Dewetting of S1-Pocket via Water Channel upon Thrombin–Substrate

Article pubs.acs.org/JPCB

Dewetting of S1-Pocket via Water Channel upon Thrombin− Substrate Association Reaction Ikuo Kurisaki,†,‡ Chantal Barberot,†,‡ Masayoshi Takayanagi,†,‡ 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: Upon protein−substrate association reaction, dewetting of the substrate-binding pocket is one of the rate-limiting processes. However, understanding the microscopic mechanism still remains challenging because of practical limitations of experimental methodologies. We have addressed the problem here by using molecular dynamics (MD) simulation of the thrombin−substrate association reaction. During the MD simulation, ArgP1 in a substrate accessed thrombin’s substrate-binding pocket and formed specific hydrogen bonds (H-bonds) with Asp189 in thrombin, while the catalytic serine of thrombin was still away from the substrate’s active site. It is assumed that the thrombin−substrate association reaction is regulated by a stepwise mechanism. Furthermore, in the earlier stage of ArgP1 access to the pocket, we observed that ArgP1 was spatially separated from Asp189 by two water molecules in the pocket. These water molecules transferred from the pocket, followed by the specific Hbond formation between thrombin and the substrate. Interestingly, they were not evacuated directly from the pocket to the bulk solvent, but moved to the water channel of thrombin. This observation indicates that the channel plays functional roles in dewetting upon the association reaction.

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INTRODUCTION Dewetting of the substrate-binding pocket is one of the ratelimiting processes in protein−substrate association reactions,1,2 although understanding the microscopic mechanism has remained challenging. This is essentially due to the practical limitation of experimental observations in spatiotemporal resolution. Under such circumstances, molecular dynamics (MD) simulations have drawn attention as a useful alternative to investigate the mechanism of dewetting upon the reaction at the atomic level.3,4 However, knowledge about such a dewetting process is still restricted, so that the mechanism we know should be only a small part of all the possible ones. At the atomic level, the mechanism of such dewetting can be described as water molecule transfer from the protein− substrate binding pocket. The previous simulation studies demonstrated a mechanism in which water molecules are directly evacuated from the binding pocket to the bulk solvent upon protein−substrate association reaction.3,4 Besides this suitable mechanism, we can assume another one, namely, water molecule transfer via a channel in protein. Actually, the presence of channels, which are connected with the substratebinding pocket, has been widely known among enzymes.5−8 However, it remains elusive whether such channels are actually involved in dewetting upon association reactions. In this context, understanding the mechanism of the thrombin−substrate association reaction can be a paradigm to © 2015 American Chemical Society

elucidate functional roles of channels in dewetting of a substrate-binding pocket. Thrombin, a serine protease, is a key player in blood coagulation. By reflecting the physiological role, the enzymatic activity is maximized under the presence of Na+.9 Actually, thrombin possesses a Na+-binding cavity consisting of 186- and 220-loops (Figure 1A).10 Of note, the Na+-binding cavity and the substrate-binding pocket, S1-pocket, form the channel which connects their entrances on the surface of thrombin (Figure 1A and Figure S1).5 The channel is occupied by 14 water molecules in the X-ray crystal structure (Figure 1C). Meanwhile, thrombin recognizes natural substrates with trypsin-like specificity.10 Asp189 inside the S1-pocket forms hydrogen bonds (H-bonds) with Arg at the P1 site in a substrate, referred to as ArgP1, and Tyr60a and Trp60d in a 60loop interact with a hydrophobic residue at the P2 site, for example, proline (Figure 1B). The S1-pocket is buried inside the protein (Figure 1A)10 and occupied by water molecules,5 thus being wetted in the absence of substrate binding. The Hbond formation should be regulated by dewetting of the S1pocket, accordingly. Furthermore, the mutations of ArgP1 significantly reduce the enzymatic activity of thrombin.10 It is, Received: October 1, 2015 Revised: December 3, 2015 Published: December 4, 2015 15807

DOI: 10.1021/acs.jpcb.5b09581 J. Phys. Chem. B 2015, 119, 15807−15812

Article

The Journal of Physical Chemistry B

Figure 1. Thrombin structure. (A) Overview of the thrombin-Na+ complex. (B) Vicinity of the S1-pocket. (C) Water channel of thrombin. 186- and 220-loops are distinguished by magenta and yellow tubes, respectively. The S1 pocket is illustrated with orange surface (solid in panel (A); transparent in panels (B) and (C)). In panel (B), ArgP1 and ProP2 in the model substrate are represented by the yellow and blue stick, respectively. The remaining parts of the substrate are shown by the finer blue stick. The 60-loop is represented by the gray ribbon. In panel (C), oxygen atoms in water molecules are shown by blue balls. The atomic coordinates were derived from thrombin-Na+ complex structure (PDB entry: 1SG8).11

Figure 2. Analyses of the thrombin−substrate association reaction. (A) Interatomic distance between CγAsp189 and CζArgP1. (B) Number of hydrogen bonds formed between Asp189 and ArgP1. (C) Representative snapshots of the structures upon the association reaction. In panels A and B, the open and closed arrows indicate the timing of substrate entering the S1-pocket and hydrogen bond formation between Asp189 and ArgP1, respectively. In panel C, Asp189 and S1-pocket are illustrated with gray stick and transparent orange surface, respectively. ArgP1 and the remaining part of the substrate are illustrated with yellow and blue sticks, respectively. The two water molecules, separating Asp189 and ArgP1 around the time of Asp189ArgP1 hydrogen bond formation, are annotated as Wat-A and Wat-B. The former and latter are represented by red and green vdW spheres, respectively. Transparent blue vdW spheres denote other water molecules observed around the S1-pocket.

previous work.12 All the MD simulations were executed by AMBER 14 pmemd or pmemd.cuda modules.15,16 With the structure restraints on thrombin-Na+ complex the positions of ions and substrates were energetically relaxed at 300 K, and randomized through heat diffusion at 500 K. After equilibration under NVT condition at 300 K, production NPT MD simulations at 1 bar and 300 K were executed for 50 ns without any restraints. This procedure was repeated 50 times with different initial atomic velocities, resulting in 50 independent MD trajectories. Then, we focused on the trajectory where ArgP1 in a tripeptide partially enters thrombin’s substrate binding pocket during the simulation. The trajectory was extended to 500 ns aiming to observe thrombin−substrate association reaction. All the images of protein structures were drawn with VMD.17 Interatomic distances and H-bonds were calculated by the AmberTools 1.5 cpptraj module.18 Grid points representing the interior of thrombin were generated by the POVME program,19 and water transfer was analyzed by an in-house program. More

therefore, possible to say that formation of the H-bond in the S1-pocket should be a key factor for the association reaction. Keeping in mind these observations, we elucidated functional roles of thrombin’s channel in dewetting of the substratebinding pocket. The thrombin−substrate association reaction was simulated in explicit 140 mM NaCl aqueous solution. In accordance with the thrombin’s substrate specificity, Pro-Arg tripeptide was employed as a model substrate. Analyzing an MD simulation trajectory undergoing the reaction, we examined how water molecules transfer from the substratebinding pocket upon H-bond formation.

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MATERIALS AND METHODS

The initial thrombin-Na+ complex structure was prepared according to our previous work12 and immersed in the periodic boundary box with 11 595 water SPC/E water molecules,13,14 and 32 Na+ and 48 Cl−. In addition, 12 Pro-Arg tripeptides were set to the surface of periodic boundary box, initially. The MD simulation force field parameters were identical to our 15808

DOI: 10.1021/acs.jpcb.5b09581 J. Phys. Chem. B 2015, 119, 15807−15812

Article

The Journal of Physical Chemistry B details of the Materials and Methods are described in the Supporting Information.

Meanwhile, our thrombin−substrate simulation shows that these water molecules transfer from the S1-pocket into the interior of thrombin, accompanied by H-bond formation. It is, therefore, possible to say that we found an alternative mechanism of dewetting, that is, water molecule transfer in a water channel. Furthermore, we observed that the water molecules separating Asp189 and ArgP1 were repeatedly replaced by the timing of the H-bond formation (Figure 2C provides an illustration as for the two water molecules). This observation indicates that water molecules continually transfer in the channel. To provide quantitative insights into water molecule transfer in the water channel, at first, we structurally characterized the channel by using grid points. As reported in the earlier study, this channel includes two regions, S1-pocket and Na+-binding cavity.5 Furthermore, we defined two more regions around them by observing the position of crystal water11 (see Figure 1C) and water molecule transfer found in our MD simulation. One is located in the vicinity of the S1-pocket and the Na+binding cavity (Figure 4A,B, and Figure S1A). We name it

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RESULTS AND DISCUSSION Dewetting of S1-Pocket Is Followed by Hydrogen Bond Formation between Asp189 and ArgP1. Among the 50 simulations we carried out, we found one MD trajectory undergoing the specific H-bond formation between Asp189 and ArgP1. In the trajectory, the interatomic distance between CγAsp189 and NζArgP1 shows remarkable changes around 19.12 ns, and at 237.27 ns (Figure 2A). Around 19.12 ns, ArgP1 in the substrate enters the S1-pocket, although at this stage, this residue does not form H-bonds with Asp189 (Figure 2B). Water molecules in the S1-pocket spatially separate these two residues (Figure 2C, left and right middle panels), so that the S1-pocket is still wetted. Although ArgP1 transiently recedes from Asp189 around 80 ns, it returns and stays there until just before the H-bond formation. At 237.27 ns, the two residues form H-bonds. This is accompanied by transfer of the water molecules separating them (annotated by Wat-A and Wat-B in Figure 2C). After this dewetting process, the H-bonds are stably formed during this simulation (Figure 2B). Meanwhile, distance between OγSer195 and CArgP1 remains to be ca. 7 Å during this simulation (Figure 3A and B). In the Michaelis complex, however, the distance

Figure 4. Illustration of thrombin’s water channel. (A) Frontal view of the water channel. (B) Lateral view of the water channel. 186- and 220-loops are distinguished by magenta and yellow tubes, respectively. The 166−171-helix and S1-pocket are illustrated with green helix and transparent orange surface, respectively. The spaces forming S1pocket, Na+-binding cavity, DYA cavity, and HELP pocket are represented by green, blue, pink, and orange balls, respectively.

Figure 3. Structural characterization of thrombin active site. (A) Interatomic distances between OγSer195 and CArgP1. The open and closed arrows indicate the timing of substrate entering the S1-pocket and hydrogen bond formation between ArgP1 and Asp189, respectively. (B) Snapshot of the structure at 500 ns. Asp189 and S1-pocket are illustrated with gray stick and transparent orange surface, respectively. ArgP1 and the remaining part of the substrate are illustrated with yellow and blue sticks, respectively. OγSer195 and CArgP1 are highlighted by red and blue balls.

“Asp-Tyr-Ala cavity” or “DYA cavity” because Asp189, Tyr228, and Ala183 are found in the region. Interestingly, this triad can form H-bonds with a water molecule (see Figure S1B), thus possibly influencing transfer of water molecules between this region and others. The role of the H-bonds will be investigated in a future study. The other region is found as a space between 166−171-helix and the 220-loop. We named it the “HELP pocket” because this pocket is located between the “helix” and the “loop” (see Figure 4). Keeping in mind the four regions, we examined how frequently water molecules move in the channel (Table 1). We focused here on the water molecules undergoing transfer between the S1-pocket and the Na+-binding cavity, in particular. Frequencies of water molecule transfer are nearly balanced between the two regions so that it is possible to say that transfer of water molecules in thrombin is in equilibrium, thus occurring continually in our simulation. After the H-bond formation, these frequencies apparently decrease. This could be explained by ArgP1 occupying the S1-pocket, which allows fewer water molecules to be located inside the S1-pocket. Figure 5A shows that the number of water molecules in the S1pocket is gradually reduced after H-bond formation, actually.

should be smaller than 3.0 Å for the acylation process to proceed.20 This observation indicates that the complex formation is still incomplete in our simulation. According to the above analyses, we propose a three-step reaction mechanism with respect to the thrombin−substrate association reaction. First, ArgP1 in a substrate enters the S1pocket. Next, this residue forms the specific H-bonds with Asp189. Finally, the backbone of ArgP1 reorients to react with the catalytic residues in thrombin. Similarly to the earlier studies,21−23 our simulation also suggests that an association reaction starts from the formation of a nonspecific interaction, and then shifts to formation of a specific one. Water Molecules Continually Transfer within Thrombin Channel. It should be noted that, in the second step of the reaction, the two water molecules separating Asp189 and ArgP1 transferred from the S1-pocket (see Figure 2C). In the previous simulation studies on protein−drug binding, water molecules are directly evacuated from the binding pocket to the bulk solvent upon the formation of a specific interaction.3,4 15809

DOI: 10.1021/acs.jpcb.5b09581 J. Phys. Chem. B 2015, 119, 15807−15812

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the space at 163.69 ns, moves to Na+-binding cavity at 237.27 ns, and is evacuated to the bulk solvent from Na+-binding cavity at 361.36 ns (Figure 6B,D, and Figure 7B). Of note, these two water molecules are found in the bulk solvent at the beginning of the MD simulation, and approach the space between Asp189 and ArgP1 in the middle of the simulation. Furthermore, one of the two, Wat-B, is evacuated from the channel to the bulk solvent during the simulation. Their behavior, therefore, can be explained from the above insights that water molecules continually transfer in the channel and they are repeatedly replaced with each other. It is, therefore, possible to say that Wat-A and -B happened to be around Asp189 and ArgP1 at the time of the H-bond formation. This observation suggests that continual transfer of water molecules in the channel itself is a hidden but important factor for dewetting of the S1-pocket and, thus, for the thrombin−substrate association reaction rate. Concluding Remarks. In the present study, we simulated the thrombin−substrate association reaction and observed the specific intermolecular H-bond formation accompanied by water molecule transfer in the water channel. This observation provides new insights that the water channel plays functional roles in dewetting of the S1-pocket, and continual transfer of water molecules in the water channel is an alternative factor influencing the reaction. Actually, the presence of water channels, which are located in the vicinity of substrate-binding pockets and lead to surfaces of enzymes, has been widely discovered among serine proteases5 and also other enzymes.6−8 The insight obtained from our study could be extended into general cases of protein−substrate association reaction, accordingly. The insight we obtained also suggests the possibility that the association reaction rate is tuned by controlling the frequency of water molecule transfer in the water channel. This possibility lets us assume an innovative strategy with respect to new drug design, new application of drugs, and adverse-drug-effect prediction, where protein functions are allosterically regulated by a drug which modifies the frequency of water molecule transfer in a water channel. Meanwhile, earlier studies demonstrated the possibility that water channels play roles in allosteric regulation.24−26 It is therefore concluded that the present study broadened the understanding the potential roles of water channels for expression of protein function.

Table 1. Frequency of Water Molecule Transfer between Regions in Thrombin Channel frequency of water molecule transfer [ns−1]

a

former and later regions

0−237.26 [ns]

237.27−500 [ns]

1→2 2→l 1→3 3→1 1→4 4→1 2→3 3→2 2→4 4→2 3→4 4→3

12.51 12.51 1.04 1.08 53.03 52.98 4.80 4.79 34.12 34.14 0.08 0.11

5.36 5.34 0.17 0.19 0.08 0.08 1.95 1.94 15.62 15.62 0.05 0.06

1: S1-pocket; 2: Na+-binding cavity; 3: HELP pocket; 4: DYA cavity.

We also observed that the number of water molecules in S1pocket and Na+-binding fluctuates during the MD simulation (see the red line in Figure 5A). The pocket and the cavity can have the greater number of water molecules than those found in the X-ray crystal structure (see Figure 1C and the related discussion). This should be explained by the following two reasons. First, both the cavity and the pocket are structurally flexible under thermal fluctuation, thus allowing a greater number of water molecules to access them. Second, our analyses can take into account thermally unstable water molecules, which appear around entrances of the pocket and the cavity, and thus cannot be resolved experimentally (see Figure 5B for the representative snapshot structure, where the pocket and the cavity possess 36 water molecules). These observations clarify that water molecules in the channel do not occupy specific positions, but move actively. Thrombin Channel Is Involved in Dewetting of S1Pocket. We here revisited analyses of the two water molecules which separate Asp189 and ArgP1 around the time of Asp189ArgP1 H-bond formation (Figure 6). As denoted in Figure 2C, we refer to these water molecules as Wat-A and Wat-B. Wat-A enters the channel via S1-pocket at 75.68 ns, approaches the space between Asp189 and ArgP1 at 158.16 ns, moves to DYA cavity at 237.27 ns and stays in the channel during this MD simulation (Figure 6A,C, and Figure 7A). Meanwhile, Wat-B enters the channel via Na+-binding cavity at 67.5 ns, approaches

Figure 5. Water molecules inside S1-pocket and Na+-binding cavity (A) Number of water molecules. (B). Snapshot of the structure at 10.31 ns. In panel (B), 186- and 220-loops are distinguished by magenta and yellow tubes, respectively. The S1 pocket is illustrated with transparent orange surface. Oxygen atoms in water molecules are shown by light blue balls. 15810

DOI: 10.1021/acs.jpcb.5b09581 J. Phys. Chem. B 2015, 119, 15807−15812

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Figure 6. Location of the water molecules separating Asp189 and ArgP1. (A) and (C) Wat-A. (B) and (D) Wat-B. Wat-A and -B are the water molecules discussed in Figure 2C. The black arrow indicates the timing of the hydrogen bond formation. The red and blue arrows indicate the timing of water entering and exiting from the water channel of thrombin. Panels (C) and (D) highlight the time duration around the timing of Asp189ArgP1 hydrogen bond formation and the vicinity.

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Figure 7. Interatomic distance between water molecule separating Asp189 and ArgP1 and these two residues. Panels (A) and (B) are for Wat-A and Wat-B, the water molecules discussed in Figure 2C and Figure 5, respectively. The green arrow indicates the timing when the water molecules approach the space between Asp189 and ArgP1.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b09581. More details for Materials and Methods, and Supporting Figure as noted in the text (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was partially 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 and by a Grant-in-Aid for Science Research from the Ministry of Education, Culture, Sport, Science, and Technology in Japan. The calculations were partially performed using several computing systems at the Information Technology Center in Nagoya University. I.K. also thanks Japan Society for the support via Promotion of Science (JSPS) by the Research Fellowship for Young Scientist. 15811

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DOI: 10.1021/acs.jpcb.5b09581 J. Phys. Chem. B 2015, 119, 15807−15812