Cooperation of Hydrophobic Gating, Knock-on Effect, and Ion Binding

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Cooperation of Hydrophobic Gating, Knock-On Effect, and Ion Binding Determines Ion Selectivity in the p7 Channel Siladitya Padhi, and U. Deva Priyakumar J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b00684 • Publication Date (Web): 25 Apr 2016 Downloaded from http://pubs.acs.org on April 27, 2016

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The Journal of Physical Chemistry

Cooperation of Hydrophobic Gating, Knock-on Effect, and Ion Binding Determines Ion Selectivity in the p7 Channel

Siladitya Padhi and U. Deva Priyakumar*

Centre for Computational Natural Sciences and Bioinformatics, International Institute of Information Technology, Hyderabad 500032, India

*

Corresponding author

Email: [email protected] Telephone: +91-40-66531161 Fax: +91-40-66531413

ACS Paragon Plus Environment

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Abstract Ion channels selectively allow certain ions to pass through at much higher rates than others, and thereby modulate ionic concentrations across cell membranes. The current molecular dynamics study elucidates the intricate mechanisms that render ion selectivity to the viral channel p7 by employing free energy calculations. Free energy barriers of 5.4 kcal mol-1 and 19.4 kcal mol-1 for K+ and Ca2+, respectively, explain the selectivity of the channel reported in experiments. Initially, the permeating ions encounter a hydrophobic barrier followed by stabilization in an ionbinding site. Electrostatic repulsion between the permeating ions propels one of the ions out of the binding site to complete the process of permeation. K+ and Ca2+ are seen to exhibit different modes of binding toward a ring of asparagine residues, which serves as the binding site. The findings illustrate how the overall selectivity of a channel can be achieved by a combination of subtle differences.


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Introduction Ion channels occur in membranes of all cells, and they possess a hydrated pore that allows the rapid permeation of ions.1-4 These channels show selectivity toward some ions over others, exhibiting unique ways of differentiating ions.1 The residues that line the pore dictate the selectivity of the channel toward certain ions, which implies that channels possess a set of unique residues in their pore lumen, depending on what ion they conduct. One interesting characteristic of ion channels is that they remain rigid during the process of ion transport, and do not undergo large scale conformational changes during the process.1-4 Ion channels play a critical role in regulating the concentration of ions in cells, and, consequently, an understanding of ion transport and selectivity mechanisms is necessary for modulating ion concentrations in diseased states, and in drug design.

The present study focuses on the ion selectivity mechanism of the p7 channel from hepatitis C virus (HCV).5 The channel, found in HCV-infected host cells, is cation-selective,6-8 and is more permeable to K+ compared to Ca2+ ions.8 The activity of the channel is important for the infectivity of the virus, and it plays a role in a number of phases in the virus life cycle.9-12 While some reports have suggested the role of p7 in an early phase in the virus life cycle, others have suggested that the protein is important for the later stages of the life cycle.5,10,11 The interaction of p7 with other viral factors, in particular the NS2 protein, enhances the infectivity of the virus.10,11 The role of the channel in virus propagation has prompted a number of drug design studies targeting the channel.6-8 Among inhibitors identified for the channel are hexamethylene amiloride, amantadine, and long alkyl-chain iminosugar derivatives.6-8


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NMR studies on the monomeric form have shown that the protein is predominantly α-helical, with two transmembrane segments being connected by a loop.13-16 Due to the difficulty in obtaining transmembrane protein structure, several computational methodologies have been proposed.17-27 There have been a number of attempts to computationally model the oligomeric structure of the p7 protein.28-31 The first complete oligomeric structure was reported in an electron microscopy study, which revealed a flower-shaped structure of the channel.32 Such a shape of the channel was later confirmed in an NMR study, which showed a hexamer with a funnel-shaped pore.33,34 Each monomeric unit possesses three α-helices, and the different monomeric units are intertwined with one another. Figure 1A shows the protein embedded in a solvated lipid bilayer environment, and Figure 1B shows the residues lining the pore. The entrance of the pore has a ring of arginine residues, and is followed by a hydrophobic stretch made of W21, L24, and L28. There is a hydrophilic stretch further inside the pore, and a ring of isoleucine residues at the exit of the pore (Figure 1B).

Umbrella sampling free energy calculations are invaluable for obtaining atomistic detailed insights into mechanisms that render selectivity to ion channels.35-42 Typically, it is seen that the conduction of certain specific ions is carried out swiftly in a concerted fashion, while prohibiting the permeation of other ions. Roux and coworkers were the first to study multi-ion permeation using umbrella sampling simulations, and they showed that ion conduction through the K+selective KcsA channel proceeds via an ion-ion knock-on.35 A very recent study showed that knock-on in K+ channels proceeds via direct interactions between the ions, rather than via bridging water molecules.43 Multi-ion permeation studies have also been performed on the viral


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ion channel Vpu, which was shown to be a weakly conducting ion channel, conducting only one ion at a time.42

Single ion permeation studies employing molecular dynamics (MD) simulations had identified the hydrophobic stretch in p7 as being responsible for the selectivity of the channel, with the permeation of the doubly charged Ca2+ being unfavorable.44 However, the small difference in energy barriers corresponding to the transport of K+ and Ca2+, and why the permeating ion does not get trapped in the ion-binding site, are not clear. In the current study, the ion permeation and selectivity mechanisms are revisited by modeling multi-ion permeation process using umbrella sampling simulations. The present study elucidates a novel cooperation of at least three independent factors that control ion permeation, and identifies “knock-on” as a requisite phenomenon for ion conduction through the channel.

Figure 1. (A) p7 embedded in a fully hydrated lipid bilayer environment. The three helices from a given monomer are shown in different colors. (B) Pore-lining residues of the p7 channel. A structure corresponding to only two out of the six protomeric units of the hexameric channel is shown for clarity.


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Methods As described previously,44 the structure of the p7 ion channel (PDB ID: 2M6X) was equilibrated in a fully solvated lipid bilayer environment. The system had a total of 68092 atoms, and the dimensions of the simulation box were 88 Å x 88 Å x 84 Å. The equilibrated structure was used to perform umbrella sampling simulations to model the permeation of K+ ions and Ca2+ ions. Two ions (two K+ or two Ca2+) were placed in the pore region, and, for every position of a given ion along the pore axis, windows corresponding to different separations between the two ions were defined. Hence, the two dimensional PMF profiles that are calculated are with respect to the position of upper ion, and the separation between the two ions as the reaction coordinates (see later). The separation between the two ions was defined as the z-axis component of the distance between the two ions (the pore is aligned along the z-axis). A total of 43 x 9 = 387 windows each were sampled for K+ and Ca2+, with the separation between the two ions in the pore varying from 4 Å to 12 Å (at intervals of 1 Å). A harmonic potential of force constant 10 kcal mol-1 Å-2 was applied on the two ions to restrain their position along the reaction coordinates, and the entry of other ions into the pore region was prevented by applying a repulsive force with force constant 20 kcal mol-1 Å-2. For windows corresponding to the ion in bulk water, a cylindrical restraint of radius 8 Å was applied to restrict the lateral displacement of the ion. A restraint with force constant 10 kcal mol-1 Å-2 was applied on the center of mass of the protein to prevent an overall drifting.

Each window was sampled for 400 ps, with the first 100 ps being regarded as equilibration period, so that the total simulation time was ~ 155 ns each for K+ and Ca2+. Lennard-Jones interactions were truncated by using a smoothing function between 10 Å and 12 Å, and the


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particle mesh Ewald method was used for treating long-range electrostatics.45 Covalent bonds involving hydrogen atoms were constrained using the SHAKE algorithm.46 The simulations were performed in the NPAT ensemble, with the position of the ion being saved every 0.2 ps, and that of the system being saved every 2 ps. All simulations were performed with the NAMD program47 using the CHARMM36 all-atom force field for proteins and lipids48-51, the TIP3P water model52, and optimized parameters for ions.53 The CHARMM program was used for trajectory analysis,54 and the VMD program was used for rendering structural images.55 The free energy profile for two-ion permeation was calculated using the 2D-WHAM implementation of the WHAM program, which reads a distribution of the positions along the pore axis of the two ions.56-58

The minimum free energy path (MFEP) for the permeation of two ions through the channel was calculated using a smoothening algorithm, which is described as follows. For every bin along the first reaction coordinate (position of upper ion along pore axis), the minimum energy configuration was identified among all configurations along the second reaction coordinate (separation between two ions). Following this, all configurations that had an energy difference of less than 0.5 kcal mol-1 relative to the minimum energy configuration for this bin were extracted. Among these extracted structures, a configuration was assigned as being a part of the MFEP such that, in moving from one bin along the first reaction coordinate to the adjacent bin, the change in the separation between the two ions is a minimum.


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Results and Discussion Ions pass one at a time through the hydrophobic stretch. Throughout this discussion, the two ions in the pore will be referred to as “upper” and “lower” ions on the basis of their relative position in the pore. The upper ion is closer to the N-terminal opening of the pore (and therefore appears at the top in the figures shown here). Free energy profiles with respect to the position of the upper ion and the separation between the two ions are shown in Figure 2. For both K+ and Ca2+, the permeating ions maintain a high separation (~ 11 Å) as they enter the pore from the Cterminal side. The ring of positively charged Arg35 residues, followed by the hydrophobic stretch, are regions where the accommodation of more than one cation is highly unfavorable, and, consequently, only one cation passes through this region at a time. While the energy barrier along the MFEP is 5.4 kcal mol-1 for K+, that for Ca2+ is 19.4 kcal mol-1. This accounts for the selectivity of the channel toward K+ over Ca2+ reported in experimental studies.8 It must also be noted that, although the energy barrier for K+ seen here is almost the same as that observed previously in the single ion permeation study,44 the energy barrier for Ca2+ is significantly higher. This suggests that, while multiple K+ ion transport through the hydrophobic stretch is possible, permeation of more than one Ca2+ ion is not favorable, further confirming the greater permeability of the channel toward K+. Once the upper ion crosses the hydrophobic stretch and binds to the ring of Ser12 residues, the lower ion starts moving from the ring of Leu24 residues. The upper ion remains bound to Ser12 until the lower ion enters the hydrophilic region and binds to Asn16, so that the two ions are very close together. While the separation between the two K+ ions is ~ 5 Å, that between the Ca2+ ions is ~ 7 Å when the two ions are bound to Ser12 and Asn16. As will be shown later, further movement of the upper ion toward the N-terminal end is facilitated by repulsion between the two ions. This is followed by a stable intermediate in which


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one ion is bound to Asn9 while the other is bound to Asn16. Further movement of the two ions is expected to be driven by a new incoming ion. It must be mentioned that, although the MFEP for Ca2+ suggests that the two ions are close together as they exit the pore region, the free energy values corresponding to different separations between the ions at this point are almost the same, and, hence, any of these states is equally likely to occur.

Figure 2. PMF for the permeation of two ions through the channel as a function of the position of the upper ion and the separation between the two ions. The minimum free energy paths (MFEP) are shown by black dashed lines. Representative configurations along the MFEP are also shown.

Ion passage through the hydrophilic region is driven by knock-on. The initial barrier in the PMFs due to the Arg35 residues and the hydrophobic stretch is expected (Figure 2). However,


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the local minimum corresponding to the ion binding sites become shallower compared to the single ion permeation.44 To explore the role of knock-on effect, the interaction energy between the two permeating ions was calculated. Figure 3A shows the interaction energy between the two ions averaged over bins along the two reaction coordinates. Near the ring of positively charged arginine residues and in the hydrophobic region, the two ions avoid unfavorable interactions by maintaining a large separation. When the ions are inside the hydrophilic region formed by Asn9, Ser12, and Asn16, interionic repulsion becomes significant, with the interaction energy between the two permeating ions going up to more than 20 kcal mol-1. For Ca2+, although the interionic repulsion is seen only for a very short stretch along the MFEP, the magnitude of the repulsion is higher than that of K+, explaining the greater separation between the permeating Ca2+ ions compared to the K+ ions. The favorable interaction between the permeating ion and the binding site is countered by ion-ion repulsion, which further facilitates the movement of ion. Such a knock-on process has previously been shown to drive ion transport in the K+-selective KcsA channel.35 In the present study, knock-on is seen to involve direct repulsion between the two ions, without any bridging water molecule in between (data not shown), which is much like the knock-on mechanism reported recently for the KcsA channel.43


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Figure 3. (A) Interaction energy between the two permeating ions with respect to the position of the upper ion and the interionic separation. MFEP is shown as dashed lines. (B) Interaction energy between the upper ion and the protein. Important configurations along the MFEP are encircled, and the approximate locations of the two ions (relative to pore-lining residues) for these configurations are shown at the bottom.

The interactions between the permeating ion and the protein were further analyzed by calculating the interaction energies (Figure 3B). Once the upper ion crosses the hydrophobic stretch and is inside the hydrophilic region, it interacts strongly with the pore-lining residues; this behavior is observed for K+ and Ca2+ alike. It can be seen that the upper ion is bound to Ser12 residue as the lower ion moves from the hydrophobic region to Asn16. The strong interaction between the upper ion and the serine is overcome only after the lower ion binds to the ring of Asn16 residues,


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and reaches a position where it repels the upper ion (this corresponds to the minimum separation between the two ions along the MFEP). Thus, ion-ion repulsion makes possible further movement of the upper ion towards the exit of the pore. Another stable configuration is seen where the upper ion is bound to Asn9 while the lower ion is still bound to Asn16. Further movement of the upper ion from this configuration is likely to be driven by complete solvation of the ion as it approaches bulk water.

It is to be noted that the knock-on mechanism facilitates ion conduction by ensuring that ions do not remain bound in the hydrophilic region. In other words, this mechanism leads to a shallow energy minimum corresponding to the binding site, thereby preventing the system from getting trapped in this local minimum. The mechanism does not, however, lower the energy barrier for the ion transport process, and, in fact, the energy barrier for Ca2+ reported here is higher than that observed previously for single Ca2+ ion permeation. This suggests that it is highly unlikely for two Ca2+ ions to pass through the hydrophobic stretch at the same time. However, as far as the hydrophilic region is concerned, the existence of two Ca2+ ions in this region becomes a necessary phenomenon for knock-on to occur. It is with this knock-on that ions are driven out of the binding site, thereby ensuring a continuous flow of ions across the channel.

K+ and Ca2+ exhibit different modes of binding in the binding site. Free energy calculations that were previously performed to investigate single ion permeation in p7 had reported Asn9 as a major binding site, and Asn16 and Ser12 as minor binding sites.44 In the present study, a particularly stable configuration that is seen is one with the upper ion bound to Asn9, and the lower ion bound to Asn16. The mode of binding of K+ and Ca2+ to these sites has been elucidated


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in detail here based on a number of analyses. Figure 4A shows a probability distribution of the distance between the permeating ions and the closest oxygen atom of the ring of asparagine residues for configurations corresponding to the two ions in the respective binding sites (the distance is measured between Asn9 and the upper ion, and between Asn16 and the lower ion). For both the upper ion and the lower ion, K+ is seen to occur closer to any of the amide oxygens in comparison to Ca2+. In spite of the lower size of Ca2+, it is seldom seen to occur at a distance of less than 3 Å of the amide oxygens. It is also to be noted that, while a few configurations are seen where K+ is not in the vicinity (> 4 Å) of the amide oxygens, thereby broadening the probability distribution, Ca2+ ions are seen to have a narrow distribution, suggesting that the latter sample a smaller space while they are inside the ring of asparagine residues. Such behavior is evident in Figure 4B, which shows snapshots of the K+ and Ca2+ ions interacting with the ring of Asn9 residues. Inspection of the trajectories shows that Ca2+ is completely solvated, and its interaction with the asparagines is via bridging water molecules only. K+, on the other hand, is able to interact directly with the asparagines, as shown in Figure 4C, thereby explaining why it is seen in the vicinity of Asn9. Higher desolvation penalty expected for the dication and the size differences between the two ions are possible factors that necessitate this kind of behavior.


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Figure 4. (A) Probability distribution of the distance between the permeating ion and the closest Asn9 residue. The data is taken from a window that corresponds to the upper ion near Asn9 and the lower ion near Asn16. (B) Snapshots of the upper ion in the binding site formed by the ring of Asn9 residues. All positions sampled by the ion in the window are shown. The color gradient from red to blue varies with the timestep of the frames in the trajectory. (C) Representative snapshots for the upper ion in the vicinity of the Asn9 are shown with coordinating water molecules.


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Conclusions In summary, this study provides a thermodynamic and structural basis for the ion selectivity of the p7 channel by employing umbrella sampling MD simulations. Initially, discrimination between the ions is made by a hydrophobic stretch, which imposes a high energy barrier to the conduction of multiple Ca2+ ions. This is followed by the binding of an ion in the hydrophilic region, where the ion is trapped until another incoming ion approaches it and causes a knock-on, thereby driving the bound ion out of the hydrophilic region. While K+ binds directly to the asparagines forming the binding site, Ca2+ interacts with the asparagines via bridging water molecules. Thus, a novel cooperation of different phenomena and subtle differences in the response of the two ions resolve ion selectivity.

Acknowledgments The authors are grateful to the Department of Atomic Energy, Government of India for financial assistance (grant no. 37(2)/14/05/2015/BRNS/20046).

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Cooperation of Hydrophobic Gating, Knock-on Effect, and Ion Binding

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