Atomistic Detailed Mechanism and Weak Cation-Conducting Activity of HIV-1 Vpu Revealed by Free Energy Calculations

<div><p>The viral protein U (Vpu) encoded by HIV-1 has been shown to assist in the detachment of virion particles from infected cells. Vpu forms cation-specific ion channels in host cells, and has been proposed as a potential drug target. An understanding of the mechanism of ion transport through Vpu is desirable, but remains limited because of the unavailability of an experimental structure of the channel. Using a structure of the pentameric form of Vpu – modeled and validated based on available experimental data – umbrella sampling molecular dynamics simulations (cumulative simulation time of more than 0.4 µs) were employed to elucidate the energetics and the molecular mechanism of ion transport in Vpu. Free energy profiles corresponding to the permeation of Na⁺ and K⁺ were found to be similar to each other indicating lack of ion selection, consistent with previous experimental studies. The Ser23 residue is shown to enhance ion transport via two mechanisms: creating a weak binding site, and increasing the effective hydrophilic length of the channel, both of which have previously been hypothesized in experiments. A two-dimensional free energy landscape has been computed to model multiple ion permeation, based on which a mechanism for ion conduction is proposed. It is shown that only one ion can pass through the channel at a time. This, along with a stretch of hydrophobic residues in the transmembrane domain of Vpu, explains the slow kinetics of ion conduction. The results are consistent with previous conductance studies that showed Vpu to be a weakly conducting ion channel.</p></div

Conformations with the permeating ion inside the channel.

<p>Snapshots of the Na⁺ ion near the ring of (A) Ser23 residues and (B) Val12 residues, respectively.</p

The structure of Vpu.

<p>(A) Schematic illustration of the monomer. (B). The pentameric channel set up in a hydrated lipid bilayer. The monomeric unit on the front has been omitted to reveal the interior of the pore. Pore water molecules can be seen in the pore lumen.</p

The average ion-protein interaction energy and average solvation energy for different positions of the permeating ion.

<p>(A) Na⁺; (B) K⁺. Values for each window have been averaged over the trajectory for the respective window, and are shown with error bars. The pore-lining residues are also shown at their respective position in the plot.</p

The PMF shown as a function of the positions of the two permeating ions.

<p>The x-axis labels at the bottom show the position of the top ion along the channel axis, while the labels at the top show the pore-lining residues at their respective positions along the channel axis. The pathway with the lowest free energy barrier is highlighted in maroon color. Images of ion positions corresponding to the pathway with the lowest free energy barrier are shown below the plot, together with the distance between the two ions at these positions.</p

The potential of mean force (PMF) for the transport of permeating ions along the channel axis.

<p>(A) Na⁺; (B) K⁺. Residues facing the pore have been shown at their appropriate positions. The coordinates of these residues were calculated by determining the center of mass of their side chains. Error bars are shown as bands. The soft minimum around Ser23 is marked with an arrow.</p

The PMF for ion transport for different sampling time periods.

<p>(A) Na⁺ (B) K⁺.</p

Electrostatic profile of pore-lining residues.

<p>(A) The pentamer model used in the study shown with the Ser23 residue in van der Waals representation. (B) A representation of the electrostatic surface of pore-lining residues in the channel. The monomer unit on the front has been omitted for clarity. Red indicates negatively charged regions, blue indicates positively charged regions, and white indicates nonpolar regions. Only the sidechain atoms are shown in color, and the backbone atoms are in white to allow a clear illustration of the nature of pore-lining residues.</p

Average hydration number for permeating ions along the channel axis.

<p>(A) Na⁺; (B) K⁺. The hydration number was calculated by determining the number of water molecules within a certain cutoff distance of the permeating ion. The cutoff used was 2.8 Å for Na⁺ and 3.2 Å for K⁺. The hydration number values shown here have been averaged over the trajectory for the respective window. Error bars are shown in pink.</p