Dielectric Allostery of Protein: Response of Myosin ... - ACS Publications

Nov 7, 2016 - ABSTRACT: Protein uses allostery to execute biological function. ... ATP binding which is a crucial step in force-generating function, f...
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Dielectric Allostery of Protein: Response of Myosin to ATP Binding Takato Sato, Jun Ohnuki, and Mitsunori Takano* Department of Pure and Applied Physics, Waseda University, Okubo 3-4-1, Shinjuku-Ku, Tokyo 169-8555, Japan S Supporting Information *

ABSTRACT: Protein uses allostery to execute biological function. The physical mechanism underlying the allostery has long been studied, with the focus on the mechanical response by ligand binding. Here, we highlight the electrostatic response, presenting an idea of “dielectric allostery”. We conducted molecular dynamics simulations of myosin, a motor protein with allostery, and analyzed the response to ATP binding which is a crucial step in force-generating function, forcing myosin to unbind from the actin filament. We found that the net negative charge of ATP causes a large-scale, anisotropic dielectric response in myosin, altering the electrostatic potential in the distant actin-binding region and accordingly retracting a positively charged actinbinding loop. A large-scale rearrangement of electrostatic bond network was found to occur upon ATP binding. Since proteins are dielectric and ligands are charged/polar in general, the dielectric allostery might underlie a wide spectrum of functions by proteins.

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INTRODUCTION It is generally understood that a protein molecule as an enzyme provides a binding site that is sterically complementary to the substrate molecule to be catalyzed, achieving stereospecificity for the substrate.1 If the binding affinity of the protein to the substrate is altered by the presence of another molecule (ligand) that is sterically different from the substrate, the protein is thought to possess the property of “allostery”.2 The allosteric property, therefore, implies that the binding site of the ligand is spatially different from that of the substrate. It is not unusual that the ligand and the substrate binding sites are distantly positioned: for example, in molecular motor dynein, the microtubule binding site is separated from the ATP-binding site by more than 10 nm.3 Then, what is interesting and has been actively studied for half a century is the molecular mechanism of how the physicochemical signal of the ligand binding is transmitted to the distant site in the protein molecule.4,5 The classic mechanism of allostery is explained by ligandbinding-induced large-scale sequential motions of rigid elements (e.g., helices and domains), as was first proposed for the allosteric mechanism of hemoglobin.4 Recently, another mechanism based on the intrinsic flexibility of protein has attracted much attention.5−8 For example, binding of cAMP to a transcription factor was shown to alter the fluctuation of a distant binding region for another cAMP in this DNA-binding protein.9 The fluctuation change at the binding region alters the binding affinity to the target molecule. This fluctuation-based allosteric mechanism is different from the classic one because it does not require such structural changes as assumed in the classic mechanism. The above-mentioned allosteric mechanisms, however, are based solely on the mechanical property of a protein. It is worthwhile to remember that a protein molecule possesses not © XXXX American Chemical Society

only the mechanical property but also the dielectric property (i.e., atomic, dipolar, and ionic polarizability) in general, and that ligand and substrate molecules are usually polar and often carry electric charges: ATP and cAMP are polar and negatively charged, and so are microtubule and DNA. Therefore, it can be thought that the electrostatic signal of the ligand/substrate binding is transmitted to a distant region of the protein via a long-range dielectric response. Indeed, the role of the electrostatics in allostery was recognized early by Perutz.4,10 The dielectric property of protein was considered in the seminal work by Warshel and Levitt,11 and has been extensively studied since then: notably, Simonson et al. studied the heterogeneity of the dielectric constants within a protein and the dielectric response to charge perturbation.12,13 However, in the context of allostery, little attention has been paid to the dielectric property so far. Myosin is a well-known motor protein where allostery is utilized for force-generating function.14,15 The force generation cycle is coupled with the cycle of the ATP hydrolysis that is catalyzed by myosin;16 during the hydrolysis of a single ATP molecule, myosin unbinds from actin and again binds to actin. Unbinding from actin is caused by the binding of ATP to myosin,16 which was recognized early through the study of the “clearing response” that the reconstituted actomyosin exhibits upon ATP addition.17−19 The myosin dissociation from actin plays an important role in the force generation.14−16,20 Particularly, it is important for myosin to be raised to the high-energy, dynamically disordered state upon ATP binding, as was demonstrated by EPR measurement;20 the initial stage of the force generation occurs in the course of rebinding of Received: October 3, 2016 Revised: November 1, 2016 Published: November 7, 2016 A

DOI: 10.1021/acs.jpcb.6b10003 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B myosin to actin,20,21 in which myosin undergoes disorder-toorder transition20 and the actin−myosin interaction energy is gradually decreased.22 Because the ATP-binding region is wellseparated from the actin-binding region (by more than 30 Å), the allosteric mechanism should exist in the ATP-bindinginduced dissociation from actin. Structural studies suggested that the actin-binding cleft is opened upon ATP binding via a leverage-like motion of the actin-binding domains,23−25 assuming the classic allosteric mechanism as mentioned above. Spectroscopic studies presented the results that are consistent with the ATP-binding-induced opening of the actinbinding cleft.26−28 It is expected that this cleft-opening spoils the stereospecific interaction between myosin and actin,23,24 which leads myosin to dissociate from actin. In addition to this mechanical response of the cleft, given that myosin is dielectric and ATP is charged, together with our previous computational study indicating the involvement of the actin-myosin electrostatic interaction in the force generation,22,29 it is also likely that the dielectric response of myosin contributes to the allosteric mechanism behind the ATP-binding-induced dissociation from actin. In this study, based on the computational results of molecular dynamics simulation of myosin as a representative allosteric molecular machine, we demonstrate that myosin exhibits a substantial dielectric response to the ATP binding, which leads us to the novel idea of “dielectric allostery”. We also show that the dielectric allostery is likely to contribute to the physical mechanism of the ATP-induced dissociation from actin, which raises our expectation that the dielectric allostery may be utilized by a wide range of molecular machines.

During the minimization, the heavy atoms in ADP and magnesium were positionally restrained by applying restoring forces (force constant of 5 kcal/mol/Å2). Atomic (partial) charges for ADP and magnesium were eliminated at this stage. In the second step, after superimposing the ADP-VO4-bound structure on to the ADP-implanted energy-minimized structure (superposition was done in the same way as described above), VO4 was implanted into the ADP-bound energy-minimized structure as PO4 (the vanadium atom was simply renamed as the phosphorus atom). Then, by treating ADP-PO4 as a covalently bonded single ATP molecule, energy minimization was conducted in the presence of the positional restraints applied to the heavy atoms in the newly created ATP molecule and magnesium (force constant of 5 kcal/mol/Å2). This twostep ATP-implantation procedure was applied to the eight ATP-free structures. The minimization and the subsequent MD simulation were carried out by using AMBER38 and the ff03 force field.39 For ATP and ADP, the parameters by Meagher et al.40 were used. Note that the charges for ATP and magnesium were kept eliminated at this stage and at the subsequent initial stage of preparation MD. The ATP-free and ATP-bound myosin structures thus prepared were then immersed in a truncated octahedral cell containing 33000 TIP3P41 water molecules and 50 mM KCl, respectively, resulting in a 111400-atom system. The periodic boundary condition was applied to the system, and the particle mesh Ewald method was employed with the direct space cutoff of 8 Å. The system was energy minimized first in the presence of the positional restraints applied to the heavy atoms of myosin and MgATP (force constant of 500 kcal/mol/Å2) and second in the absence of the restraints. Next, the system was heated up to 310 K (the Langevin thermostat42 was used with the collision frequency of 1 ps−1; the volume of the system was kept constant) by applying the positional restraints to the heavy atoms of myosin and MgATP [the force constant was gradually decreased in four consecutive 10 ps periods (10, 5, 2.5, and 1.25 kcal/mol/Å2)]. Then the volume of the system was relaxed for 1 ns at 1 bar without restraints (the Berendsen barostat43 was used with the pressure relaxation time of 50 ps). After the volume relaxation was finished, we turned on all of the atomic charges of ATP and magnesium for the ATP-bound structure. By conducting 1.6-μs MD simulation at 1 bar (the pressure relaxation time was set at 5 ps) and 310 K for each of the ATPfree and the ATP-bound states (i.e., eight independent 1.6-μs simulations were conducted for each state), we observed the myosin’s response to the bound ATP. The time step was set at 2 fs by fixing the bonds involving hydrogen atoms. Electrostatic Potential. Electrostatic potential, denoted as Φ, was obtained by numerically solving the Poisson− Boltzmann equation using APBS;44 the standard finite difference method was applied to the linearized Poisson− Boltzmann equation under the single Debye−Hückel boundary condition, with the grid-spacing of 3 Å, the interior and exterior dielectric constants of 1 and 78.5, respectively, the salt concentration at 50 mM, temperature at 310 K, and the solvent radius of 1.6 Å. Φ was calculated for each snapshot structure of myosin extracted from the MD trajectories at 1 ns intervals during the last 0.6 μs (1.0−1.6 μs). Then Φ was averaged over the calculated 4800 snapshot potentials for the ATP-free state and those for the ATP-bound state, respectively. The averaged Φ for the ATP-free state is denoted as ⟨ΦMF⟩ and that for the ATP-bound state as ⟨ΦMA⟩. Before averaging, translational and rotational movements and domain motions of

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MATERIALS AND METHODS Molecular Dynamics Simulation. As the initial structure for MD simulation, we used the crystal structure of scallop striated muscle myosin II in the nucleotide-free state (PDB ID: 1SR6).30 This structure is considered to be in the “post rigor” state31 (“rigor” means the strong actin-binding, and the post rigor state corresponds to the state just after ATP binding that causes the transition from the strong to weak actin-binding states). We used a minimum-sized myosin (C-terminal residues after E776 were removed) that retains the force-generating function.32 Missing residues (residues 1−5, 201−212, 627− 642, and 731−733) were complemented by MODELLER,33 and eight structures with different loop 2 conformations were adopted (note that residues 627−642 constitute the major part of loop 2). H357, H581, H664, and H689 were considered to be protonated according to the evaluation by ProPKA34 and H ++.35 The ATP-bound structure was prepared by implanting ligands in the ADP-VO4 (ADP-Pi analogue)-bound crystalline structure (PDB ID: 1QVI)36 into the ATP-free structure that was modeled as mentioned above. The preparation was conducted in a stepwise manner as employed in the study by Ovchinnikov et al.37 In the first step, after superimposing the ADP-VO4 bound structure onto the ATP-free structure, ADP and magnesium, which are observed in the ATP-binding site of the crystal structure,36 were implanted into the ATP-free structure. The superposition was conducted by best-fitting the ATP-binding region (residues 175−182, 230−242, and 460− 471) of the two structures. The ADP-implanted structure was then energy minimized (1000-step steepest-descent minimization followed by 1000-step conjugate-gradient minimization, which was applied to the subsequent energy minimizations). B

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The Journal of Physical Chemistry B myosin were removed by superimposing each snapshot structure onto the reference structure (mentioned below) on a domain basis. For this domain-based superposition (Cα atom superposition), we divided myosin into four domains in the same way as employed by Ovchinnikov et al.37 (i.e., N-terminal domain, 6−201 and 665−695; upper 50 kDa domain, 213− 358, 375−401, 413−471, and 605−625; lower 50 kDa domain, 472−563, 578−604, and 643−664; and converter domain, 696−775). Note that the domain-based superposition before averaging Φ can eliminate the false detection of Δ⟨Φ⟩ due to translational or rotational motion of the domain and can extract Δ⟨Φ⟩ due to the dielectric response that occurs inside the domain. Note also that the relatively large grid spacing of 3 Å was sufficient for obtaining the averaged potential from a large number of snapshot structures with thermal fluctuations (we confirmed that smaller grid-spacing of 1 Å yields the same results within statistical errors). The use of the large number of snapshot structures that were thermally equilibrated also allows us to explicitly take into account the dipolar and the ionic polarizations in myosin [the dielectric response due to the atomic polarization, which was not considered in this study (the dielectric constant of unity was used for protein interior), is generally smaller than that due to the dipolar and ionic polarizations11−13]. Note also that the atomic charges in ATP and magnesium were eliminated in calculating Φ for the ATPbound state to observe the response of myosin; the electrostatic potential by ATP and magnesium was instead examined separately. The electrostatic potentials coming from highly flexible regions on the surface [i.e., loop 1 (residues 202−212), loop 2 (626−642), loop 3 (564−577), loop 4 (359−374), cardiomyopathy loop (402−412), and N-terminus (1−5)] were also removed. Then the myosin’s electrostatic potential response to the bound ATP, Δ⟨Φ⟩, was obtained by subtracting ⟨ΦMF⟩ from ⟨ΦMA⟩, i.e., Δ⟨Φ⟩ = ⟨ΦMA⟩ − ⟨ΦMF⟩. The false positive responses were eliminated on the basis of the statistical errors:

Figure 1. (a) Electrostatic potential in the ATP-free state ⟨ΦMF⟩ on the surface of myosin. The electrostatic potentials due to ATP and surface loops (loops 1, 2, 3, and 4, and cardiomyopathy loop) were excluded. (b) Electrostatic potential change Δ⟨Φ⟩ upon ATP binding. (c) Δ⟨Φ⟩ inside myosin seen on a cross-sectional surface; the cutting plane (green) contains ATP, E263, and the acidic cluster region.

K635/K636/K638). The response of loop 2 upon ATP binding, Δ⟨ρ⟩, was obtained by subtracting ⟨ρF⟩ from ⟨ρA⟩. The false positive responses were eliminated in rendering the figures in the same way as employed for the electrostatic potential analysis. Note that in addition the reference structure used for the domain-based superimposition in averaging the snapshot electrostatic potentials (see above) is the average structure of the 48000 snapshot structures obtained here for the ATP-free state. Electrostatic Bond Network. The rearrangement of the electrostatic bond network was analyzed in the same way as in our previous work.47 An electrostatic bond (ionic or hydrogen bond between charged and polar groups), which we refer to as “EB”, was judged formed when the distance between H and O (or H and N) is less than 3 Å. Using the snapshot structures for the ATP-free state extracted from the MD trajectories at 0.1 ns intervals during the last 0.6 μs (1.0−1.6 μs), the average number of EBs, ⟨nF⟩, was calculated for each bond-forming residue pair. Likewise, the average number of EBs for the ATPbound state, ⟨nA⟩, was calculated. We then extracted EBs that showed substantial change in response to ATP binding, i.e.,

i.e., response with |Δ⟨Φ⟩| < δ ΦMA 2 + δ ΦMF 2 was considered as false positive and was excluded in rendering Figure 1. δΦ denotes the standard error at the 95% confidence interval and was calculated by using bin-averages (bin-width is 0.1 μs) for each of eight independent trajectories (thus, the number of bin-averages for estimating the standard error is 48). The resulting Δ⟨Φ⟩ was mapped on the reference structure and rendered by Chimera.45 Spatial Distribution of Actin-Binding Loop. To determine the position and orientation of myosin relative to the actin filament, we used the recent cryo-EM structure of the actin−myosin complex.46 First, the amino-acid sequence of human nonmuscle myosin IIc46 was replaced by that of scallop striated muscle myosin II using MODELLER33 (except for the missing N-terminal domain). Then a snapshot structure was superimposed onto this modeled structure with the scallop sequence; least-squares fit was done locally with respect to the residues in the upper and lower actin-binding domains (residues 215−242, 268−446, 513−555, and 601−625).23,24 This superimposition was conducted for all of the snapshot structures extracted from the MD trajectories at 0.1 ns intervals during the last 0.6 μs (1.0−1.6 μs). Using those superimposed snapshots (48000 snapshots for each of the ATP-free and the ATP-bound states), the spatial distribution (i.e., occurrence probability density) of loop 2 was calculated, with loop 2 represented by the Nζ atoms of five lysines (K633/K634/

those satisfying |Δ⟨n⟩| > max(0.05, δnA 2 + δnF 2 ), with Δ⟨n⟩ = ⟨nA⟩ − ⟨nF⟩ and δnA and δnF being the standard errors at the 68% confidence interval for the ATP-bound and the ATP-free states, respectively (the number of averages for estimating the standard error is 48). We showed those EBs with the magnitude of the response (i.e., |Δ⟨n⟩|).

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RESULTS Dielectric Response to ATP binding. To explore the possibility that the dielectric response of myosin may contribute to the allosteric mechanism behind the ATPbinding-induced unbinding from actin, we first examined the electrostatic potential change upon ATP binding. Figure 1a shows the electrostatic potential on the surface of myosin in the ATP-free (unbound) state. The potential was averaged using 4800 snapshots extracted from the MD data at 1 ns intervals, which is denoted as ⟨Φ⟩. Myosin has two actin-binding

C

DOI: 10.1021/acs.jpcb.6b10003 J. Phys. Chem. B XXXX, XXX, XXX−XXX

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The Journal of Physical Chemistry B domains, the upper and lower 50 kDa domains,14,15,23 with the lower domain offering the major actin-binding interface.23 Figure 1a shows that the actin-binding region in the lower domain presented highly negative potential. Note that this region contains a cluster of acidic residues (E534/E535/E536) that are highly conserved and was shown to play an important role in the actin binding48 (we refer to this cluster as the “acidic cluster”). Then, we calculated the difference of the electrostatic potential, Δ⟨Φ⟩, between the ATP-free and ATP-bound states, as shown in Figure 1b. Since the same initial structure30 was employed for both states, it is obvious that the observed Δ⟨Φ⟩ is due to the myosin’s response to the bound ATP. Figure 1b shows that myosin exhibited large potential changes on the surface in response to ATP binding, with |Δ⟨Φ⟩| larger than kT/e (kT represents the thermal energy at 310 K, and e is the elementary charge). Focusing on the actin-binding region, we can recognize the positive potential change in the acidic cluster region mentioned just above. Negative potential change observed at the “strut”49,50 in the actin-binding cleft is also prominent (the positively changed region in the actin-binding cleft is situated deep inside the cleft). Weak negative Δ⟨Φ⟩ can be recognized at around E263 which is adjacent to the actinbinding region. We then studied how these surface potential changes were induced by the bound ATP. Since the direct electrostatic potentials by ATP and magnesium were excluded in calculating the potential (and the effect of the domain motion was also eliminated by applying domain-based fitting before averaging), the potential changes shown in Figure 1b should be simply due to the charge redistribution in myosin that occurred upon ATP binding. Actually, large-scale charge redistribution was observed in myosin, and the charge redistribution, denoted as Δ⟨q⟩, correlates well with Δ⟨Φ⟩ inside myosin (Figure S1). Furthermore, the cross-sectional image of Δ⟨Φ⟩ (Figure 1c) makes it clear that the addition of the net negative charge of ATP (ATP4− and accompanying Mg2+) caused the attraction of the surrounding positive charges, which propagated through the interior of myosin and finally reached the surface. Thus, in the same way as dielectric materials, myosin exhibited a “dielectric response” to the electric charge of ATP. The electrostatic potential on the surface directly due to the electric charge of ATP (Figure 2), ⟨ΦATP⟩, was smaller than that due to the induced surface charges of myosin Δ⟨Φ⟩. Response of Actin-Binding Cleft. It is important whether or not the observed dielectric response has relevance to molecular function; in the present case of myosin, the important function is the ATP-binding-induced dissociation from actin. Since it is widely held that ATP binding causes opening of the actin-binding cleft, which disrupts the stereospecific myosin−actin interaction and drives the dissociation,23−28 we first examined the response of cleft. We paid attention to the domain-level motion between the two actinbinding domains, as inferred from the structural model,23−25 so we calculated the distance between the geometric centers of the upper and the lower actin-binding domains. The calculated interdomain distance showed large thermal fluctuations, as seen in the wide distribution in Figure 3. Then, from the difference in the distributions of the ATP-bound and ATP-free states, we can recognize the tendency that the cleft is opened in the ATP-bound state. Note again that we employed the same initial structure30 for both states, so the observed cleftopening is due to the effect of the bound ATP. Note also that the employed initial structure (the corresponding distance is

Figure 2. (a) Electrostatic potential due to the charges of ATP (ATP4− and Mg2+), ⟨ΦATP⟩ (= ⟨ΦMF + ΦATP⟩ − ⟨ΦMF⟩), on the surface. The view angle is the same as in Figure 1a,b. (b) ⟨ΦATP⟩ inside myosin seen on the same cross-sectional surface as in Figure 1c.

Figure 3. Probability distribution of the width of the actin-binding cleft as measured by the distance between the geometric centers of the Cα atoms of the upper and the lower actin-binding domains.

32.5 Å) is considered to be in the post rigor state where the cleft is opened,31 so the result in Figure 3 suggests that the cleft becomes more opened in aqueous solution in the presence of the bound ATP (three trajectories contributed mainly to the distribution around 36 Å). In addition, a small population at ∼29 Å in the ATP-free distribution indicates the closure of the cleft in the absence of ATP. Later, we will see the involvement of the dielectric response in the cleft-opening, and also discuss the dynamical aspect of this response of the cleft.28 Response of ATP-Binding Loop (Loop 2). Loop 2 is a major actin-binding loop51,52 and positively charged due to a high content of basic residues (mainly lysines). The electrostatic interaction between loop 2 and the acidic residues in actin has been shown to be involved in the actin−myosin complex.23,25,46,51−56 Our previous computational studies have also elucidated the importance of the electrostatic interaction involving loop 2 in the force generation.22,29,57 Since loop 2 is located in the actin-binding region, it is expected that loop 2 responds to the surface potential change as observed in Figure 1b. As shown in Figure 4a, loop 2 is so mobile and widely distributed in the actin-binding region. By taking the difference between the distributions of the ATP-bound and ATP-free states, it was revealed that loop 2 actually responded to the surface potential change (Figure 4b); loop 2 tended to be D

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Figure 4. (a) Spatial distribution (occurrence probability density) of loop 2 as represented by the Nζ atoms of five lysines (K633−K636, K638). The distribution in the ATP-free state, ⟨ρF⟩, is displayed (contour surface at ⟨ρF⟩ = 0.05 nm−3). The position of actin in the complex46 is depicted for reference (gray). (b) Change of ρ upon ATP binding. Contour surface at ⟨Δρ⟩ = +0.05 is shown in blue and that at ⟨Δρ⟩ = −0.05 in red. (c) Probability distribution of the distance between loop 2 (geometrical center of the five lysines) and E534, (d) that between loop 2 and D600 in the strut, and (e) that between loop 2 and E263.

repelled from the acidic cluster region (Figure 4c) where Δ⟨Φ⟩ was positive, and instead pulled toward the strut (Figure 4d) where Δ⟨Φ⟩ was negative, and also pulled toward E263 (Figure 4e) where the negative potential change due to Δ⟨Φ⟩ was augmented by ⟨ΦATP⟩ (Figure 2). Since the acidic cluster region of myosin is located near the acidic residues in actin (particularly in the N-terminus),23,25,46,53−56 it seems to be possible that the positive charges in loop 2 act as the electrostatic bridge between the negative charges in the acidic cluster of myosin and the same negative charges of actin. It is noteworthy that the response of loop 2 was in a direction away from the actin (Figure S2). Rearrangement of Electrostatic Bonds Network and Dielectric Pathway. The charge redistribution in myosin, Δ⟨q⟩, that occurred upon ATP binding (Figure S1) indicates that the intramolecular network of the electrostatic bonds (referred to as “EB”), such as ionic and hydrogen bonds between charged and polar groups, underwent a large-scale rearrangement in response to ATP binding. In the previous work, we observed such a large-scale rearrangement of the EB network in myosin in response to the mechanical input applied to the converter domain (lower-left domain in Figure 2a).47 In the present study, to address the response to the electrostatic input of ATP binding, we analyzed the rearrangement of EB network. As expected, we found that the EB network was extensively and concertedly rearranged in the region where large electrostatic potential changes were observed (Figure 5). Highly conserved residues were found to be involved in this rearrangement network. In particular, E534 in the acidic cluster48 and D600 in the strut50 were involved in this network. E534 strengthened EBs with neighboring residues including R650 (R650 is often substituted by lysine and was shown to interact with actin46,58,59). This strengthened EB between E534 and R650 was found to contribute largely to the positive Δ⟨Φ⟩ in the acidic cluster region (Figure 1b). D600 exhibited a large response, as indicated by the significantly weakened D600−

Figure 5. Rearrangements of electrostatic bonds that occurred upon ATP binding. Bonds are drawn between the key residues, with the strengthened and weakened bonds shown in blue and red, respectively. The thickness of the bonds reflects the magnitude of the response. (a) Rearrangement network involving E534, D600, and E263, and (b) that involving R242, E465, and D600. Charged residues involved in the network are shown.

K269 bond (see the thick red bond between D600 and K269). The D600−K428 bond was also weakened. Accordingly, D600 became more exposed to solvent; solvent accessible surface area of D600 was increased by 11 Å2. This response of D600 is consistent with the observed negative potential change in the strut (Figure 1b). Furthermore, the rearrangement network shows that D600 was connected to R242, which is the essential residue in the ATP-binding region, located close to the γphosphate of ATP; indeed, we found R242 formed EB with the γ-phosphate of ATP. E263, though not so highly conserved, E

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by von der Ecken et al. (R663 in nonmuscle myosin IIc).46 In our study, it was observed that loop 2 moves in the direction away from actin in response to ATP binding (Figure S2). This response of loop 2 therefore could contribute to weakening of the binding affinity with actin. We note that the ATP-bindinginduced response of loop 2 was actually observed in myosin V by cryo-EM25 (loop 2 of myosin V is much longer than that of myosin II studied here). In addition, since it has been suggested that loop 2 interacts with the strut in the actin-binding cleft,46,50 the response of loop 2 and that of the actin-binding cleft might be correlated and work cooperatively for the ATP-bindinginduced dissociation. Pathways of the Dielectric Response. We showed that the movement of loop 2 was caused by the surface potential change, and the surface potential change was caused by the ATP-binding-induced dielectric response, i.e., the large-scale rearrangement of the electrostatic bond (EB) network in myosin. In the EB rearrangement network, the detailed pathways of the dielectric response can be identified (Figure 5). Importantly, we can recognize the pathways that connect the ATP-binding region and the acidic cluster region in the lower actin-binding domain. One of the most probable dielectric response pathways seen in Figure 5b is as follows: First, the γ-phosphate of ATP formed an electrostatic bond with R242 (not depicted in Figure 5b). Second, the R242− E268 bond was weakened. Third, the E268−K269 bond was strengthened. Fourth, K269−D600 was weakened; and last, the pathway extended to E534 in the lower actin-binding domain via EBs involving D600 and neighboring residues (e.g., K599). It is noteworthy that the K269−D600 bond, the response of which was significant, has been identified as a key EB in the structural study by Yang et al.31 where breaking and reforming of EBs upon cleft-opening was discussed. Breaking of EBs in the cleft region was observed in the restrained targeted MD simulation of myosin V.37 In Figure 5a, another dielectric response pathway from the ATP-binding region to the acidic cluster region can be recognized as the pathway via R248 (adjacent to the ATP-binding region), E263, and K655, which appears to extend to R650 and further to E534 (R650−E534 bond would also contribute to the reduction of the binding affinity with actin, because R650 was shown to form EB with the acidic residues of actin46,58,59). We note that the pathway from the ATP-binding region to E534 was also predicted from the analysis of conserved residues in myosin.63 Regarding the ATP-binding-induced response of myosin, there have been arguments on the roles of switch I and switch II loops.14,15 Since R242 is in the switch I loop, the dielectric response pathway we identified above indicates that switch I is actually involved in the ATP-binding-induced response of myosin that should lead to the myosin−actin dissociation, which is in agreement with the general understanding.14,15,24,30,31,37,57,60,61 Figure 5b further shows that R242− E465 bond, known as the “back door”,14,15,64 was weakened upon ATP binding, as observed by an earlier MD study by Lawson et al.64 E465 is in the switch II loop, and it appears that the response of E465 was extended to the actin-binding cleft. Therefore, switch II is likely to play a role in the ATP-bindinginduced dissociation from actin, which is also consistent with the experimental and theoretical studies.37,65 In addition to the opening of the actin-binding cleft, ATP binding was shown to induce the bending of the relay helix,66,67 which leads to the swinging back of the converter domain (switch II is considered to play a crucial role in this response14,15). Therefore, it is also

weakened EBs with K655 and R440, yielding a weak negative potential shift (Figure 1b) that contributed to pull loop 2. We thus revealed that the large-scale EB rearrangement network underlies the observed dielectric response that myosin exhibited upon ATP binding. It is expected that this rearrangement network bears close relevance to the allosteric pathways. In the following, we will discuss the observed dielectric response from both the myosin-specific and more generic viewpoints of allostery in the light of related studies.

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DISCUSSION ATP-Binding-Induced Cleft-Opening. The original structural model of the actomyosin complex by Rayment et al.23 suggested that the ATP-binding-induced opening of the actinbinding cleft causes myosin to dissociate from actin, which has been reinforced by cryo-EM,24,25 fluorescence spectroscopy,26,27 and EPR.28 It is important to note that our MD simulation also captured the tendency that the cleft is opened in response to the bound ATP, in accordance with the experimental studies.25−28 An EPR study by Klein et al.28 further indicated that the cleft is so dynamic that the coupling between the structural state of the cleft (opening or closing) is not tightly coupled with the chemical state of the ATP-binding site, in contrast to the tight coupling as assumed previously.16 Our study is consistent with this EPR study; as seen in Figure 3, the cleft showed considerable fluctuations both in the ATPbound and ATP-free states, and the two distributions substantially overlapped with each other (in addition, there appears to be intermediate states between fully open and fully closed states). Our study is also in line with the previous computational studies37,57,58,60,61 which indicated that the cleft is opened in the ATP-bound state. It should be noted that the cleft-opening observed in this study is due to the myosin’s natural response to the bound ATP, which is the consequence of having conducted plain (conventional) MD simulations on the microsecond time scale, whereas in the previous studies, the two end-point structures (cleft open and closed structures) were assumed57,58,61 or approximations were applied to the myosin’s natural dynamics.37,60 Mechanism for the ATP-Binding-Induced Dissociation. The ability of myosin for strong actin-binding is lost when the cleft is kept opened by inserting an artificial strut (crosslinker) in the cleft62 or introducing an insertion mutation in the strut.50 These experimental results strongly suggest that the opening of the actin-binding cleft is physically essential for myosin to dissociate from actin. In addition to the observed cleft-opening (Figure 3), we found that loop 2 exhibits remarkable response to the ATP binding (Figure 4). Since loop 2 is known to be involved in the interaction with actin,23,25,46,51−56 it is likely that the observed response of loop 2 contributes to the ATP-binding-induced dissociation, in addition to the cleft-opening. In the absence of the bound ATP, loop 2 was located in the vicinity of the highly conserved acidic cluster48 in the lower actin-binding domain (Figure 4). Furch et al.48 showed that the negative charge in this acidic cluster region enhances the binding affinity with actin, which is apparently against our expectation because actin is also negatively charged. While the positively charged local region in actin may interact with the negative charges in the acidic cluster,46 it can also be considered that positively charged basic residues in loop 2 act as an electrostatic bridge between the negatively charged residues of myosin and those of actin: in fact, such a bridge can be found in the recent cryo-EM structure F

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highly important to examine the response of the converter and analyze the dielectric response pathways. In the present study, however, we focus on the responses of the actin-binding region and the response pathways to the actin-binding region. We will address the ATP-binding-induced response of the converter and the dielectric response pathways separately in future work (preliminary analysis indicates the converter response consistent with the experimental observation66,67). “Dielectric Allostery”. The concerted response of the EB network we detailed just above is typical of the response of a dielectric material: an electric charge placed in a dielectric material causes concerted responses of constituent dipoles, which eventually induces polarization charges on a distant surface. At the same time, the dielectric response observed in myosin is nothing other than an allosteric response. Therefore, this kind of dielectric response in protein may as well be called “dielectric allostery”. Even though the dielectric response and the allosteric response have long been studied as important subjects of biophysical chemistry, there has been no study that highlights the connection between the two. We believe that presenting the concept of the dielectric allostery would be the important role of this study from a generic point of view of biophysical chemistry. The molecular mechanism of the dielectric allostery is essentially different from that of the classic allostery4,5 and instead bears some resemblance to the fluctuation-based allostery;5−9 the dielectric allostery does not require largescale mechanical movements of rigid elements, and is based on the dipolar and ionic polarizations that require thermal fluctuations (note that the atomic polarization, which was not considered in this study, is generally smaller than the other two11−13). The dielectric allostery, together with the piezoelectric allostery that we recently proposed,47 highlights the importance of the electrostatic aspect of the allostery, and should add a new perspective to the physical mechanisms of allostery. While we have shown the consistency between our computational results and experimental results in the literature, more direct experimental validation of the dielectric allostery, particularly measurement of the surface potential change upon binding of ATP (ATP analogues or pyrophosphate), would be needed. To this end, site-specific EPR measurement using charged paramagnetic relaxation agents68 would be one of the powerful tools. Finally, it is noteworthy that the triphosphate moiety of ATP frequently acts as the trigger of allosteric regulation when ATP is used as an allosteric modulator.69

Article

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b10003. Correlation plot between the net-charge density change and the electrostatic potential change upon ATP binding, and the probability distribution of the distance between loop 2 and the N-terminal acidic residues of actin (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Takato Sato: 0000-0002-8510-978X Jun Ohnuki: 0000-0002-7351-3427 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Drs. Koji Umezawa, Taro Q. P. Uyeda, and Makoto Suzuki for valuable discussions. This work was supported by Grants-in-Aid for Scientific Research by MEXT (Japan).

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REFERENCES

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CONCLUSIONS Our computational study indicated that myosin exhibits substantial dielectric response to the binding of ATP, from which the idea of “dielectric allostery” was derived. The dielectric allostery should advance the understanding of how molecular machines execute and regulate their functions, and also contribute to the fundamental understanding of the physical mechanisms of allostery. In the present case of myosin, we showed that the dielectric allostery, in addition to the conventional (mechanical) allostery, is likely to play an important role in the ATP-binding-induced dissociation from actin. We expect that the dielectric allostery found in myosin would be utilized in a wide range of allosteric proteins with various biochemical functions, ranging from other molecular motors that use ATP/GTP to the signal processing molecular machines that use electric charges (cAMP, GTP, calcium ions, phosphorylation, etc.) as the signals for regulation. G

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