Bound Na+ is a Negative Effecter for Thrombin-Substrate

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The Bound Na is Negative Effecter for ThrombinSubstrate Stereospecific Complex Formation Ikuo Kurisaki, Masayoshi Takayanagi, and Masataka Nagaoka J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b00976 • Publication Date (Web): 10 May 2016 Downloaded from http://pubs.acs.org on May 10, 2016

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The Journal of Physical Chemistry

The Bound Na+ is Negative Effecter for Thrombin-Substrate Stereospecific Complex Formation

Ikuo Kurisaki†,‡, Masayoshi Takayanagi†,‡, 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

* Corresponding author Professor Masataka Nagaoka; Email: [email protected]; phone and fax: +81-52-789-5623.

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Abstract Thrombin has been studied as a paradigmatic protein of Na+-activated allosteric enzymes.

Earlier

structural studies suggest

that

Na+-binding

promotes the

thrombin-substrate association reaction. However, it is still elusive because (1) the structural change, driven by Na+-binding, is as small as the thermal fluctuation, and (2) the bound Na+ is close to Asp189 in the primary substrate binding pocket (S1-pocket), possibly preventing substrate access via repulsive interaction. It still remains a matter of debate whether Na+-binding actually promotes the reaction. To solve this problem, we examined the effect of Na+ on the reaction by employing molecular dynamics (MD) simulations. By executing independent 210 MD simulations of apo and holo systems, we obtained 80 and 26 trajectories undergoing substrate access to S1-pocket, respectively. Interestingly, Na+-binding results in a 3-fold reduction of the substrate access. Furthermore, we examined works for the substrate access and release, and found that Na+-binding is disadvantageous for the presence of the substrate in S1-pocket. These observations provide the insight that the bound Na+ is essentially negative effecter in thrombin-substrate stereospecific complex formation. The insight rationalizes an enigmatic feature of thrombin, relatively low Na+-binding affinity. This is essential to reduce disadvantage of Na+-binding in the substrate-binding.

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Introduction Numerous enzymes show the highest activity under the presence of specific cations. The roles of cations are classified into two types.1 One is a cofactor, and the other is an allosteric effecter. Human thrombin has been studied as a paradigmatic protein of the latter, namely, cation-activated allosteric enzymes. This enzyme is the serine protease playing central roles in blood coagulation by activating substrate molecules, for example fibrinogen, protein activated receptor 1, factor XIII,2 and shows maximum activity under the presence of Na+.3 The allosteric regulation by Na+-binding is attributed to the presence of Na+-binding cavity, which is located in the vicinity of the primary specific pocket, S1-pocket (Figure 1A). The bound Na+ is energetically stabilized by interactions with backbone oxygen atoms in Arg221 and Lys224, water molecules in the cavity and carboxyl side chain of Asp189 at the bottom of S1-pocket (Figure 1B).4,5 Asp189 also recognizes Arg at P1 site in a substrate (referred to as ArgP1, hereafter) (Figure 1C). The hydrogen bond formation between Asp189 and ArgP1 is involved in stability of the Michaellis complex.6

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Figure 1. Thrombin and substrate structures. (A) Overview of thrombin-Na+ complex structure. (B) Oxygen coordination around the bound Na+. (C) Interaction between ArgP1 in the substrate and Asp189, and Ser195. (D) Overview of the model peptide, Pro-Arg tripeptide. The bound Na+ is shown as a blue sphere. 186- and 220-loops are highlighted by magenta and yellow tubes, respectively. S1-pocket is illustrated by transparent orange surface. In the panel (A), thrombin is colored in gray. In panel (C), the residues in the active site but for Asp189 and Ser195 (Ala190, Cys191, Glu192, Gly193 and Asp194) are represented by gray tube. Hydrogen bonds between Asp189 and ArgP1 are illustrated by dotted blue lines. 4


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In the context of the Na+-activation mechanism, slow-fast transition has been the central concept for the last 20 years.3 This states that Na+-binding promotes thrombin’s structural change from slow form with relatively low activity to fast form with relatively high activity. Assuming that slow and fast forms correspond to Na+-free thrombin and thrombin-Na+ complex, namely, apo and holo forms of thrombin, respectively, Pineda and his colleagues investigated effects of the bound Na+ at the atomic level. They solved structures of apo and holo forms, and found that Na+-binding optimizes the orientation of Asp189 and Ser195 for substrate-binding.7 Finally, they concluded that Na+-bound thrombin has advantage in the Michaellis complex formation via thrombin-substrate association reaction. In general, protein-substrate association reactions consist of two steps, that is, the encounter complex formation and the following stereospecific complex formation. Pineda’s study provided the insights as to how Na+-binding promotes the stereospecific complex formation, accordingly. Actually, their insight provides a milestone to solve the longstanding problem. Nonetheless, there is still room for further investigation. As they reported, the structural difference between apo and holo forms is smaller than 0.5 Å in terms of root mean square deviation (RMSd).7 Considering the spatial resolutions of

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these structures are around 2.8 Å, the difference could be in the range of thermal fluctuation. Furthermore, it is possible that the presence of the bound Na+ electrostatically screens ArgP1 from Asp189, effectively reducing attractive interaction acting between these two amino acid residues. In the holo form solved by Pineda et al. (PDB entry: 1SFQ), Cγ of Asp189 and Cζ of ArgP1 are close to Na+ bound to thrombin by 5.8 and 8.8 Å, respectively.7 This indicates the possibility that Na+-binding prevents substrate access to S1-pocket by repulsive interaction with ArgP1. These observations imply an opposite insight that the conformational change upon Na+-binding is essentially irrelevant to the stereospecific complex formation, or rather, Na+-binding is disadvantageous in substrate access. Accordingly, it still remains a matter of debate whether Na+-binding actually promotes thrombin-substrate stereospecific complex formation. To solve the above problem, it is necessary to gain atomic-level insights into thrombin-substrate association reaction, although it is still challenging even for state-of-the-art experimental methods. We thus addressed the problem with an alternative method, all-atom molecular dynamics (MD) simulations. We examined the effect of Na+-binding on the initial step of the stereospecific complex formation, namely, ArgP1-Asp189 hydrogen bond (HBP1-189) formation, where Pro-Arg tripeptide was

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employed as the model substrate for thrombin (Figure 1D). By analyzing MD trajectories, we compared frequencies of HBP1-189 formation between apo and holo forms of thrombin. Furthermore, the effect of Na+-binding on HBP1-189 formation was characterized, physico-chemically. According to these analyses, we discuss how Na+ affects HBP1-189 formation upon the stereospecific complex formation.

Materials and Methods Molecular dynamics simulations In all the molecular dynamics (MD) simulations, we used full-length thrombin structure consisting of 295 amino acid residues8. First, we executed MD simulations of thrombin-Na+ with 12 model substrates, Pro-Arg tripeptides, whose N- and C-terminals were capped by acetyl and N-methyl amide group, respectively, in aqueous solution containing 140 mM NaCl, and obtained one 500 ns trajectory undergoing the substrate access to Asp189. The details of the molecular modeling and MD simulation procedures are described in our earlier study.9 Using the trajectory, we picked up 210 snapshot structures during the period between 28 to 237 ns with a 1 ns interval. In each of the snapshots, ArgP1 in the substrate is found in S1-pocket but spatially separated from Asp189 by water molecules (the representative illustration is provided in our earlier

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study9). From each snapshot, the remaining 11 substrates, and 11 Cl− ions were removed (this system is referred to as the holo system, hereafter), and then the locations of the Na+ and Cl- ions except for the bound Na+ were randomized to prepare the initial structure of the holo system according to the procedure reported in our earlier study10. We also prepared 210 initial structures of the apo system from the 210 initial structures of the holo system by swapping all the ions, including the bound Na+, with water molecules in the randomization procedure. The molecular components in the systems are summarized in Table 1.

Table 1. Molecular Components for the Thrombin Systems

number of molecules

system

+

−

Cl 48

thrombin-Na with 12 aPRn

11595

Na 33

thrombin-Na with 1 aPRn

+

11595

33

37

thrombin with 1 aPRn

11595

33

37

+

a

water a

’aPRn’ denotes the model substrate Pro-Arg tripeptides, whose N- and C-terminals

were capped by acetyl and N-methyl amide group, respectively.

We executed 210 independent production constant pressure and temperature (NPT) MD simulations of both the apo and holo systems starting from the prepared 210 initial 8


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structures after equilibrating constant volume and temperature (NVT) MD simulations. The MD simulation procedure is similar to those of our earlier study10, although the length of the present production NPT simulation is 30 ns and the coupling constant for Berendsen barostat11 is 5 ps. MD trajectories were recorded at 10 ps intervals. The production NPT simulations were employed for the following analyses. As for the production NPT simulations, we employ GPU-version PMEMD module based on SPFP algorism12 with NVIDIA GeForce GTX-titan Black. The other simulations were executed with CPU-version PMEMD. Molecular modeling and MD simulations were executed by using AmberTools1413 and AMBER 14 package14, respectively.

Selection of MD trajectories undergoing ArgP1-Asp189 hydrogen bond formation Among each set of 210 MD trajectories, we selected ones which show stable formation of HBP1-189 by the following 2-step procedure. First, we focused on the time point when the number of HBP1-189 becomes higher than a threshold value, that is, 2.8 (this time point is referred to as τHB, hereafter). The threshold value was provided based on the MD simulations of thrombin-chromogenic substrate complex system (see SI-1 in Supporting Information for details). Second, we calculated the average number of HBP1-189 after τHB. If the average number is larger than the threshold value, we consider

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that the MD trajectory stably formed the HBP1-189. From such trajectories, we extracted 2.01 ns partial trajectory centered at τHB including the 1 ns time domains just before and after τHB. Of note, we choose trajectories where τHB is within the time domain between 1 and 29 ns. In each partial trajectory, τHB was assigned to 0 ns in the following analyses.

Trajectory analyses Interatomic distance, hydrogen bond (HB) formation and interaction energy were analyzed using cpptraj module in AmberTools1413. The geometrical criterion of HB formation is as follows: H-X distance was < 3.5 Å and X-H-Y angle was > 120˚, where X, Y and H denote acceptor, donor and hydrogen atoms, respectively. Intermolecular energy was calculated with non-bonded cutoff of 999 Å. These values were averaged over each of the 1 ns time domains defined above, and the statistical significance was evaluated by the 95% confidential intervals. Molecular structures were illustrated using visual molecular dynamics (VMD)15.

Steered molecular dynamics simulations From the 500 ns trajectory, we picked up the snapshots at 18.9 and 500 ns. The

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former and latter were used to calculate works for the formation and breakage of ArgP1-Asp189 hydrogen bonds. The remaining 11 substrates and 11 Cl− were removed from each of these two snapshots, referred to as holo systems, hereafter. Using each of these two initial structures of holo system, we prepared initial structures of apo system by replacing the bound Na+ with a water molecule. Finally, we obtained four initial structures. For each of these initial structures, ions and solvents were energetically relaxed by the three-step procedure: NVT (0.001 to 1 K, 0.1 ps) → NVT (1 to 300 K, 0.1 ps) → NPT (300 K, 1 bar, 100 ps). The NVT and NPT simulations were performed using the time step of 0.01 fs and 2 fs for the integration, respectively. The NVT simulations were performed using a 0.001 ps coupling constant for Berendsen thermostat11, where the reference temperature was linearly increased along the time course. In the NPT simulations, temperature and pressure were regulated using Langevin thermostat with a 1 ps−1 of collision coefficient and Berendsen barostat11 with a 2 ps coupling constant, respectively. During these relaxation processes, the atomic coordinates of thrombin, the substrate and the bound Na+ were positionally frozen. Using each of these four initial structures, we executed 96 and 192 steered molecular dynamics (SMD) simulations as for HB formation and breakage, respectively. For each

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SMD simulation, the initial atomic velocities were randomly assigned from Maxwellian distribution at 300K. The simulation procedure is similar to that of the production NPT simulation above, although the simulation length is 500 ps and the coupling constant for Berendsen barostat11 is 0.1 ps. A harmonic potential is imposed on the distance between CζArgP1 and CγAsp189 with the force constant of 5 kcal/mol/Å2. The target distances for HB formation and HB breakage SMD simulations were set to 3.2 and 7.0 Å, respectively. The values of work accumulated during the SMD simulations were averaged over the 96 and 192 simulations, and the statistical significance was evaluated by the 95% confidential intervals.

Results and Discussion From the independent 210 MD simulations of holo and apo systems, we obtained 80 and 26 trajectories undergoing stable HBP1-189 formation, respectively. Interestingly, the frequency of HBP1-189 formation of apo system is 3-fold higher than that of holo system. This suggests that Na+-binding prevents ArgP1 from HBP1-189 formation with Asp189, thus implying a disadvantage in HBP1-189 formation. To verify this observation, we analyzed these 80 and 26 trajectories focusing on the time domain centered at the moment of the HBP1-189 formation (τHB). According to the procedure described in

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Materials and Methods, 2.01-ns partial trajectories were extracted and used for the following analyses. Time domains just before and after 1 ns of τHB are referred to as Pre-HB and Post-HB, respectively. Figure 2A shows the number of HBP1-189 ensemble-averaged over the 80 and 26 partial trajectories of apo and holo systems, respectively. Interestingly, the time average of the number during Pre-HB are non-zero, 0.67 ± 0.20 and 0.88 ± 0.44 for apo and holo systems, respectively. These non-zero values mean that stable HBP1-189 formation does not complete instantaneously from the situation without HBP1-189, but it gradually forms by repeating transient formation of HBP1-189 (see SI-2 in Supporting Information for representative cases). Since the number of HBP1-189 is larger in holo system during Pre-HB, Na+-binding seems to promote HBP1-189 formation. However, we suppose Na+-binding is, on the contrary, disadvantageous in HBP1-189 formation. Remembering the suggested electrostatic repulsive interaction between ArgP1 and the bound Na+, Na+-binding should prevent ArgP1 from access to Asp189. It is thus possible to say that frequent preliminary HBP1-189 formation in holo system is necessary for stable HBP1-189 formation to overcome the additional repulsion compared to apo system.

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Figure 2. Ensemble average of the number of hydrogen bond (HB). (A) HB between ArgP1 and Asp189. (B) HB between ArgP1 and water molecules. (C) HB between Asp189 and water molecules.

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Meanwhile, the time average during Post-HB are 2.84 ± 0.01 and 2.82 ± 0.01 for apo and holo systems, respectively. Considering that the number of HBP1-189 is similar between the two systems during Post-HB, it can be assumed that the Na+-binding does not affect the number of HBP1-189 once HBP1-189 stably forms. Nonetheless, Na+-binding is disadvantageous in preservation of HBP1-189 formation. HBP1-189 is temporally broken under thermal fluctuation (see SI-2 in Supporting Information for a representative case), while the bound Na+ repulsively interacts with ArgP1. The Na+-binding possibly diminishes the chance of HBP1-189 reformation. That would promote dissociation of the substrate from S1-pocket. This observation is supported by the earlier experimental study2, which reported that kinetic rate constant of thrombin-substrate dissociation reaction is maximized under the presence of Na+. In addition to the above discussed repulsive interaction between the bound Na+ and ArgP1, changes of solvation by the Na+-binding can also be another factor affecting the HBP1-189 formation. To obtain insights into the solvation effect, we calculated the number of hydrogen bonds with water molecules for ArgP1 (HBP1-Wat, Figure 2B) and Asp189 (HB189-Wat, Figure 2C). The differences between apo and holo systems are not large. During Pre-HB, both

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HBP1-Wat and HB189-Wat are smaller in holo system by ca. 0.2. This should reflect the compensation of the 0.2 increase in holo system in HBP1-189 during Pre-HB to keep the total numbers of hydrogen bond of ArgP1 and Asp189. Meanwhile, a relatively large difference 0.4 was found in HB189-Wat during Post-HB. This is explained by the solvation of the bound Na+. Water molecules coordinate to the Na+ by their oxygen atoms (Figure 1B). Hence, water molecules between Na+ and Asp189 are constrained to enhance the hydrogen bond network Na+･･･O-H･･･O (a representative illustration is shown in SI-3 in Supporting Information) and, as a result, increase the HB189-Wat. However, the number of HBP1-189 during Post-HB is not affected by Na+-binding (Figure 2A), so that it is possible to say the increase is irrelevant to HBP1-189 formation. We conclude that changes of solvation by the Na+-binding are ineffective in HBP1-189 formation, accordingly. In summary, we suppose that Na+-binding is disadvantageous in stable thrombin-substrate complex formation and the mechanism is attributed to repulsive interaction between ArgP1 and the bound Na+. Keeping in mind this scenario, we will physico-chemically characterize the effect of Na+-binding in the following sections.

Na+-binding prevents ArgP1-Asp189 hydrogen bond formation

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Focusing on Pre-HB, we analyzed intermolecular interaction acting on the substrate (Table 2). The substrate is energetically stabilized by interaction with the remaining system (the first row in Table 2). This energetic stabilization is mainly derived from the interaction with ArgP1 (the second row in Table 2). However, the interaction energies are similar between apo and holo systems. Meanwhile, the interaction energy between thrombin and ArgP1 in holo system is lower by ca. 12 kcal/mol than that in apo system (the third row in Table 2). Both the functional regions of thrombin (FRT) and the outward of FRT, shown in Figure 3, contribute to this energetic stabilization (the fourth and fifth rows in Table 2, respectively). As for FRT, the relative energetic stabilization of ArgP1 is ca. 8 kcal/mol. This is mainly attributed to the interaction with S1-pocket and 220-loop (Table 3). In particular, interaction energies between Asp189 and ArgP1 are −67.6 ± 3.0 and −71.1 ± 6.3 for apo and holo systems, respectively. As suggested by Pineda et al.,5 Na+-binding could optimize the orientation of Asp189 to yield additional energetic stabilization with interaction with ArgP1. Meanwhile, Figure 4 shows that apo and holo systems are similar in interaction energy between ArgP1 and the other residue in thrombin. The remaining energetic stabilization of 4 kcal/mol, thus, could not be assigned to a few specific residues. Rather, accumulation of local interaction energies should result in the total energetic stabilization of 4 kcal/mol. Similarly, as for

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interaction with the outward of FRT, the energetic stabilization of 4 kcal/mol would be attributed to accumulation of local interaction energies.

Table 2. Interaction energies acting on the substrate, in Pre-HB.

interaction energy [kcal/mol]

interaction pair

+

apo (without Na ) substrate-the remaining

−221.2 ± 1.5

−220.7 ± 3.0

ArgP1-the remaining

−168.6 ± 1.1

−166.7 ± 2.1

−97.0 ± 3.6

−109.7 ± 7.2

−172.4 ± 3.2

−180.7 ± 6.5

75.4 ± 1.8

71.0 ± 2.4

a

ArgP1-thrombin ArgP1-thrombin (FRT)

b

ArgP1-thrombin (outward of FRT) a

+

holo (with Na )

'The remaining' consists of thrombin, ions and water molecules.

b

The functional

region of thrombin (FRT) consists of S1-pocket, 186- and 220-loops, and the active site (Asp189, Ala190, Cys191, Glu192, Asp194 and Ser195), while the outward of FRT consists of the rests of residues in thrombin.

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Figure 3. The functional regions of thrombin (FRT) and the outward of FRT (transparent yellow, and blue, respectively). FRT consists of S1-pocket, 186- and 220-loops, and the active site (Asp189, Ala190, Cys191, Glu192, Asp194 and Ser195), while the outward of FRT consists of the rests of residues in thrombin. 186- and 220-loops, and S1-pocket are illustrated by yellow tubes and surface, respectively.

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Table 3. Interaction energies between ArgP1 and thrombin’s functional regions, in Pre-HB.

functional region

+

apo (without Na )

+

holo (with Na )

S1-pocket

−64.9 ± 1.2

−69.7 ± 2.0

186-loop

28.5 ± 0.4

28.1 ± 0.6

−33.8 ± 1.2

−37.7 ± 1.9

−156.9 ± 2.7

−158.1 ± 5.3

220-loop the active site a


a

‘The active site’ consists of Asp189, Ala190, Cys191, Glu192, Asp194 and Ser195.

S1-pocket shares some residues with the active site and 220-loop.

Figure 4. Interaction energies between ArgP1 and residue of thrombin, in Pre-HB. Residue number is given with sequential numbering.8 Error bars denote 95% confidential intervals.

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The above observations suggest that Na+-binding enhances stabilization of thrombin-ArgP1 interaction. However, it should be noted that the bound Na+ is located in the vicinity of Asp189. Actually, the bound Na+ energetically destabilizes ArgP1 in S1-pocket by ca. 38 kcal/mol. This overcomes the energetic stabilization discussed above. Na+-binding could energetically stabilize interaction between Asp189 and ArgP1. However, that is still insufficient to completely cancel out the repulsive interaction acting between ArgP1 and the bound Na+. It is therefore suggested that Na+-binding prevents ArgP1 access to S1-pocket, thus supporting the observation that the substrate in holo system forms HBP1-189 less frequently than that in apo system. To elucidate this suggestion, we calculated the work for HBP1-189 formation. The work values are 18.99 ± 1.00 and 21.88 ± 0.84 kcal/mol for apo and holo systems, respectively (additionally, time course analyses of works are provided in SI-4 in Supporting Information). This clearly indicates that Na+-binding prevents HBP1-189 formation. The energetic difference is in the range of a few kcal/mol, thus seeming to be modest. Nonetheless, this could be rationalized considering that allosteric interaction modestly affects the enzymatic activity.16 As shown above, formation of HBP1-189 can occur in both apo and holo systems. It is, thus, possible to say that Na+-binding does not completely block HBP1-189 formation, but modestly prevent it to some extent. In

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summary, we clarified that Na+-binding is disadvantageous in HBP1-189 formation. This insight reminds us of the studies on murine thrombin17. This enzyme cannot bind to Na+ because Lys222 is protruded into Na+-binding cavity, and the enzymatic activity of murine thrombin is higher than that of human thrombin. Considering the characteristic location of Lys222, murine thrombin is similar to human thrombin bound to Na+ in that one positive charge is present inside the Na+-binding cavity. However, the distance between the bound Na+ and Cγ of Asp189 is 6 Å in human thrombin7 (PDB entry: 1SG8), while the distance between Nζ of Lys222 and Cγ of Asp189 is 9 Å in murine thrombin (PDB etnry: 2OCV) (Figure 5).17 This suggests that the interference by the Lys222 on HBP1-189 formation should be weaker than that by the bound Na+. The structural study on murine thrombin concluded that Lys222 in murine thrombin is mimicry of the bound Na+.17 However, the above discussion indicates an opposite insight that the Lys222 is an inhibitor of Na+-binding. We, therefore, consider that the absence of Na+-binding promotes thrombin-substrate association, then enhancing the enzymatic activity.

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Figure 5. Na+ mimicry by Lys222 in murine thrombin. Na+-binding cavity and Asp189 are colored by blue and pink for human and murine thrombins, respectively. S1-pocket of human and murine thrombins are illustrated transparent orange and yellow surface, respectively.

Na+-binding promotes ArgP1-Asp189 hydrogen bond breakage Similarly, in Post-HB, we analyzed intermolecular interaction acting on the substrate (Table 4). The substrate is energetically stabilized by interaction with the remaining system (the first row in Table 4), although holo system is less stabilized by ca. 4 kcal/mol than apo system (see the first and second columns in Table 4). As shown in Figure 6A and 6B, thrombin-substrate complexes obtained from our simulations are still in the process of Michaelis complex formation, so that ArgP1 and Ser195 in the active site should reorient to complete the complex formation. It is, therefore, possible that

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Na+-binding is disadvantageous in the Michaellis complex formation due to energetic destabilization of substrate’s presence in S1-pocket.

Table 4. Interaction energies acting on the substrate, in Post-HB.


interaction pair

+

apo (without Na ) substrate-the remaining

−228.9 ± 1.3

−224.4 ± 1.7

ArgP1-the remaining

−175.7 ± 1.0

−169.6 ± 1.5

−125.7 ± 1.8

−140.3 ± 2.9

−197.4 ± 1.6

−207.9 ± 2.5

71.8 ± 1.5

67.5 ± 2.2

a

ArgP1-thrombin ArgP1-thrombin (FRT)

b

ArgP1-thrombin (outward of FRT) a

+

holo (with Na )

'The remaining' consists of thrombin, ions and water molecules.

b

The functional

region of thrombin (FRT) consists of S1-pocket, 186- and 220-loops, the active site (Asp189, Ala190, Cys191, Glu192, Asp194 and Ser195), while the outward of FRT consists of the rests of residues in thrombin.

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Figure 6. Structural characterization of incomplete thrombin-substrate complex derived from our simulations. (A) Representative conformation illustrating positional relationship between CArgP1 and Oγ195. (B) Histograms of distance between CArgP1 and Oγ195. In panel-(B), the green arrow indicates the value derived from the X-ray crystallographic structure of thrombin-fibrin complex18 (PDB entry: 1DM4). Error bars denote 95% confidential intervals.

Meanwhile, interaction energy between thrombin and ArgP1 in holo system is lower by ca. 15 kcal/mol than that in apo system (the third row in Table 4). Both FRT and the outward of FRT (Figure 3) contribute to this energetic stabilization (the fourth and fifth rows in Table 4, respectively). As for FRT, the relative energetic stabilization of ArgP1 is 11 kcal/mol. As shown in Table 5, this is attributed to the interaction with S1-pocket and 220-loop. However, Figure 7 shows that apo and holo systems are similar in 25



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interaction energy between ArgP1 and residues in thrombin. The difference of 11 kcal/mol, thus, could not be assigned to a few specific residues. Rather, accumulation of local interaction energies should result in the total energetic stabilization of 11 kcal/mol. The same observation would hold to the energetic stabilization of 4 kcal/mol with respect to the outward of FRT. As is the case of Pre-HB, however, the energetic stabilization of 15 kcal/mol is overcome by the interaction energy with the bound Na+. Actually, the bound Na+ energetically destabilizes ArgP1 in S1-pocket by ca. 42 kcal/mol. Remembering energetic destabilization of the substrate in the holo system, these results suggest that Na+-binding promotes ArgP1’s release from S1-pocket.

Table 5. Interaction energies between ArgP1 and thrombin functional sites, in Post-HB.

functional region

+

apo (without Na )

+

holo (with Na )

S1-pocket

−69.3 ± 1.2

−76.8 ± 1.3

186-loop

31.0 ± 0.4

30.4 ± 0.6

220-loop

−38.1 ± 1.0

−43.2 ± 1.0

−179.2 ± 1.0

−179.8 ± 1.4

the active site a


a

‘The active site’ consists of Asp189, Ala190, Cys191, Glu192, Asp194 and Ser195.

S1-pocket shares some residues with the active site and 220-loop.

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Figure 7. Interaction energies between ArgP1 and residue of thrombin, in Post-HB. Residue number is given with sequential numbering8. Error bars denote 95% confidential intervals.

To elucidate this suggestion, we calculated the work for HBP1-189 breakage. The work values are 16.06 ± 0.47 and 14.48 ± 0.43 kcal/mol for apo and holo systems, respectively (additionally, time course analyses of the works are provided in SI-4 in Supporting Information). This clearly indicates that Na+-binding promotes HBP1-189 breakage. Although the energetic difference is in the range of a few kcal/mol, this modest difference could be reasonable considering that allosteric interaction modestly affects the enzymatic activity.16

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In summary, we clarified that Na+-binding promotes breakage of HBP1-189. As referred above, this insight is consistent with that by the earlier kinetics study of thrombin.3 Actually, the dissociation rate constant of thrombin-substrate complex is maximized under the presence of Na+. The present result indicates that the maximization of the kinetic constant is attributed to Na+-binding. HBP1-189 is transiently broken under thermal fluctuation (see SI-2 in Supporting Information). After such transient breakage, HBP1-189 can be immediately reformed, while it is also possible that HBP1-189 remains to be broken. Considering the above observations that repulsive interaction between the bound Na+ and ArgP1 interferes with ArgP1 access to Asp189, HBP1-189 breakage would be sustained longer in holo system than in apo system. We suppose that this situation promotes ArgP1 release from S1-pocket, that is, dissociation of thrombin-substrate stereospecific complex, accordingly.

Low Na+-binding affinity is advantageous to thrombin-substrate association reaction Our results indicate the disadvantage of Na+-binding in the initial step of thrombin-substrate stereospecific complex formation, that is, interference with HBP1-189 formation. Meanwhile, Na+-binding could be advantageous in dissociation of

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thrombin-substrate stereospecific complex due to promotion of HBP1-189 breakage. This might similarly contribute to the product release from S1-pocket and enhance catalytic efficiency as a result. It is therefore possible to say that Na+-binding destabilizes thrombin-substrate stereospecific complex totally. This insight could shed new light on understanding of low Na+-binding affinity of thrombin. The Na+ dissociation constant of substrate-free thrombin is estimated to be 110 mM, thus meaning that only 60% of thrombin forms complex with Na+ under physiological condition.6 Considering the importance of Na+ for thrombin’s enzymatic activation, the relatively low affinity seems to be enigmatic. However, remembering the disadvantage of Na+-binding in thrombin-substrate stereospecific complex formation, this could be rationalized: remaining 40% of thrombin can form complex with substrates faster than the other 60%. Meanwhile, Na+-binding affinity is similar between substrate-free thrombin and thrombin-substrate complex2, so that Na+ can rebind to thrombin-substrate stereospecific complex, then promoting the dissociation. Besides the low Na+-binding affinity, time scale separation between reactions might allow thrombin to bind substrates efficiently. Remembering the kinetics studies3,19, the thrombin-substrate association reaction could be slower than the thrombin-Na+ complex formation reaction, while it possibly occurs as slow as Na+-dissociation from thrombin.

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It is, therefore, assumed that the bound Na+ is released from a thrombin within the period of a substrate access to S1-pocket, so that Na+-binding does not prevent HBP1-189 formation, virtually. Besides structure design of proteins, design of dynamics regulation might be also an important factor to understand the mechanism of protein functions. Meanwhile, it should be noted that Pozzi and colleagues reported such a chimera thrombin that shows both higher Na+-binding affinity and enzymatic activity than those of the wild type.20 According to the above discussion, in the chimera, higher frequency of Na+-binding should result in reduction of the enzymatic activity. Although these two observations seem to create a dilemma, higher enzymatic activity of the chimera could be explained as follows. The chimera possesses another negative charge in the autolysis loop besides S1-pocket (Figure 8). Considering that thrombin specifically recognizes ArgP1 in a substrate, the negative charge should contribute to attract a substrate around S1-pocket. This would promote formation of encounter complexes which lead to stereospecific complex. Furthermore, our simulations indicate that Na+-binding does not completely block substrate access. Na+-binding to the chimera would thus prevent substrate access to Asp189 relatively strongly compared to the wild type, but the disadvantage should be overcome by the promotion of the encounter complex formation. This conjecture is supported by our earlier study, which discusses the importance of the

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encounter complex formation upon thrombin-substrate association reaction.10 We, therefore, assume that the chimera possesses the higher enzymatic activity not by the higher Na+-binding affinity, but rather by the enhancement of active encounter complex formation. According to the above discussions, we proposed that the bound Na+ is, essentially, a negative effecter in thrombin-substrate stereospecific complex formation: thrombin is designed to possess lower Na+-binding affinity to optimize efficiency of the reaction under physiological condition.

Figure 8. Chimera thrombin structure (PDB entry: 3R3G)19. Difference in amino acid sequence between human and chimera is shown above. Lys149e and Glu149e are 31



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highlighted by bold character. In the structure illustration, S1-pocket is represented with orange surface. Autolysis, 186- and 220-loops are distinguished by green, magenta and yellow tubes, respectively.

Concluding Remarks In this study, we examined the effect of Na+-binding on the thrombin-substrate association reaction. It has long been believed that Na+-binding promotes thrombin-substrate stereospecific complex formation.3,7 However, we revealed that the bound Na+ plays the opposite role, namely, interference with the substrate access to S1-pocket. According to the insight obtained, we proposed the role of relatively low Na+-binding affinity of thrombin, which reduces the negative effect of the bound Na+ on the stereospecific complex formation. Meanwhile, other cation-activated allosteric enzymes are also known to show relatively low cation binding affinity.21-23 Although the functional roles would be different depending on the enzymes, relatively low cation-binding affinity might be an important factor to activate enzymatic reaction for such enzymes. Meanwhile, it still remains elusive how the efficiency of thrombin-substrate association reaction is maximized under the presence of Na+. Considering that

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Na+-binding cavity is located in the vicinity of S1-pocket (see Figure 1B), it could be assumed that Na+-binding affects structural fluctuation of S1-pocket and then expands the volume. Earlier X-ray crystallographic studies resolved thrombin structures whose S1-pocket and Na+-binding cavity collapsed simultaneously.24-28 These observations support the possibility for interaction between S1-pocket and Na+-binding. Although Pineda’s study showed that S1-pocket is stably formed with no regard to Na+-binding under thermal equilibrium,7 it is possible that S1-pocket transiently collapses by thermal fluctuation within the timescale for the blood coagulation reaction, which interferes with the substrate access. Under the circumstances, keeping S1-pocket earlier than the substrate access would arrange for the chance of the substrate access to the pocket and promote the stereospecific complex formation as a result. On the other hand, remembering association reactions consist of two steps, the presence of Na+ would also be involved in the encounter complex formation. This insight is supported by our earlier study10, where we suppose that unbound Na+ surrounding thrombin contribute to optimal formation of thrombin-substrate encounter complex ensemble. It should be noted that these two scenarios refer to the role of Na+ in the previous step of the thrombin-substrate stereospecific complex formation. Actually it has been considered that the bound Na+ promotes the stereospecific complex

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formation. However, the results we obtained encourage a reconsideration of this conventional scenario. By elucidating the alternative scenarios, we will address to clarify the comprehensive mechanism of Na+ activation of thrombin.

ACKNOWLEDGMENT 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 “The Strategic Program for Innovation Research (SPIRE)”. The calculations were partially performed using the computing systems at the Information Technology Center (ITC) in Nagoya University. I.K. also thanks Japan Society for the support via Promotion of Science (JSPS) by the Research Fellowship for Young Scientist.

Supporting Information More details for Materials and Methods, and Supporting Materials as noted in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

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Bound Na+ is a Negative Effecter for Thrombin-Substrate

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