Na+ Binding Is Ineffective in Forming a Primary ... - ACS Publications

Oct 26, 2016 - Thrombin is a serine protease involved in the blood coagulation reaction, and it shows maximum enzymatic activity in the presence of Na...
0 downloads 0 Views 3MB Size
Download PDF
Article pubs.acs.org/JPCB

Na+ Binding Is Ineffective in Forming a Primary Substrate Pocket of Thrombin Ikuo Kurisaki†,‡ and Masataka Nagaoka*,†,‡ †

Graduate School of Information Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, Honmachi, Kawaguchi 332-0012, Japan

‡

S Supporting Information *

ABSTRACT: Thrombin is a serine protease involved in the blood coagulation reaction, and it shows maximum enzymatic activity in the presence of Na+. It has been supposed that Na+ binding promotes conversion from the inactive form, with a collapsed primary substrate pocket (S1 pocket), to the active form, with a properly formed S1 pocket. However, the evidence supporting this activation mechanism was derived from the X-ray crystallographic structures solved under nonphysiological conditions and using thrombin mutants; thus, it still remains elusive whether the activation mechanism is actually attributed to Na+ binding. To address the problem, we employed all-atom molecular dynamics simulations for both active and inactive forms of thrombin in the presence and absence of Na+ binding and examined the effect of Na+ binding on S1-pocket formation. In contrast to the conventional supposition, we revealed that Na+ binding does not prevent S1-pocket collapse virtually, but rather, the bound Na+ can move to the S1 pocket, thus blocking substrate access directly. Additionally, it was clarified that Na+ binding does not promote S1-pocket formation. According to these insights, we concluded that Na+ binding is irrelevant to the interconversion between the inactive and active forms of thrombin.

■

INTRODUCTION Numerous enzymes show their maximum activities on interacting with a specific monovalent cation.1 Thrombin activation is one of representative paradigms to understand the mechanism of cation-specific activation.2 Thrombin is a serine protease involved in the blood coagulation reaction, and it shows maximum enzymatic activity in the presence of Na+.3 This protein has a Na+-binding cavity consisting of 186 and 220 loops (Figure 1), and it recognizes ArgP1, Arg at the P1 site of a substrate molecule, with Asp189 inside the primary substratebinding pocket (S1 pocket). As the bound Na+ is 15 Å away from Ser195 in the active site, Na+ binding has been supposed to regulate thrombin activation allosterically.4 Over the last 20 years, the thrombin activation mechanism has been studied on the basis of two-phase kinetics consisting of inactive−active conversion and the following slow−fast transition (Scheme 1).5 The inactive form of thrombin (E*) is structurally characterized by a collapsed S1 pocket (Figure 2A), which blocks the binding of the substrate to thrombin. The active form of thrombin, which stably forms the S1 pocket (Figure 2B), is classified into the Na+-unbound active form (E) and Na+-bound active form (E:Na+). In the context of activation of thrombin by Na+, E and E:Na+ show relatively low and high activities and are originally referred to as the slow and fast forms, respectively. © 2016 American Chemical Society

Figure 1. Structure of the thrombin−chromogenic substrate (Phe-ProArg−p-nitroanilide (FPR)) complex. The bound Na+ is shown as a blue sphere. The loops (186 and 220) are presented as magenta and yellow tubes, respectively. The S1 pocket is illustrated by a transparent orange surface. ArgP1 in FPR and the remaining residues are colored dark and light blue, respectively. The hydrogen bonds between Asp189 and ArgP1 are illustrated by dotted lines.

Received: August 3, 2016 Revised: October 5, 2016 Published: October 26, 2016 11873

DOI: 10.1021/acs.jpcb.6b07827 J. Phys. Chem. B 2016, 120, 11873−11879

Article

The Journal of Physical Chemistry B

Furthermore, we examined the effect of Na+ binding on the reverse process, that is, S1-pocket formation, by executing simulations for the inactive form of thrombin. Interestingly, for both MD simulations starting from the active and inactive forms, we observed that conversion between the two forms occurs irrespective of Na+ binding. We then discussed the validity of the conventional supposition that Na+ binding stabilizes the active form of thrombin.

Scheme 1. Thrombin Activation via Two-Phase Kinetics

■

MATERIALS AND METHODS System Setup. We prepared substrate-free thrombin systems of the Na+-unbound active form (E) and Na+-bound active form (E:Na+) and thrombin−chromogenic substrate (Phe-Pro-Arg−p-nitroanilide (FPR)) complex systems of the Na+-unbound active form (E:S) and Na+-bound active form (E:Na+:S), where “S” denotes the substrate-binding condition. The initial atomic coordinates of E:Na+, E:Na+:S, and E:S were built as described in our previous studies.7,13 Those of E were built according to a procedure similar to that for E:Na+ (see SI1 for details). For each system, thrombin or the thrombin−FPR complex is solvated in a 140 mM NaCl aqueous solution (see Table S1 for the molecular components in the systems). Using the initial atomic coordinates for each system, 100 sets of atomic coordinates were generated by randomizing the positions of the ions and water molecules (the detailed procedure is described in our previous study7). To calculate the forces acting among atoms, AMBER force field 99SB,14 the SPC/E water model,15,16 and JC ion parameters adjusted for the SPC/E water model17,18 were applied for the amino acid residues, water molecules, and ions, respectively. The set of force field parameters for FPR is identical to that developed in our previous study.13 Production Run. For each system, using the 100 sets of initial atomic coordinates, we executed 100 independent MD simulations. A constant volume and temperature (NVT) MD simulation with structure restraint was followed by a constant pressure and temperature (NPT) MD simulation without any structure restraint. The NPT MD simulations were extended to 20 and 25 ns for the substrate-free thrombin systems and thrombin−FPR complex systems, respectively. Further details of the MD simulation procedure can be found in SI-2. All of the MD simulations were executed using AMBER 12 pmemd or pmemd.cuda modules with GeForce GTX-680.19,20 Furthermore, we executed two sets of 100 independent MD simulations for the Na+-bound and Na+-unbound inactive forms of thrombin. The initial atomic coordinates for the substrate-free thrombin system of the Na+-bound inactive form (E*:Na+), where the S1-pocket volume is 0 Å3, were derived from one of the MD trajectories for E:Na+. The initial atomic coordinates for the substrate-free thrombin system of the Na+unbound inactive form (E*) were derived from those for E*:Na+ by swapping the bound Na+ with a water molecule. Using each of these two initial atomic coordinates, we generated 100 sets of independent atomic coordinates by randomizing the positions of the ions by a similar procedure as discussed above.13 The MD simulation procedure is similar to that for E and E:Na+, although the length of the present production NPT MD simulation is 10 ns. All of the MD simulations were executed using AMBER 14 pmemd or pmemd.cuda modules with NVIDIA GeForce GTX-780 Ti.20,21 Calculation of the Potential of Mean Force (PMF). We calculated the PMF for S1-pocket collapse using representative active forms of thrombin. We selected snapshot structures with

Figure 2. Structures of the inactive and active forms of thrombin. (A) Structure of the inactive form of thrombin (PDB entry: 2AFQ). (B) Structure of the active form of thrombin (PDB entry: 1SFQ). The loops (186 and 220) are represented as magenta and yellow tubes, respectively. The S1 pocket is illustrated by an orange surface.

In Scheme 1, it is postulated that Na+ binding enhances thrombin−substrate Michaelis complex formation. Thus, characterizing slow−fast conversion by clarifying the effect of allosteric Na+ binding on the thrombin structure was a central problem. However, an X-ray crystallographic study of these two active forms revealed that the difference between them is smaller than 0.5 Å in terms of the root-mean-square deviation (RMSD) for the backbone Cα carbon (see Figure S1 in the Supporting Information (SI)).6 The difference is in the range of thermal fluctuation or smaller so that Na+ binding possibly has no advantage in thrombin−substrate Michaelis complex formation. Meanwhile, our previous study clarified that Na+ binding prevents complex formation, with repulsive interactions acting on ArgP1 in the substrate.7 The above observations therefore indicate that Na+ binding is not advantageous but rather disadvantageous in complex formation. Taking these circumstances into consideration, the remaining possibility for the role of Na+ binding in thrombin activation is promotion of the other step, that is, inactive−active conversion. The above scheme suggests that the active form of thrombin can return to its inactive form only in the absence of Na+ binding. In other words, the active form of thrombin is thermodynamically stabilized by Na+ binding. As per the structures of the inactive form of thrombin resolved by X-ray crystallography, Na+ is not bound to thrombin,8−12 thus supporting the above scenario. Meanwhile, it should be noted that these structures were resolved under nonphysiological conditions10 and using thrombin mutants.8,9,11,12 It still remains elusive whether such structures of the inactive form of thrombin simply result from the absence of Na+ binding. However, answering the question is currently outside the reach of experimental methodologies due to a technical limitation: they cannot directly observe the effect of Na+ binding on the inactive−active conversion of thrombin with sufficiently high spatial and temporal resolutions. To overcome this technical limitation, we performed all-atom molecular dynamics (MD) simulations for the active form of thrombin, both in presence and absence of Na+ binding. Analyzing structural fluctuations of the S1 pocket, we evaluated the effect of Na+ binding on the stability of the active form of thrombin. 11874

DOI: 10.1021/acs.jpcb.6b07827 J. Phys. Chem. B 2016, 120, 11873−11879

Article

The Journal of Physical Chemistry B the maximum S1-pocket volume from MD trajectories of E:Na+ as representative structures of the Na+-bound active form in a 140 mM NaCl aqueous solution. The representative structure of the Na+-unbound active form of thrombin in a 140 mM NaCl aqueous solution was derived from that of the Na+-bound active form of thrombin by swapping the bound Na+ with a water molecule. For each of the two representative structures, we executed steered MD (SMD) simulations, in which the interatomic distance between CαCys191 and CαGly216 was gradually changed through the simulations by imposing a harmonic potential with a force constant of 20 kcal/mol Å2. This distance was selected as the reaction coordinates because of good correlation with the S1-pocket volume (see Figure S2 for details). An SMD simulation was executed for 1 ns under NPT conditions (300 K; 1 bar). The initial atomic velocities were randomly assigned from Maxwellian distribution at 300 K. The trajectory was recorded at every 5 ps interval. The temperature and pressure were regulated using a Langevin thermostat, with a 1 ps−1 collision coefficient, and Berendsen barostat,22 with a 0.1 ps coupling constant, respectively. The target distance of the SMD simulations was set to 3.5 Å. From the SMD simulations, we picked up 16 snapshot structures with different values in the reaction coordinate, ranging from 4.2 to 8.5 Å. Using each of these snapshots, we executed the relaxation simulation and the following umbrella sampling (US) MD simulation. The relaxation simulation consists of four steps: NVT (1−300 K, 30 ps, 10 kcal/mol Å2) → NVT (300 K, 10 ps, 10 kcal/mol Å2) → NVT (300 K, 40 ps, 5 kcal/mol Å2) → NVT (300 K, 40 ps, 1 kcal/mol Å2). The initial atomic velocities were randomly assigned from Maxwellian distribution at 1 K. Thrombin and the bound Na+ were restrained by the harmonic potential around the initial atomic coordinates. In the first NVT MD simulation, the reference temperature was linearly increased along the time course. The US MD simulation was executed under NVT conditions (300 K) and extended to 6 ns, where a harmonic potential is imposed on the reaction coordinate. The minima of the harmonic potentials and the corresponding force constants for each US MD simulation window are summarized in Table 1. In each of these simulations, the temperature was regulated using a Langevin thermostat with a 1 ps−1 collision coefficient, and the interatomic distance between CαCys191 and CαGly216 was recorded at every simulation step. We confirmed 5% or greater overlap between the samplings of neighboring US MD simulation windows. Finally, we used a set of 16 US MD simulation windows to calculate the PMF using the weighted histogram analysis method,23,24 in which we employed the partial US trajectories during the period between 4 and 6 ns. For each of the two systems, the US combined with SMD procedure was executed 10 times, and we calculated the ensemble average of the PMFs using the 10 independent PMFs, where the statistical significance was evaluated with 95% confidence intervals. The convergence of PMFs was evaluated by changing the time domains (see Figure S3 for details). The SMD and US MD simulations were executed using the GPUversion PMEMD module based on the single-precision floatingpoint (SPFP) algorism in the AMBER 14 package with NVIDIA GeForce GTX Titan Black.20,21 Analyses of MD Trajectories. Assuming the equilibrium of ensemble-averaged RMSDs, we used a set of partial MD trajectories of the last 5 ns for the following analyses (see Figure S4 for further details of the RMSD analyses). The

Table 1. Minima of the Harmonic Potentials and Corresponding Force Constants for US MDa Simulations minima of biasing potentials [Å]a

force constant [kcal/mol Å2]

4.2 4.5 4.75 5 5.25 5.5 5.8 6 6.25 6.5 6.8 7 7.25 7.5 8 8.5

30 10 30 20 30 30 30 20 30 20 30 20 30 30 10 10

a

The reaction coordinate of the US simulations is distance between CαCys191 and CαGly216.

interatomic distances and RMSDs were calculated using the cpptraj module in AmberTools 15.25 The volume of the S1 pocket was defined as described in our previous study,26 and the volumes were calculated using the POVME program.27 Distribution of the S1-pocket volume was calculated using each MD trajectory, and the distributions were ensemble-averaged over 100 MD trajectories. The statistical significance was evaluated with 95% confidence intervals. All images of the protein structures were illustrated with visual molecular dynamics (VMD).28 Similarly, a density distribution of the bound Na+ for E:Na+ was calculated and visualized using the Volmap plugin of VMD.28 Values of the bound Na+ distribution density at each grid point were scaled using the bulk density of the Na+ molecules.

■

RESULTS AND DISCUSSION Na+ Binding Is Ineffective in Stabilizing the Active Form of Thrombin. For both E and E:Na+, the S1-pocket volume ranges from 0 to 135 Å3 (Figure 3A). Figure 4 shows the representative conformation with an S1-pocket volume of 0 Å3, which is derived from a simulation of E:Na+. We find that the S1 pocket completely collapses, thus preventing substrate binding. Meanwhile, for both E:S and E:Na+:S, the value ranges from 30 to 115 Å3 (Figure 3B). We then define the inactive form of thrombin as the thrombin structure with an S1-pocket volume 6 kcal/mol at 3.8 Å, at which the S1 pocket collapses (see Figure 6B), to 0 kcal/mol at the minimum point, 7.8 Å. It can therefore be supposed that the active form of thrombin is thermodynamically more stable than the inactive form of thrombin under physiological 11875

DOI: 10.1021/acs.jpcb.6b07827 J. Phys. Chem. B 2016, 120, 11873−11879

Article

The Journal of Physical Chemistry B

Figure 3. Distribution of the S1-pocket volume, which is calculated from MD simulations starting from the structure of the active form of thrombin. (A) Substrate-free thrombin system. (B) Thrombin−chromogenic substrate complex system. The black and red lines represent the Na+-unbound and Na+-bound conditions, respectively.

Figure 6. Representative structures showing the relationship between S1-pocket formation and the interatomic distance between CαCys191 and CαGly216. Panels A and B are derived from the snapshot structures picked up from an SMD simulation. The orange surface illustrates the S1 pocket. The right and left green spheres are for CαCys191 and CαGly216.

Figure 4. Representative structure of the inactive form of thrombin, derived from the MD simulation for the substrate-free thrombin system of the Na+-bound active form (E:Na+). The molecular illustration is similar to that in Figure 2.

similar in the thermodynamic stability of the active form of thrombin. This indicates that Na+ binding is irrelevant to the stabilization of S1-pocket formation. Rather, the value of PMF at 3.8 Å could be smaller in E and E:Na+, implying that Na+ binding promotes S1-pocket collapse. Meanwhile, we observed that the bound Na+ is distributed in both the Na+-binding cavity and the S1 pocket (Figure 7A). This means that the bound Na+ can move from the Na+binding cavity to the S1 pocket. As shown in Figure 7B, the bound Na+ typically interacts with OArg221a. However, it can move into the S1 pocket under thermal fluctuation and interact with the carboxyl group of Asp189 (Figure 7C). If Na+ is present in the S1 pocket, it would directly prevent ArgP1 in a substrate from accessing the S1 pocket. Nonetheless, such a move was observed only in 8% of our MD simulations, and there is no experimental evidence that Na+ is bound in the S1 pocket. It could thus be supposed that Na+ staying in the S1 pocket is energetically unstable, thus being a minor population at thermal equilibrium. Accordingly, such a move of the bound Na+ is possibly disadvantageous to the thrombin−substrate

Figure 5. PMF for S1-pocket collapse. The black and red lines represent the Na+-unbound and Na+-bound conditions, respectively.

conditions, that is, in a 140 mM NaCl aqueous solution at room temperature. The above observation is supported by the previous experimental study, which reported that structure of the active form thrombin occupies >99% of all conformations under thermal equilibrium.29 Additionally, the two PMFs are quite 11876

DOI: 10.1021/acs.jpcb.6b07827 J. Phys. Chem. B 2016, 120, 11873−11879

Article

The Journal of Physical Chemistry B

Figure 7. Location of Na+ bound to thrombin. (A) Spatial distribution of the bound Na+. (B) Frequency of the distance between the bound Na+ and OArg221a. (C) Frequency of the distance between the bound Na+ and CγArg221a. In (A), the locations of the bound Na+ >5 and >50 Å−3 are drawn with a wire frame and solid surface, respectively. In (B) and (C), the green arrow heads point to the values derived from the X-ray crystallographic structure (PDB entry: 1SFQ).6 The molecular illustration is similar to that in Figure 1.

Michaelis complex formation, although the effect could be virtually insignificant. In our previous study, we clarified that Na+ binding represses substrate access into the S1 pocket by a repulsive interaction with ArgP1 in the substrate.7 Here, we provide the insight that the bound Na+ would potentially prevent substrate access into the S1 pocket by moving into the S1 pocket. These observations support the scenario that Na+ binding is a negative effecter in thrombin−substrate Michaelis complex formation. Additionally we showed that S1-pocket collapse occurs similarly in E and E:Na+; therefore, it can be supposed that Na+ binding is ineffective in stabilizing the S1 pocket. In other words, the S1 pocket could remain thermodynamically stable irrespective of Na+ binding once it forms. Inactive−Active Conversion Occurs irrespective of Na+ Binding. The remaining question is whether Na+ binding promotes the reverse process, namely, inactive−active conversion or S1-pocket formation. To elucidate the problem, we executed additional MD simulations starting from the structure of the inactive form of thrombin, whose S1-pocket volume is 0 Å3. For both E* and E*:Na+, 100 sets of 10 ns MD simulations were executed using the procedure described in the Materials and Methods section. We obtained 109 and 100 MD trajectories for E* and E*:Na+, respectively. For E*, 9 among all trajectories show Na+ binding during the simulations; thus, we first analyzed the 100 trajectories to be under the Na+unbound condition and then analyzed the 9 remaining trajectories separately. As shown in Figure 8, inactive−active conversion occurs similarly in both the systems. Actually, 15 and 14% of all conformations assume the active form with regard to E* and E*:Na+, respectively. Thus, this observation suggests that Na+ binding does not necessarily promote conversion from the inactive form to the active form, and the suggestion is verified by analyses of the PMFs shown in Figure 5. Comparison of the PMFs indicates that Na+ binding does not destabilize the inactive form of thrombin. To confirm the above insight that Na+ binding is irrelevant to S1-pocket formation, we examined another possibility that the Na+-binding process perturbs the structure of the inactive form of thrombin and triggers S1-pocket formation. Actually, it is widely discussed that the ligand-binding process induces

Figure 8. Distribution of the S1-pocket volume, which is calculated from MD simulations starting from the structure of the inactive form of thrombin. The black and red lines represent E* and E*:Na+, respectively.

protein conformational changes.30,31 To verify the possibility, we analyzed the other nine MD trajectories for E*, which undergo Na+-binding during simulations. The analyses simply indicate that inactive−active conversion occurs irrespective of the Na+-binding process. The representative case is shown in Figure 9 (the other eight cases are discussed in Figure S5 in SI-3). Na+ binding occurs at around 9.65 ns (Figure 9A), although inactive−active conversion starts around 4.1 ns before it (Figure 9A,B). This analysis apparently illustrates that Na+ binding is uncorrelated with inactive−active conversion. Finally, we conclude that Na+ binding is irrelevant to inactive−active conversion: thrombin spontaneously converts from the inactive form to the active form irrespective of Na+ binding. Thrombin Is Activated via Two Different Kinetic Pathways. Keeping in mind the above insights, we revisited the activation mechanism of thrombin, where E and E* again denote the active and inactive forms of thrombin, respectively. The mechanism has been considered as two-phase kinetics, indicating that Na+ binding finally completes the activation via slow−fast conversion (see Scheme 1). However, our previous study clarified that Na+ binding prevents substrate access into the S1 pocket;7 thus, it is suggested that the “fast form” and “slow form” should be redefined as E and E:Na+, respectively. Furthermore, the present study revealed that Na+ binding is irrelevant to the interconversion between the active and inactive forms of thrombin. It is therefore supposed that 11877

DOI: 10.1021/acs.jpcb.6b07827 J. Phys. Chem. B 2016, 120, 11873−11879

Article

The Journal of Physical Chemistry B

For unbound Na+-activation mechanisms, we have two scenarios. One is the selective stabilization of the active form of thrombin at thermal equilibrium. This is supported by the experimental fact that in the presence of Li+ in aqueous solution thrombin assumes the inactive form at thermal equilibrium.10 The other is the optimization of the thrombin−substrate encounter complex ensemble, proposed in our earlier study.13 We then speculate that Na+ molecules surrounding thrombin prevent substrate access less than that by other monovalent cations. As we clarified that Na+ binding is irrelevant to S1pocket formation, the verification of these two scenarios can now be important in understanding the activation mechanism of thrombin. We will therefore address this problem by executing additional MD simulations in the future. Figure 9. Inactive−active conversion, followed by Na+ binding. In (A), the black and gray lines are for the S1-pocket volume and the distance between CγAsp189 and CζArgP1, respectively. In (A), the red arrow head on the abscissa axis indicates the timing of Na+ binding. In (B), the molecular illustration is similar to that in Figure 2.

■

CONCLUDING REMARKS

■

ASSOCIATED CONTENT

Over the last 20 years, the thrombin activation mechanism has been discussed in the context of allosteric Na+ binding, that is, site-specific thrombin−Na+ interaction. However, our previous7 and present studies clarified that, contrary to the conventional discussion, site-specific interaction has no advantage in S1pocket formation but rather interferes with thrombin function by preventing thrombin−substrate Michaelis complex formation. This insight raises the following questions: “Why does thrombin possess a Na+-binding cavity?” and “How does Na+ activate thrombin?”. The first question could be answered by our previous study,26 which revealed that the Na+-binding cavity plays roles in dewetting of the S1 pocket upon thrombin−substrate Michaelis complex formation. We think that the presence of the Na+-binding cavity is important for promotion of complex formation rather than for the Na+-binding affinity itself. On the other hand, the second question is still unanswered. As sitespecific Na+-binding interactions are irrelevant to S1-pocket formation, Na+ solvated in an aqueous solution possibly plays important roles in stabilizing S1-pocket formation and optimizing thrombin−substrate encounter complex formation, as discussed in our earlier study.13 This should be examined in the context of non-site-specific interactions between thrombin and Na+. It is still challenging to characterize non-site-specific interactions at an atomic level, even if we employ state-ofthe-art experimental methodologies. Instead, molecular simulations are now becoming useful counterparts of experimental methodologies, thus enabling us to examine the atomic details with extended spatial and temporal resolution. Utilizing molecular simulations should enable us to gain a further understanding of the thrombin activation mechanism within the new context of allostery. We will address the problem in our future studies and elucidate the hidden roles of unbound Na+ in thrombin activation.

E:Na+ is an intermediate state in thrombin activation rather than the final state. Finally, these observations provide two insights: (1) E has the highest activity in thrombin−substrate Michaelis complex formation and (2) E* could directly convert into E. Considering them, we propose that thrombin is activated via both one- and three-phase kinetics, that is, ‘E* → E’ and ‘E* → E*:Na+ → E:Na+ → E’, respectively (Scheme 2). Although Scheme 2. Thrombin Activation via Both One- and ThreePhase Kinetics

thrombin can specifically bind Na+ in the Na+-binding cavity, the Na+-binding affinity is relatively low, 110 mM in terms of the equilibrium constant, Kd, for Na+ dissociation.32 This means that Na+ binding would prevent substrate binding, but 40% of thrombin conformations assume the Na+-unbound active form, that is, the fast form, under physiological conditions, thus being ready for substrate binding. It can therefore be supposed that the low Na+-binding affinity is an important factor in thrombin activation, as discussed in our previous study.7 Over the last 20 years, it has been discussed that Na+ binding plays key role in thrombin activation, that is, S1-pocket formation, and promotes the following thrombin−substrate Michaelis complex formation. However, contrary to the conventional discussion, our previous and present studies clarified that Na+ binding is totally disadvantageous to complex formation. This insight implies that unbound Na+ in aqueous solution plays important roles in thrombin activation as a result.

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b07827. More details on the materials and methods, a related discussion, and supporting figures as noted in the text (PDF) 11878

DOI: 10.1021/acs.jpcb.6b07827 J. Phys. Chem. B 2016, 120, 11873−11879

Article

The Journal of Physical Chemistry B

■

(15) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. Comparison of Simple Potential Functions for Simulating Liquid Water. J. Chem. Phys. 1983, 79, 926−935. (16) Kusalik, P. G.; Svishchev, I. M. The Spatial Structure in Liquid Water. Science 1994, 265, 1219−1221. (17) Joung, I. S.; Cheatham, T. E. Determination of Alkali and Halide Monovalent Ion Parameters for Use in Explicitly Solvated Biomolecular Simulations. J. Phys. Chem. B 2008, 112, 9020−9041. (18) Joung, I. S.; Cheatham, T. E., III Molecular Dynamics Simulations of the Dynamic and Energetic Properties of Alkali and Halide Ions Using Water-Model-Specific Ion Parameters. J. Phys. Chem. B 2009, 113, 13279−13290. (19) Case, D. A.; Darden, T. A.; Cheatham, T. E., III; Simmerling, C. L.; Wang, J.; Duke, R. E.; Luo, R.; Walker, R. C.; Zhang, W.; Merz, K. M.; et al. Amber12; University of California: San Francisco, CA, 2012. (20) Le Grand, S.; Gotz, A. W.; Walker, R. C. SPFP: Speed without Compromise-A Mixed Precision Model for GPU Accelerated Molecular Dynamics Simulations. Comput. Phys. Commun. 2013, 184, 374−380. (21) Case, D. A.; Berryman, J. T.; Betz, R. M.; Cerutti, D. S.; Cheatham, T. E., III; Darden, T. A.; Duke, R. E.; Giese, T. J.; Gohlke, H.; Goetz, A. W.; et al. Amber14; University of California: San Francisco, CA, 2015. (22) Berendsen, H. J. C.; Postma, J. P. M.; Vangunsteren, W. F.; Dinola, A.; Haak, J. R. Molecular-dynamics with Coupling to an External Bath. J. Chem. Phys. 1984, 81, 3684−3690. (23) Grossfield, A. WHAM: The Weighted Histogram Analysis Method, version 2.0.9; University of Rochester Medical Center: Rochester, NY, 2013. http://membrane.urmc.rochester.edu/content/wham (accessed Sept 7, 2014). (24) Kumar, S.; Bouzida, D.; Swendsen, R. H.; Kollman, P. A.; Rosenberg, J. M. The Weighted Histogram Analysis Method for FreeEnergy Calculations on Biomolecules.1. The Method. J. Comput. Chem. 1992, 13, 1011−1021. (25) Case, D. A.; Berryman, J. T.; Betz, R. M.; Cerutti, D. S.; Cheatham, T. E., III; Darden, T. A.; Duke, R. E.; Giese, T. J.; Gohlke, H.; Goetz, A. W.; et al. AmberTools14; University of California: San Francisco, CA, 2015. (26) Kurisaki, I.; Barberot, C.; Takayanagi, M.; Nagaoka, M. Dewetting of S1-Pocket via Water Channel upon Thrombin-Substrate Association Reaction. J. Phys. Chem. B 2015, 119, 15807−15812. (27) Durrant, J. D.; de Oliveira, C. A. F.; McCammon, J. A. POVME: An algorithm for measuring binding-pocket volumes. J. Mol. Graphics Modell. 2011, 29, 773−776. (28) Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual molecular dynamics. J. Mol. Graphics 1996, 14, 33−38. (29) Bah, A.; Garvey, L. C.; Ge, J. P.; Di Cera, E. Rapid Kinetics of Na+ Binding to Thrombin. J. Biol. Chem. 2006, 281, 40049−40056. (30) Ikeguchi, M.; Ueno, J.; Sato, M.; Kidera, A. Protein Structural Change upon Ligand Binding: Linear Response Theory. Phys. Rev. Lett. 2005, 94, No. 078102. (31) Cai, L.; Zhou, H. X. Theory and Simulation on the Kinetics of Protein-ligand Binding Coupled to Conformational Change. J. Chem. Phys. 2011, 134, No. 105101. (32) Di Cera, E.; Dang, Q. D.; Ayala, Y. M. Molecular Mechanisms of Thrombin Function. Cell. Mol. Life Sci. 1997, 53, 701−730.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel/Fax: +81-52-7895623. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the Core Research for Evolutional Science and Technology (CREST) “Establishment of Molecular Technology towards the Creation of New Functions” of the Japan Science Technology Agency (JST); by a Grant-in-Aid for Science Research from the Ministry of Education, Culture, Sport, Science and Technology (MEXT) in Japan; and also by the MEXT program “Elements Strategy Initiative for Catalysts and Batteries (ESICB)” and FLAGSHIP2020 within the priority study5 (Development of new fundamental technologies for high-efficiency energy creation, conversion/storage, and use). The calculations were partially performed using several computing systems at the Information Technology Center in Nagoya University. I.K. also thanks the Japan Society for the support via Promotion of Science (JSPS) by the Research Fellowship for Young Scientist.

■

REFERENCES

(1) Page, M. J.; Di Cera, E. Role of Na+ and K+ in Enzyme Function. Physiol. Rev. 2006, 86, 1049−1092. (2) Di Cera, E. A Structural Perspective on Enzymes Activated by Monovalent Cations. J. Biol. Chem. 2006, 281, 1305−1308. (3) Wells, C. M.; Dicera, E. Thormbin Is a Na+-Activated Enzyme. Biochemistry 1992, 31, 11721−11730. (4) Zhang, E.; Tulinsky, A. The Molecular Environment of the Na+ Binding Site of Thrombin. Biophys. Chem. 1997, 63, 185−200. (5) Di Cera, E. Thrombin. Mol. Aspects Med. 2008, 29, 203−254. (6) Pineda, A. O.; Carrell, C. J.; Bush, L. A.; Prasad, S.; Caccia, S.; Chen, Z. W.; Mathews, F. S.; Di Cera, E. Molecular Dissection of Na+ Binding to Thrombin. J. Biol. Chem. 2004, 279, 31842−31853. (7) Kurisaki, I.; Takayanagi, M.; Nagaoka, M. Bound Na+ is a Negative Effecter for Thrombin-Substrate Stereospecific Complex Formation. J. Phys. Chem. B 2016, 120, 4540−4547. (8) Carter, W. J.; Myles, T.; Gibbs, C. S.; Leung, L. L.; Huntington, J. A. Crystal Structure of Anticoagulant Thrombin Cariant E217K Provides Insights into Thrombin Allostery. J. Biol. Chem. 2004, 279, 26387−26394. (9) Pineda, A. O.; Chen, Z. W.; Caccia, S.; Cantwell, A. M.; Savvides, S. N.; Waksman, G.; Mathews, F. S.; Di Cera, E. The Anticoagulant Thrombin Mutant W215A/E217A Has a Collapsed Primary Specificity Pocket. J. Biol. Chem. 2004, 279, 39824−39828. (10) Johnson, D. J.; Adams, T. E.; Li, W.; Huntington, J. A. Crystal Structure of Wild-type Human Thrombin in the Na+-free State. Biochem. J. 2005, 392, 21−28. (11) Niu, W.; Chen, Z. W.; Bush-Pelc, L. A.; Bah, A.; Gandhi, P. S.; Di Cera, E. Mutant N143P Reveals How Na+ Activates Thrombin. J. Biol. Chem. 2009, 284, 36175−36185. (12) Niu, W.; Chen, Z. W.; Gandhi, P. S.; Vogt, A. D.; Pozzi, N.; Pelc, L. A.; Zapata, F.; Di Cera, E. Crystallographic and Kinetic Evidence of Allostery in a Trypsin-like Protease. Biochemistry 2011, 50, 6301−6307. (13) Kurisaki, I.; Takayanagi, M.; Nagaoka, M. Toward Understanding Allosteric Activation of Thrombin: A Conjecture for Important Roles of Unbound Na+ Molecules around Thrombin. J. Phys. Chem. B 2015, 119, 3635−3642. (14) Hornak, V.; Abel, R.; Okur, A.; Strockbine, B.; Roitberg, A.; Simmerling, C. Comparison of Multiple Amber Force Fields and Development of Improved Protein Backbone Parameters. Proteins 2006, 65, 712−725. 11879

DOI: 10.1021/acs.jpcb.6b07827 J. Phys. Chem. B 2016, 120, 11873−11879