1­(NTD)-NPC2 complex were studied using 86 ns MD simulations. Significant differences in the cholesterol binding mode and the overall structure of the two proteins compared to the crystal structures of the cholesterol binding separate units were obtained. Relevant residues for the binding are identified using MM/GBSA calculations and the influence of the mutations analyzed by modeling them <i>in silico</i>, rationalizing the results of previous mutagenesis experiments. From the calculated energies and the NEB (nudged elastic band) evaluation of the cholesterol transfer mechanism, an atomistic model is proposed of the transfer of cholesterol from NPC2 to NPC1­(NTD) through the formation of an intermediate NPC1­(NTD)-NPC2 complex

Protocol for ANM-restrained-MD simulations and β₂AR conformations.

<p><b>A.</b> Structure of β₂AR. Transmembrane helices 1–7 are labeled by numbers and colored in red, orange, yellow, green, blue, purple, and pink, respectively. Cytoplasmic helix 8 and the short extracellular helix below the binding cavity in extracellular loop 2 are colored in cyan. Ligand- and G-protein binding sites are shown by arrows. The palmitolyl group that is anchored to the membrane from the H8 is also shown in cyan. <b>B.</b> The protocol for generating the ensemble of conformations by ANM-restrained-MD algorithm. <b>C.</b> Ribbon diagrams of β₂AR conformations. Front view (top) and back view (bottom) of β2AR conformers generated by ANM-restrained-MD are shown.</p

Binding of salmeterol to β₂AR.

<p><b>A.</b> Interactions of salmeterol at the binding site of an ANM-restrained-MD conformation; <b>B.</b> The location of salmeterol tail forming a ‘lid’ with the extracellular loop 2 (EC2) of β₂AR from the extracellular site view. The flexible tail of salmeterol folds parallel to the EC2 and stabilizes the rest of the ligand forming a lid at the extracellular site. The green arrow shows the direction that the salmeterol tail runs similar to a beta-sheet structure. <b>C.</b> The residues lining the salmeterol binding pocket; <b>D.</b> The stabilization of the aromatic ring of salmeterol by Val114, Thr118 at H3, Ser203 and Ser207 at H5, and Phe290 atH6. Carbon, oxygen, nitrogen and hydrogen atoms of salmeterol is colored green, red, blue and white; respectively. ANM-restrained-MD conformation that is able to accommodate inside the ligand binding pocket is shown in ribbon diagram with transparent helices. The residues with the majority of atoms within the 3.5 Å making specific interactions with salmeterol are displayed. The EC2 that closely interacts with salmeterol is also show in green ribbon diagram.</p

Microdomains of β₂AR stabilized by new interactions and water molecules.

<p><b>A</b>. Water molecules stabilizing the conserved NPXXY motif (left) and the Asn-Asp pair (right) within the transmembrane region, and (<b>B</b>) the critical catecholamine binding Ser203 and Ser207 residues at H5 in conformations where they both point to the ligand binding pocket and connected through a water molecule. <b>C</b>. The motion of extracellular loop two (EC2) and the residues that form the salt bridge at the EC site. The motion found by ANM-restrained-MD to break the salt bridge at the extracellular site and the opening of the extracellular site is shown. ANM-restrained-MD conformation and the carazolol-bound structures are in solid and transparent colors, respectively. The side chains of Asp192 at EC2 and Lys305 at H7 that form the salt bridge at the inactive state of β₂AR are displayed on both structures. The motion of the EC2 including the short helix is depicted by red arrows.</p

β₂AR residues that are shown to be interacting with agonists.

<p>β₂AR residues that are shown to be interacting with agonists.</p