Molecular Dynamics Simulations of Orai Reveal How the Third

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Molecular Dynamics Simulations of Orai Reveal How the Third Transmembrane Segment Contributes to Hydration and Ca Selectivity in CRAC Channels 2+

Azadeh Alavizargar, Claudio Berti, Mohammad Reza Ejtehadi, and Simone Furini J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b12453 • Publication Date (Web): 30 Mar 2018 Downloaded from http://pubs.acs.org on March 31, 2018

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The Journal of Physical Chemistry

Molecular Dynamics Simulations of Orai Reveal how the Third Transmembrane Segment Contributes to Hydration and Ca2+ Selectivity in CRAC Channels Azadeh Alavizargar1,2, Claudio Berti3, Mohammad Reza Ejtehadi1,4 and Simone Furini2,*

1

School of Nano Science, Institute for Research in Fundamental Sciences (IPM), Tehran, Iran

2

Department of Medical Biotechnologies, University of Siena, Siena, Italy

3

Department of Molecular Biophysics and Physiology, Rush University Medical Center, Chicago, Illinois,

United States 4

Department of Physics, Sharif University of Technology, Tehran, Iran

*Corresponding author: Department of Medical Biotechnologies University of Siena Viale Mario Bracci, 16 53100 Siena, ITALY Phone: +39 0577585297 Email: [email protected].

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Abstract Calcium release-activated calcium (CRAC) channels open upon depletion of Ca2+ from the endoplasmic reticulum, and when open, they are permeable to a selective flux of calcium ions. The atomic structure of Orai, the pore domain of CRAC channels, from Drosophila Melanogaster has revealed many details about conduction and selectivity in this family of ion channels. However, it is still unclear how residues on the third transmembrane helix can affect the conduction properties of the channel. Here, Molecular Dynamics and Brownian Dynamics simulations were employed to analyse how a conserved glutamate residue on the third transmembrane helix (E262) contributes to selectivity. The comparison between the wild-type and the mutated channel revealed a severe impact of the mutation on the hydration pattern of the pore domain and on the dynamics of residues K270, and Brownian Dynamics simulations proved that the altered configuration of residues K270 in the mutated channel impairs selectivity to Ca2+ over Na+. The crevices of water molecules revealed by Molecular Dynamics simulations, are perfectly located to contribute to the dynamics of the hydrophobic gate and basic gate, suggesting a possible role in channel opening and in the selectivity function.


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1. Introduction Calcium release-activated calcium (CRAC) channels are proteins of the plasma membrane, which are activated by the depletion of Ca2+ from the endoplasmic reticulum 1. The opening of these channels leads to a sustained increase of intracellular Ca2+ concentration, which in turn regulates numerous cellular functions, such as secretion, gene transcription, and cell proliferation 2–4

. Although the process of calcium entry across CRAC channels has been recognized for

decades, their molecular components have been identified only recently 5–10. CRAC is composed of two modules: Orai, which constitutes the pore domain of the channel; and STIM (STromal Interaction Molecule), which regulates the process of calcium activation. STIMs are single-pass endoplasmic reticulum transmembrane proteins. As the concentration of Ca2+ in the endoplasmic reticulum decreases, STIM proteins aggregate and translocate into discrete junctions, where they interact with Orai, opening the ion-conducting pore

2,4,11

. In mammalians, the Orai family

includes three homologous proteins, named Orai1-3 3,12. Interestingly, the sequence of Orai does not share any relevant similarity with other pore forming proteins, suggesting that this family of ion channels is likely to exhibit unique structural and functional characteristics. In 2012, the atomic structure of the first channel of the Orai family was solved by X-ray crystallography, using the sequence of Drosophila Melanogaster, which shares 73% identity with human Orai1 in the transmembrane region 13. Because of the role of CRAC in signalling cascades, understanding how ions move across Orai might have important implications in cell physiology and drug development. The Orai channel is made of six subunits, arranged in threefold symmetry around the axis of the pore. Each subunit consists of four transmembrane helices, named TM1 to TM4, with TM1 helices defining the internal boundaries of the ion-conducting pore (Figure 1 and S1)

14,15

.

Proceeding from the extracellular towards the intracellular compartment, the pore of Orai presents first a ring of six glutamate residues (E178, corresponding to E106 in human Orai1), followed by three consecutive rings of hydrophobic residues (V174, F171 and L167), and finally, three rings of basic residues (K163, K159 and R155). The diameter of the hydrophobic region at the center of the pore ranges between 8 Å and 10 Å, and consequently, it represents an energy barrier for the permeation of fully hydrated ions. Furthermore, the high density of basic residues at the intracellular side of the pore is expected to block the passage of cations by two (non3 ACS Paragon Plus Environment


exclusive mechanisms): i) creating an electrostatic barrier for the permeation of cations; and ii) strongly binding anions that physically occlude the pore 13. Therefore, the experimental structure of Orai from Drosophila Melanogaster is likely to be representative of a closed state of the channel, in accordance with the crystallographic conditions. The block of cation fluxes by the basic residues K163, K159, and R155, might be suppressed by a radial movement of the intracellular part of helices TM1 in the outward direction 13,16. This gating-model is supported by the presence of a region along TM1 (between residues S162 and S166) that is rich in residues usually involved in helix-bending (serine and threonine). An alternative gating-model that involves the hydration/de-hydration of the central section of the channel was also proposed. The role of the hydrophobic residues V174 and F171 on channel gating is supported both by experimental data and Molecular Dynamics (MD) simulations. Mutations of the residue analogous to V174 in human Orai1 (V102) to alanine resulted in a constitutively active channel 17,18

. In agreement, MD simulations revealed that the mutation V174A causes a marked decrease

in the energy barrier for the permeation of Na+ across the pore with respect to the wild-type channel

19

. The configuration of residue F171 (corresponding to F99 in human Orai1) was also

proved to regulate hydration and dewetting, and consequently the ion-permeability of the channel

20

. According to this gating-model, the opening of the channel is due to a 20-degrees

rotation of helices TM1 around their longitudinal axis, which reorients F171 residues away from the pore. Frischauf et al. 21, proposed a different atomic mechanism for channel opening, where both the hydrophobic and the basic gate are controlled by a network of local interactions, involving hydrogen bonds among residues on helices TM1 and TM2, with no need for global movements of helices TM1. Despite these different atomic details, a coherent picture is emerging from experimental data and MD simulations, where both the hydrophobic and the basic gate contribute to the opening of the channel, with hydrophobic gating having a dominant role. However, it is important to remember that the current atomic models of gating are almost entirely based on MD simulations of constitutively active channels that do not require STIM binding for channel opening, and consequently more experimental data and computational models are needed for a complete description of gating in wild-type CRAC channels at the atomic level. Noteworthy, in MD simulations of constitutively active channels, the opening of both the hydrophobic and the basic gate does not involve the extracellular portion of the pore; i.e.


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the structure of the ring of glutamate residues at the extracellular entrance of Orai (E178) is conserved between the closed and the open state. The main function of the glutamate residues, E178, is to confer selectivity to calcium ions. When extracellular Ca2+ concentration is in the mM range, CRAC channels select Ca2+ over monovalent ions, such as Na+ or K+ by more than 1000:1, classifying them among the most highly-selective Ca2+ channels

22

. Instead, when Ca2+ is missing from the extracellular

environment, CRAC channels are permeable to monovalent ions

22,23

. In the presence of a fixed

concentration of Na+ and increasing concentrations of Ca2+, the conductance of the channel changes non-monotonically, first decreasing and then increasing

24

. A possible explanation for

this anomalous mole fraction effect of CRAC is that residues E178 define a binding site that is highly selective for Ca2+

over Na+

8,9,25–28

.

In

agreement

with

this

hypothesis,

electrophysiological experiments proved that even the conservative mutation of these residues to aspartate decreases the selectivity to Ca2+ over Na+ significantly 26 . The side chain of aspartate is one carbon atom shorter that the side chain of glutamate. Thus, mutating glutamate to aspartate increases the radius of the pore and decreases the density of negative charges. This decrease in charge density might readily explain the effect on the selectivity mechanism, as the more densely packed negative charges of the wild-type channel are better suited to select divalent cations over monovalent cations. More surprisingly, the selectivity mechanism is also impaired by mutations of another ring of glutamate residues, E262 (analogous to E190 in human Orai1), located on helix TM3 (Figure 1). The mutation of this residue to glutamine severely impacts the selectivity to Ca2+ over Na+, while a more conservative mutation to aspartate is tolerated

8,9,25

. The

immediate consequence of these mutagenesis studies is that a ring of negative charges at the position occupied by residues E262 is crucial for the selectivity mechanism. However, a direct electrostatic effect of E262 on conduction and selectivity does not fit with the experimental structure of the channel. E262 residues are embedded within the protein core, among helices TM1, TM2 and TM3 (Figure 1). The distance between the side chains of E262 and E178 is more than 12 Å, which excludes the idea that the negative side chain of E262 finely regulates the binding of cations to the selectivity filter. An alternative explanation is that E262 exerts an indirect effect on the selectivity filter mediated by water molecules, ions, or other protein residues. The significant role of E262 on the structure/function of Orai is supported by the high 5 ACS Paragon Plus Environment


conservation of this residue among different channels of the Orai family (Figure S2). Furthermore, E262 is in close proximity of residues V174 and F171 that are involved in hydrophobic gating (Figure 1), and mutations of these residues not only impact channel gating, but they might also impair the selectivity to Ca2+ over Na+. Therefore, an intriguing hypothesis is that E262 is part of a network of atomic interactions that affects the behavior of the selectivity filter, and that it might also be involved in gating processes. The role of the residues at position 262 was here investigated by MD simulations of the wildtype Orai channel and of the E262Q mutant. The simulations revealed a previously undescribed crevice of water molecules extending in the region between the internal ring of helices TM1 and the external ring of helices TM2 and TM3. Taking advantage of this hydrated region embedded among TM1, TM2 and TM3, ions can reach a binding site close to E262. The mutation E262Q reduces the number of water molecules at the back of TM1 and prevents the binding of ions to this region. In turn, the altered local environment affects the dynamics of E178 and of a conserved lysine residue on helix TM3 (K270, corresponding to K198 in human Orai) (Figure 1). Brownian Dynamics (BD) simulations proved that the different configuration of residues K270 between the wild-type and the mutated channel might explain the impaired selectivity of the E262Q mutant. These molecular details shed new lights on the functioning of CRAC channels, and on the possible role of residues E262, E178, and K270 in gating and selectivity.

2. Methods 2.1 Molecular Dynamics (MD) simulations The atomic coordinates of the wild-type Orai channel were taken from the Protein Data Bank entry 4HKR 13. All the residues in the crystal structure were included in the model (from T144 to H334). The missing loops (residues L181 to G190, connecting TM1 to TM2; and I220 to S235, connecting TM2 to TM3) were defined using Modeller, as implemented in the Chimera software 29,30

. To prepare the initial structure for MD simulations, CHARMM-GUI membrane builder was

employed

31

.

The

protein

was

embedded

in

1-Palmyoil-2-oleoyl-sn-glycero-3-

phosphatidylcholine (POPC) lipids, with normal vector oriented along the z-axis. The initial position of the protein in the lipid bilayer was determined from the “Orientations of Proteins in 6 ACS Paragon Plus Environment

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Membranes (OPM)” database 32. The final number of lipid molecules was 217 in the upper layer and 185 in the lower layer. The protein-membrane system was solvated by water molecules (around 40.000), and 150 mM of sodium and chloride ions were added to neutralize the system. The total number of atoms in the atomic model was close to 187000 (Figure S1). MD simulations were run with version 2.12 of NAMD 33, using the CHARMM36 force field 34, and the TIP3P model for water molecules

35

. Periodic boundary conditions were applied in all

directions. Long-range electrostatic interactions were calculated with the Particle Mesh Ewald method

36

. Van der Waals interactions were smoothly truncated between 10 Å and 12 Å. The

SETTLE algorithm was used to restrain bonds with hydrogen atom

37

. The temperature was

controlled at 310 K by coupling to a Langevin thermostat with a damping coefficient of 1 ps-1. A pressure of 1 atm was maintained by coupling the system to a Nose-Hoover Langevin piston, with a damping constant of 25 ps and a period of 50 ps 38. The equilibration processes consisted of: (i) 10000 steps of energy minimization with harmonic restraints of 2 kcal*mol-1*Å-2 on all the heavy atoms of the protein; (ii) 450 ps in the NVT ensemble with harmonic restraints of 2 kcal*mol-1*Å-2 on the backbone atoms of the protein, using a time step of 1 fs for integrating the equations of motion; (iii) 33 ns in the NPT ensemble using a time step of 2 fs, during which harmonic restraints were gradually decreased to zero. The atomic model of the wild-type channel at the end of the equilibration protocol was used to define the initial structure of the E262Q mutant. Residues E262 of the six subunits were mutated to glutamine by the VMD software 39. Then, the system was minimized by 10000 steps and equilibrated for 2 ns. Unconstrained trajectories of 150 ns were simulated in the NPT ensemble for both the wild-type channel and the E262Q mutant. Two completely independent trajectories were simulated for each atomic model, giving a total simulated time of 0.6 µs. Trajectories were analysed by python routines integrating the MDanalysis module 40. 2.2 Brownian Dynamics (BD) simulations BD simulations were run by BROWNIES

41

, using a toy-model of the selectivity filter of Orai

channel. The model was inspired by previous models of calcium channels adopted in Monte Carlo and BD simulations, and it is based on a selectivity filter made by a set of mobile particles with partial negative charge equal to -0.5 elementary charges 7 ACS Paragon Plus Environment

42

. The dynamics of the mobile


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particles describing the selectivity filter and of the ions were simulated by numerically solving the Langevin’s equation by the method proposed by van Gunsteren and Berendsen

43

. The

simulation domain consisted of a rectangular simulation box with size 40 Å x 40 Å x 125 Å. Periodic boundary conditions were used along the x-, y- and z- directions. The simulation box included an impermeable slab orthogonal to the z-axis and extending from z = -7.5 Å to z = +7.5 Å, which presented a cylindrical hole with radius equal to 4.25 Å at the centre. Short-range forces implemented as in reference

41

prevented the ions and the mobile particles of the

selectivity filter from entering this impermeable slab. The particles of the selectivity filter were further confined by short-range forces in the z-direction acting at z = ±4.5 Å. The PACO algorithm

44

was used to control the concentration of Na+, Ca2+ and Cl- at the two sides of the

impermeable slab, utilizing control cells with width equal to 0.1 Å placed respectively at z = +57.5 Å and z = -57.5 Å. The membrane potential was simulated by applying a constant electric field in the z-direction. The simulation domain is schematically depicted in Figure S3, and the parameters of the BD simulator are provided in Table S1.

3. Results 3.1 Pore structure in wild-type and E262Q mutant channel The atomic models of the wild-type channel and of the E262Q mutant were stable during the simulated MD trajectories. The Root Mean Square Deviation (RMSD) of the protein’s backbone atoms slightly increases in the second part of the trajectories, but this increase is largely due to structural changes in the loop regions (Figure S4 and S5). The RMSD of the backbone atoms in the pore region (residues T144 to V179) is close to 2.0 Å for both atomic models, and no relevant drift is observed along the trajectories (Figure 2 and S5). Previous MD simulations of Orai reported contradictory results about the simulation time needed to reach an equilibrated state of the system. In detail, Dong et. al. observed a drift in the RMSD of the alpha-carbon atoms lasting hundreds of nanoseconds

19

, while in simulations by Amcheslavsky et al. and by

Frischauf et al. the system reached an equilibrated state much more rapidly

21,45

, in better

agreement with the trajectories discussed here. The different composition of the lipid bilayer, including cholesterol, might explain the shorter equilibration time reported by Frischauf et al. 21, 8 ACS Paragon Plus Environment

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as cholesterol is known to stabilize the closed state of CRAC channels

46

. However, the same

lipid composition (pure POPC membrane) was used in this study, as well as in MD simulations by Dong et al. and by Amcheslavsky et al. 19,45,47. An alternative hypothesis is that the presence of ions and water molecules inside the channel affect the stability of the structure, and consequently, the time required to reach an equilibrated state is sensible to small differences in the initial loading state of the channel; i.e. how many ions and water molecules are present in the initial structure. In the current simulations, water molecules were present in the starting structure, and Na+ and Cl- ions moved inside the channel during the relatively long equilibration time. Therefore, at the beginning of the production runs, the channel was already hydrated, and the binding sites for Na+ and Cl- ions along the channel were already occupied. The presence of ions inside the pore counter balances the negative and positive charges due to the protein’s charged residues, which might explain the stability of the structure. The pore profile of the wild-type and of the E262Q mutant channel was calculated to explore a possible effect of the mutation. The narrowest constriction of the pore is due to residues K163 in both wild-type and mutated channels (Figures 3 and S6). The diameter of this region is not likely to be compatible with ion conduction, in agreement with the closed state reported for the experimental structure of the Orai channel. Previous MD simulations of gating events were performed using mutated channels that are constitutively active even in the absence of a gating stimulus 19,20,21. As this is not the case for the E262Q mutant, the closed state of the channel was preserved in the MD trajectories, as expected. The small differences in pore profile at the basic gate between the wild-type and the mutated channel are likely due to the random variability of MD trajectories, and indeed, they are not conserved between different set of simulations (Figure 4 and S6). On the contrary, the comparison between the wild-type and the mutated channel revealed that the radius of the pore in the region close to the selectivity filter is slightly wider for the mutant, and this difference was preserved in independent set of simulations (Figure 3 and S6). In the wild-type channel, the diameter of the pore between residues F171 and E178 is around 4 Å, in good agreement with the available experimental data

25

. The E262Q mutation

caused a limited, but significant, widening of this region. The wider radius of the selectivity filter in the mutated channel agrees with experimental data, which revealed that the E262Q mutation increases the pore diameter of CRAC channels 25. 9 ACS Paragon Plus Environment


3.2 Ions and water molecules inside the pore The pore of the wild-type channel was occupied on average by 6.2±0.5 Na+, 13.7±0.3 Cl-, and 34±2 water molecules, as calculated from two independent MD simulations (the difference between the average values in the two trajectories was adopted as an estimate for the uncertainty). Ions were considered inside the pore when above the center of mass of residues W148 and below the centre of mass of residues D184 along the z-axis (pore axis), with the upper threshold of 10 Å in the radial direction. The boundaries of this region were defined to incorporate the binding sites for cations at the extracellular entrance of the pore, close to residues D182 and D184. For water molecules, the region above the centre of mass of residues R155 and below the centre of mass of residues E178 along z-axis was considered, with the upper threshold of 5 Å in radial direction. Residues E178 and T155 were used to select only the water molecules in the narrow part of the pore, excluding the highly hydrated intracellular and extracellular entrances, while the threshold of 5 Å in the radial direction was necessary to consider only water molecules inside the central ion-conducting pore (Figure S7). Chloride ions accumulate at the intracellular side of the channel (Figure S7), attracted by the basic residues K163, K159, and R155. The accumulation of Cl- ions inside the channel is likely to contribute to sodium permeation, by decreasing the electrostatics repulsion between basic residues and permeating cations 47. Sodium ions accumulate at the extracellular entrance, attracted by the acidic residues of the selectivity filter (E178) and of the extracellular loop connecting TM1 to TM2 (D182 and D184). The peak of Na+ density extends around 5 Å in the radial direction and 10 Å in the axial direction (Figure 4). In this region, sodium ions might interact directly with the side chain oxygen atoms of the glutamate residues, losing up to three water molecules from the first hydration shell. On average, these ion-protein interactions are not stable for more than a few nanoseconds, with the consequence that sodium ions inside the selectivity filter are highly dynamic. Binding of Na+ and Cl- respectively at the extracellular and intracellular side of the pore was largely expected on the base of the experimental structure, and previous MD simulations

19,47

. The distribution of ions and water molecules along the pore was not modified

by the mutation E262Q. In the MD trajectory of the E262Q mutant, the average number of ions inside the pore was respectively 5.6±0.6 and 14.0±0.3 for Na+ and Cl- and the average number


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of water molecules was 34±2 (Figure 4 and S7), in good agreement with the values estimated for the wild-type channel. 3.3 Ions and water molecules in the region among helices TM1, TM2 and TM3 MD simulation of the wild-type channel revealed a previously undescribed hydrated region at the interface between the internal ring of helices TM1, and the external ring of helices TM2 and TM3 (Figure 5). In the following, this region among helices TM1, TM2 and TM3 is briefly referred to as back of the pore (ring highlighted by light-blue lines in Figure 5). After an initial drift, the number of water molecules in this region reached a steady state in both simulations of the wild-type channel (Figure S8), corresponding to an average of 34±11 water molecules per each subunit. The uncertainty was estimated as the standard deviation computed among the six subunits that contribute to the protein. In two out of the six subunits, water molecules spanned the entire region at the back of the pore between the intracellular and the extracellular side of the channel (Figure 6a). In the course of the MD trajectories, around 1000 complete permeation events of water molecules were sampled in both directions along this route. Instead, in the remaining 4 subunits of the channel, the hydration layer at the back of the pore was interrupted halfway between the intracellular and the extracellular side (Figure 6b). At this central section of the pore, the residues exposed to the interface among helices TM1, TM2, and TM3 are L168, L202, and F259. These hydrophobic residues, and in particular, the bulky F259, create a hydrophobic siege that blocks the passage of water molecules between the intracellular and the extracellular compartments (Figure 6b). The single point-mutation E262Q caused a dramatic effect on the distribution of water molecules at the back of the pore. In the second half of the MD trajectory (last 75 ns) of the E262Q mutant channel, the average number of water molecules that penetrated the crevices among TM1, TM2 and TM3 was around 105, roughly half the value estimated for the wild-type channel (around 200). Moreover, the hydration layer at the back of the pore was interrupted by the hydrophobic residues L168, L202, and F259 in all of the six subunits composing the channel. The average number of water molecules per chain was 17±6, and no permeation event between the intracellular and the extracellular side of the protein was observed during two independent MD simulations of the mutated channel. It is worth mentioning that the atomic models of the E262Q 11 ACS Paragon Plus Environment


mutant channel were defined using the coordinates of the wild-type model at the end of the equilibration trajectory (see Methods section for details), and consequently, the number of water molecules at the back of the pore in the initial structure of the mutant corresponds to the analogous number in the wild-type channel at the end of equilibration (approximatively 30 molecules per subunit). The E262Q mutation caused a rapid decrease in the number of water molecules, as well as structural changes of residues L168, L202, and F259, which blocked the water-passage between the intracellular and the extracellular compartments. The number of water molecules reached a new steady state after 50-75 ns in both the simulated trajectories (Figure S8), proving that the mutation E262Q has a profound impact on the distribution of water molecules at the back of the pore. The difference in hydration between the wild-type and the mutated channel is particularly surprising considering that the glutamate residues are substituted by neutral, but still hydrophilic residues, as glutamine. The presence of water molecules at the back of the pore in the wild-type channel creates a route for hydrated ions that can easily move between the extracellular compartment and a position close to residues E262. The density of sodium ions exhibits a clear peak in the proximity of the side chain of residues E262 (Figure 4a). During the MD trajectory, several Na+ were observed to move along the crevice of water molecules at the back of the pore, with binding events to E262 lasting from a few nanoseconds to 10-20 nanoseconds, but no ion was observed to move from the extracellular to the intracellular side of the channel along this route. Besides the interaction with sodium ions, residues E262 were also observed to exert an electrostatic attraction on residues K270 (from adjacent subunits). In detail, in simulations of the wild-type channel, the side chain of residues K270 switched between two alternative configurations (Figure 7a and 7b), respectively directed towards the protein-core (60% of the simulated time, Figure 7a), or exposed to the extracellular compartment, near E178 residues (40% of the simulated time, Figure 7b). The presence of these alternative configurations is clearly exemplified by the separate density peaks exhibited by the position of residues K270 (Figure 4a, blue contour plots). The distribution of ions at the back of the pore and the configuration of residues K270 are severely modified by mutation E262Q (Figure 4 and Figure S9). The binding site for cations at the back of the pore disappeared completely in the E262Q mutant channel, and the inward configuration of residues K270 became inaccessible. In the MD trajectories of the E262Q mutant, the side chain of 12 ACS Paragon Plus Environment

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residues K270 were always exposed to the water solution at the extracellular side of the channel, close to the ring of glutamate residues of the selectivity filter (Figure 7c). 3.4 Effect of K270 on selectivity to Ca2+ over Na+ The effect of K270 on conduction and selectivity was investigated by BD simulations using a toy-model of the selectivity filter of the Orai channel. The selectivity filter was represented by a set of 12 particles with electric charge equal to -0.5 elementary charges. These mobile particles were confined into a cylindrical region that extended respectively 9 Å and 8.5 Å in the axial and radial direction. This model of the selectivity filter was embedded into a membrane slab impermeable to ions, and conduction of Na+ and Ca2+ at different membrane potentials and ion concentrations was simulated (see Figure S3 for a schematic representation of the toy-model and Methods section for details). The confinement of 12 negative particles in a restricted region mimics the basic characteristics of the selectivity filter of Orai, where 12 side chain oxygens from residues E178 are confined into a limited space. This simplified model of the selectivity filter obviously misses many features of Orai channels, and it cannot be expected to reproduce experimental data in a quantitative way. However, in BD simulations with mixed concentrations of Na+ and Ca2+, the toy-model reproduced the main functional features of CRAC currents. In simulations with identical concentrations of the two ion-species, the model was selective for Ca2+ (Figure 8). Moreover, in simulations with a fixed concentration of Na+ and increasing concentrations of Ca2+, the anomalous mole fraction effect observed experimentally was reproduced

24

. In detail, when calcium concentration was increased, the total current across the

toy-model first decreased as a result of Ca2+ block to Na+ permeation, and then increased again when the Ca2+ current became dominant over the Na+ current (Figure S10). The selectivity of the toy-model is lower than the one observed experimentally, and the blockage of Na+ currents is less complete and requires higher calcium concentrations compared to electrophysiological experiments. This lower selectivity of the toy-model might have many causes, like the absence of other acidic residues at the extracellular entrance of the channel (D182 and D184), or the higher mobility of the elementary particles as compared to the carbonyl oxygens of the glutamate residues of the selectivity. However, since the toy-model reproduces the qualitative features of the selectivity filter of CRAC channels, it is possible to investigate how these features are affected by the presence of a ring of positive charges at the extracellular entrance of the pore, 13 ACS Paragon Plus Environment


representing residues K270. Residues K270 were modelled as a ring of six point-charges, placed with cylindrical symmetry at the extracellular entrance of the selectivity filter (Figure S3). The value of these point charges was gradually increased from 0 to 1, and the selectivity to Ca2+ over Na+ was estimated from BD simulations with equal concentrations of the two ion-species. The toy-model with no positive charges at the extracellular entrance of the channel was designed to mimic a condition where the side chain of residues K270 are directed towards E262. When residues K270 assume this configuration, the side chain of K270 points away from the selectivity filter, and its positive charge is counter-balanced by the negative charge of E262 (Figure 7a). Therefore, the electrostatic effect on the selectivity filter is expected to be minimal. Instead, the simulations with positive charges equal to 1.0 was designed to represent a state where the six residues K270 are all pointing to the extracellular compartment, close to the ring of glutamate residues E178 (Figure 7b). The selectivity of the toy model to Ca2+ over Na+ decreased roughly linearly when the point charges were increased from 0 to 1, disappearing almost completely when six elementary charges are present at the extracellular entrance of the pore (Figure 8).

4. Discussion The experimental structure of Orai from Drosophila Melanogaster confirmed many of the predictions based on previous functional and biochemical experiments, with a relevant exception: residue E262. Despite this glutamate residue is embedded within the protein core, at a distance of more than 12 Å from the selectivity filter of Orai, mutation to glutamine severely reduces the selectivity to Ca2+ over Na+. This apparent contradiction was here investigated by MD simulations, which revealed a major difference between the wild-type and the E262Q mutant; i.e. the interface between the inner ring of helix TM1 and the outer ring of helices TM2TM3 is much more hydrated in the wild-type channel compared to the E262Q mutant. Residue K270 at the C-terminal of helix TM3 emerged as being critical for explaining the effect of the E262Q mutation on the hydration pattern of the channel. In the wild-type channel, residues K270 rapidly move among several configurations, with the side chain respectively pointing to the extracellular or to the intracellular side of the channel. The presence of a positive (K270) and a negative (E262) residue creates a hydrophilic environment in the region among helices TM1, TM2, and TM3. Instead, in simulations of the mutated channel, residues K270 are always 14 ACS Paragon Plus Environment

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directed towards the extracellular side of the channel. Therefore, as a consequence of a single point mutation (E262Q), the interface among helices TM1, TM2, and TM3 becomes less hydrophilic (two polar residues are lost), and the positive charges of residues K270 move closer to the selectivity filter. The presence of a ring of positive charges close the selectivity filter is likely to hamper the binding of divalent calcium ions more than the binding of monovalent sodium ions, and consequently, it might affect the selectivity of the channel for the two ionspecies. In principle, one could test this hypothesis by performing atomic simulations with calcium and sodium ions. However, an accurate description of ion-protein interactions for calcium ions is a well-known shortcoming of classical (non-polarizable) force fields

48

, and,

49

despite rapid improvements , an extensive testing of polarizable force fields in ion channels is still missing. Moreover, the effect of the E262Q mutation on the conduction properties of the channel cannot be tested directly by MD simulations, as the experimental structure of Orai represents a closed state. Therefore, as an alternative to direct MD simulations with calcium ions, BD simulations of a simplified channel model were used to analyse how positive charges close to the extracellular entrance of the channel (representing residues K270) modify the selectivity to Ca2+ over Na+. The BD simulations confirmed that the altered dynamics of residues K270 explain the effect of mutation E262Q on the selectivity function. In summary, the model proposed on the base of MD and BD simulations is that mutation E262Q reduces the number of water molecules at the back of TM1 helices, and this de-hydration pushes residues K270 close to the selectivity filter where they impair the selectivity to Ca2+ over Na+. A consequence of the water crevices at the back of the pore, observed in the MD simulation of the wild-type channel, is that extracellular pH might modify the protonation state of residues E262 and the conductance of the channel. Indeed, electrophysiological experiments proved that the analogous amino acid in human Orai1, E190, is a pH sensor50. Interestingly, E190 is responsible for pH sensitivity only when Na+ is the charge carrier, while in the presence of Ca2+ the pH sensitivity is due to the glutamate residues of the selectivity filter (E106 in human Orai1). A more alkaline extracellular environment promotes the protonation of the glutamate residues in the selectivity filter. This lower density of negative charge is expected to decrease the binding affinity of calcium ions to the selectivity filter, which explain the increased conductance of the wild-type channel at high pH in the presence of Ca2+.

However, according to the model



Page 16 of 36

proposed here, this increase in extracellular pH should also increase the permeability to Na+ (as the protonated state of residues E262 favours the out-facing configuration of residues K270, which impairs Ca2+ selectivity). This increase in Na+ permeability is currently not confirmed by experimental data, but, to the best of our knowledge, the dependence of Orai conductance on extracellular pH was always characterized using impermeable cesium ions in the intracellular solution, which prevents the measurement of outward currents

50,51

. Instead, the experimental

observation that in divalent free solution, the conductance increases at basic pH is in perfect agreement with the model proposed here, as the protonated state of E262 renders a selectivity filter that is better suited for monovalent cations. The high-density of water molecules at the interface between the inner ring of helices TM1 and the outer ring of helices TM2 and TM3 observed in the MD simulation of the wild-type channel might have profound implications on the function of Orai channels, and it is also in agreement with experimental data. In detail, electrophysiological experiments proved that the residues analogous to F171 (F99) and G170 (G98), change orientation with respect to the ion-conducting pore as a result of STIM activation

20

. These experiments were executed using human Orai1-

V102A and introducing cysteine residues at the positions analogous to F171 and G170. The valine at position 102 in human Orai1 corresponds to V174 in the sequence of Drosophila Melanogaster, and when mutated to alanine, it renders a channel that is constitutively open 2+

In the absence of STIM, human Orai-V102A is not selective to Ca the selectivity to calcium ions upon STIM binding

17,18,52

17,18

.

+

over Na , while it recovers

. Therefore, human Orai1-V102A can

be used as a model to investigate how STIM-activation modifies the solvent accessibility of pore residues. These experiments proved that the residues analogous to F171 and G170 switch orientation with respect to the channel pore upon STIM binding: position 171 is sensitive to Cd2+ blockade in the STIM-free state, and it is not in the STIM-activated state; while the opposite behavior is exhibited by the residue at position 170. Despite this coherent movement of residues 170 and 171, both positions are accessible by Ag+ regardless of STIM binding. This experimental observation is readily explained if helices TM1 are surrounded by water molecules on both sides. Moreover, binding of Ag+ at position 170 increases the conductance of the channel in the STIM-activated state. This increase in conductance of a cation-channel is hardly explained if position 170 is exposed to the pore lumen, which suggests that in the STIM-activated state, 16 ACS Paragon Plus Environment

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position 170 is still solvent-accessible but not exposed to the pore. The water molecules at the back of helices TM1 observed in MD simulations might represent this alternative water-route to position 170. Moreover, the movements of residues F171 and G170 upon STIM binding, as well as other residues in the hydrophobic and basic gates, would be hampered by a tight packing of helices TM1, TM2 and TM3. Instead, the water molecules at the back of TM1 helices might act as a lubricant, reducing the energetic cost of channel gating. The experimental data on human Orai1-V102A briefly described in the previous paragraph proves the existence of a link between gating and selectivity, as binding of STIM transforms a cation non-selective channel into a highly selective calcium channel. The same phenotype was observed in other Orai mutants, further confirming that channel opening has an effect on the selectivity filter

20

. However, the molecular details of how gating modifies selectivity are still

blurred. A possible explanation is that the revolving movement observed for the central section of helix TM1 (residues 171 and 170) extends to the entire alpha helix. In this scenario, the opening of the hydrophobic gate is directly related to a structural change in the selectivity filter that boosts selectivity. While it is not possible to exclude this mechanism, it seems unlikely on the basis of the current experimental data. In the experimental structure of Orai, that represents a closed state of the channel, the ring of glutamate residues of the selectivity filter are perfectly directed towards the ion-conducting pore. It is not obvious how the displacement of these glutamate residues away from the channel axis might improve selectivity. On the contrary, the decrease in selectivity observed when glutamate residues are mutated to aspartate suggests that the tight packing of the negative charges of the selectivity filter is essential for the normal functioning of the channel 26. Thus, an outward movement of the glutamate residues, as the one associated with the revolving of helices TM1, is more likely to decrease than to increase selectivity. An alternative hypothesis for the relationship between gating and selectivity is that the interface between helices TM1, TM2 and TM3 implements this link. In agreement with this hypothesis, the computational analyses of the E262Q mutant revealed that reducing the number of water molecules at the back of helices TM1 impairs the selectivity function as a result of the altered dynamics of residues K270. Similar mechanisms might explain the general link between gating and selectivity in Orai channels. It is worth mentioning that a link between the selectivity function and the interface among helices TM1, TM2 and TM3 is in agreement with several 17 ACS Paragon Plus Environment


experimental data. Mutation of a conserved tryptophan residues at the N-terminal of helix TM3 to cysteine (W176 in human Orai1) renders a channel that conducts sustained outward current (transported by monovalent ions) in the absence of STIM 53. In a similar way, Orai3 channels are not selective to calcium ions when activated by 2-aminoethoxydiphenyl borate (2-APB), and the same phenotype can be mimicked in Orai1 channels by mutating a conserved glycine residue along helices TM3 (G183 in human Orai1) 54. The role of the TM1-TM2 interface on gating and selectivity of the Orai channel has also been proposed as being responsible for gating in a recent study by Frischauf et al.

21

, where a series of mutations on helix TM2 were analysed by

experiments and computational models. The hydrogen bonding between TM1 and TM2 was identified as a molecular determinant of channel gating. Moreover, several mutations that change the Ca2+ selectivity of the constitutively open state of Orai were identified, and characterized experimentally. Interestingly, the effect of these mutations on selectivity is strongly correlated to the change in hydrophobicity, with mutations to more hydrophobic amino acids having a more severe impact on selectivity (Figure S11). The link between the hydrophobic scale of the mutation and selectivity is in perfect agreement with the model proposed here. Indeed, the lack of water molecules at the back of the pore resembles the behaviour observed in MD simulation of the E262Q mutant, with the resulting effect on Ca2+ selectivity. These experiments are obviously not a proof that the interface between the inner ring of helices TM1 and the outer ring of helices TM2 and TM3 regulates selectivity with a molecular mechanism similar to the one observed for the E262Q mutant, and further experimental and computational analyses are certainly needed to test the hypothesis that the water molecules at the back of helices TM1 implements the link between gating and selectivity. The simulations of the wild-type channel and of the E262Q mutant presented here offers a first glimpse on this process, which might have further implications on the functioning of CRAC channels.

Acknowledgement A.A and M.R.E would like to thank Prof. Hashem Rafii-Tabar and Dr. Yousef Jamali for the scientific discussions. This work was supported by a CINECA Award under the ISCRA initiative (HP10BKVK3K).


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Supporting Information. Parameters for BD simulations (Table S1); the structure of Orai channel and the atomic model for MD simulations (Figure S1); sequence alignment of Orai from Drosophila Melanogaster with human Orai channels, highlighting the conserved residues (Figure S2); simulation domain for BD simulations (Figure S3); RMSD of different subunits of Orai channel for the first set of simulations (Figure S4); RMSD of different subunits of Orai channel for the second set of simulations (Figure S5); pore profiles for the wild-type and the E262Q mutant channel for the second set of simulations (Figure S6) ; distribution of ions and water molecules in the z- and r- plane (Figure S7); number of water molecules in time for two sets of independent simulations (Figure S8); average and standard deviation of the minimum distance between residues K270 and E178, and between residues K270 and E/Q262 for all the subunits of Orai channel (Figure S9); Ca2+ and Na+ currents in BD simulations with different concentration of CaCl2 (Figure S10); Correlation between the hydrophobic nature of the mutation and the effect on selectivity (Figure S11).



Figure legends Figure 1. Three transmembrane helices (TM1-TM3) of two opposing subunits of the Orai channel are represented using grey cartoons. The pore-facing amino acids are shown on TM1 helices in licorice representation with the exception of E178, which is shown in VDW representation. The pore is lined by a ring of glutamate residues at the extracellular side (E178), followed by three rings of hydrophobic amino acids (V174, F171 and L167), and three rings of positive residues (K163, K159 and R155). Residues E262 and K270 on the TM3 transmembrane helix are also shown in VDW representation. Aspartate residues, D182 and D184, on the TM1TM2 loop are shown in licorice representation. The analogous amino acids in human Orai1 are indicated in parenthesis. Figure 2. The Root Mean Square Deviation (RMSD) of the backbone atoms of residues T144 to V179 (TM1 helices) with respect to the X-ray structure (4HKR) is shown for the wild-type (black line) and the E262Q (blue line) channel. Figure 3. The pore profiles for the wild-type and for the E262Q mutant channel are shown in black and blue lines, respectively. The corresponding location of the pore residues (Cα atoms) are displayed on the left axis, with analogous amino acids in human Orai1 indicated in parenthesis. In order to calculate the radius of the pore, the MD simulations were sampled with a period of 1 ns. Then, for each sample, the axis of the channel was divided in slices with width of 0.25 Å, and the radius was calculated as the minimum distance in the radial direction between the center of the channel in that slice and any protein atom. The continuous lines show the average pore profile over the trajectory. The width of the shaded area corresponds to the distance between the average radius calculated over the first half of the simulations (from 0 to 75 ns) and the second half of the simulations (from 75 to 150 ns). On the left side, TM1 helices from two opposing subunits are shown as cartoons, with the amino acids lining the pore in licorice or VDW (E178) representation. Figure 4. Density of sodium ions (grey map) and position of residues E178, K270 and E262 (Q262) (red, blue and green contour plots, respectively) in wild-type (a) and E262Q mutant (b) channel. Probability histograms were calculated with a bin size equal to 0.25 Å along the z- and radial-axis. For each atomic model (wild-type and mutant channel), histograms were calculated 20 ACS Paragon Plus Environment

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combining data from two independent simulations. The units for the color bars is equal to the number of the species in each bin divided by the volume of the bin in Å3. The position of residues was measured using: atom CD for E178; atom NZ for K270; atom CD for E262 and atom CD for Q262. The analogous amino acids in human Orai1 are indicated in parenthesis. Figure 5. Top and side view of helices TM1, TM2 and TM3 and crevices of water molecules in between. The TM1-TM3 helices are represented in cartoons. The oxygen atoms of water molecules are shown as red spheres for the wild-type (a) and for the E262Q mutant (b) channel. The positions occupied by oxygen atoms in 75 snapshots are superimposed. The snapshots were taken from the second half of the simulated trajectories using a sampling period equal to 1 ns. Water molecules are selected if satisfying the following conditions: (i) below the center of mass of residues V174 and E178 along the z-axis; (ii) above the center of mass of residue K159 along the z-axis; and (iii) within 10 Å of residues T144-E178. The light-blue arrows highlight the different hydration pattern observed for the wild-type and for the mutant channel at the back of the pore (ring defined by light-blue dashed lines in top panels). Figure 6. Water crevices in the wild-type channel. In the snapshot depicted in panel (a), the configuration of the hydrophobic residues allows the permeation of a continuous layer of water molecules between the intracellular and the extracellular side of the channel; while in panel (b), the water layer at the back of the pore is interrupted by residues F259 and L168. Helices TM1, TM2 and TM3 of one chain are shown in cartoon representation. Water molecules among TM1, TM2 and TM3, and residues E262, L168, L202 and F259 are shown as VDW spheres. The analogous amino acids in human Orai1 are indicated in parenthesis. Figure 7. Possible configurations of residues K270 observed in the MD trajectories of the wildtype and the mutant channel are shown: (a) side chain of K270 pointing to the protein core, in the wild-type channel; (b) side chain of K270 exposed to the water-solution at the extracellular side of the channel, in close proximity of residues E178, in the wild-type channel; (c) side chain of K270 exposed to the water-solution at the extracellular side of the channel, in close proximity of residues E178, in the E262Q mutant channel. Fragments of helices TM1, TM3, and TM3 of the adjacent chain are shown in cartoon representation. Residues E178, E262, and K270 of the adjacent chain are shown using VDW spheres. The analogous amino acids in human Orai1 are indicated in parenthesis. 21 ACS Paragon Plus Environment


Figure 8. Ca2+ and Na+ currents in BD simulations with increasing positive charges at the extracellular entrance of the channel. Values along the x-axis correspond to the partial electric charge assigned to each of the six positive particles placed at the extracellular entrance of the channel. The charge ranged from 0 (no positive charge at the extracellular entrance) to 1 (six elementary charges at the extracellular entrance) in steps of 0.1. A schematic representation of the channel-model from the extracellular side is shown below the x-axis, with the mobile particles of the selectivity filter in red, and the positive charges turning from white to blue. NaCl and CaCl2 concentrations were set to 100 mM. Membrane potential was set to -100 mV. For each value of the positive charge, the currents were calculated using a BD trajectory of 100 µs.


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TOC Graphic


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Figure 1

ACS Paragon Plus Environment


Figure 2


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Figure 3



Figure 4


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Figure 5



Figure 6


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Figure 7



Figure 8 146x110mm (300 x 300 DPI)


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Molecular Dynamics Simulations of Orai Reveal How the Third

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