A mechanism of catalyzed GTP hydrolysis by Ras protein through magnesium ion

Chemical Physics Letters 516 (2011) 233–238

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A mechanism of catalyzed GTP hydrolysis by Ras protein through magnesium ion Qiang Lu a,f, Nicolas Nassar b, Jin Wang a,c,d,e,⇑ a

Department of Chemistry, Stony Brook University, United States Department of Physiology and Biophysics, Stony Brook University, United States c Department of Physics, State University of New York at Stony Brook, United States d Department of Applied Mathematics, State University of New York at Stony Brook, United States e State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, China f Natural Science Department, Shorter University, Rome, GA 30161, United States b

a r t i c l e

i n f o

Article history: Received 7 March 2011 In final form 30 September 2011 Available online 8 October 2011

a b s t r a c t The hydrolysis by Ras plays pivotal roles in the activation of signaling pathways that lead to cell growth, proliferation, and differentiation. Despite their significant role in human cancer, the hydrolysis mechanism remains unclear. In the present Letter, we propose a GTP hydrolysis mechanism in which the c phosphate is cut off primarily by magnesium ion. We studied both normal and mutated Ras and the cause of the malfunction of these mutants, compared the effect of Mg2+ and Mn2+. The simulation results are consistent with the experiments and support the new hydrolysis mechanism. This work will benefit both GTPases and ATPases hydrolysis studies. Ó 2011 Elsevier B.V. All rights reserved.

1. Introduction Ras, a typical GTPases, plays a central role in relaying extracellular signals to gene expressions that control cell growth, proliferation and differentiation, cytoskeletal organization, and development [1]. It cycles between a GTP-bound form (RasT) and a GDP-bound form (RasD). RasT adopts the active conformation of the switch and turns on cellular signaling pathways. GTP hydrolysis in turn turns off the switch by converting RasT to RasD. Ras mutations at hot spots, such as G12 and Q61, result in a slower GTP-hydrolysis rate that leads to longer activated signaling pathways ultimately promoting uncontrolled cell growth and are responsible for over 25% of human tumors [2]. Almost all GTPases and ATPases, including Ras and Ga, require a magnesium ion for their catalytic activities (Figure 1). People believe that magnesium ion is presumably required both for proper positioning of the c phosphate and for weakening the P–O bond that is split during catalysis, as well as for maintaining the stability of the guanine nucleotide complex [3]. Based on the existing mechanisms, hydrolysis is mainly caused by an attacking (or nucleophilic) water to the c phosphate resulting in bond breaking between the c–b phosphate groups [4]. Despite the sizable amount of work for the last two decades [4–8], the microscopic mechanism of the GTP hydrolysis still remains unclear. In the current work, we propose a new hydrolysis mechanism in which the magnesium ion is the major factor for the GTPase-cata⇑ Corresponding author at: Department of Chemistry, Stony Brook University, Stony Brook, NY 11794-3400, United States. Fax: +1 631 632 7960. E-mail address: [email protected] (J. Wang). 0009-2614/$ - see front matter Ó 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2011.09.071

lyzed reaction besides the nucleophilic water. In our hypothesis, the hydrolysis reaction pathway can be divided into two steps: pre-hydrolysis and hydrolysis. In the first step, the bridge oxygen of b–c phosphate, O3B, rotates towards the Mg2+ and becomes one of its ligands, forming a twisted octahedron complex (Figure 2). This is caused by the Ras protein and the thermal motions of the bridge oxygen. In the hydrolysis step, the twisted octahedron complex is rearranged and restored to a normal octahedron structure, meanwhile the bond between Pc and the bridge oxygen is broken during the restoration of the metal complex. There are four main reasons for us to propose this mechanism. First, all GTPases and ATPases have magnesium ion in the vicinity of c phosphate as a common similarity. Second, in many Ras PDB structures, the bridge oxygen, O3B, becomes a ligand of the magnesium ion after hydrolysis in Ras–GDP structure, but does not in Ras– GTP structure [9–21]. Besides, the symmetry between the bridge and other c phosphate oxygen are distinguishable. This give the evidence for the rotation of b phosphate before hydrolysis. The third reason is that according to this mechanism the charge shifting is only involving one c phosphate oxygen during the catalysis, comparing to the charge shifting in three c phosphate oxygen atoms in the traditional nucleophilic water mechanism [4]. Furthermore, if our hypothesis is true, the normal Ras must have the lowest transition state in free energy and the hydrolysis step should be a down hill process. Both energy and free energy calculations do confirm the idea and support this mechanism of all seven typical mutants (Q61L, Q61I, Q61K, Q61G, Q61V, G60A, G12V) and normal Ras. Minimum energy path. First, at the pre-hydrolysis step, we only focus on the rotation of O3B, the bridge oxygen, till it becomes one of the Mg2+’s ligands. The minimum energy path method in Moil

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Q. Lu et al. / Chemical Physics Letters 516 (2011) 233–238 Table 1 In the pre-hydrolysis process, energy barrier heights (kcal/mol) difference between wild type and mutants.

Figure 1. RasT structure, SI (25–40) is in yellow, SII (57–75) is in orange, P-loop (12–16) is in blue. Mg2+ is in purpleblue. While GTP is in colorful sticks and balls. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Type

Wild

Q61K

Q61L

Q61G

Q61I

Q61V

G12V

G60A

Refs. DEM-W

[24] 0.0

[25] 6.5

[25] 15.7

[7] 20.3

[25] 1.7

[25] 12.3

[26] 20.6

[27] 6.1

package [22] can provide a reasonable reaction coordinate for the free energy calculation [23] based on the known two end structures. The Chmin program is used in the Moil package to find the minimum energy path using self penalty walk (SPW) algorithm [22]. The SPW can easily handle more than 100 particles and is capable of calculating continuous and well defined low energy paths even if a reasonable adiabatic coordinate is not available. Its advantage compared to algorithms used for small molecules is that it does not require the second derivative matrix. This makes it possible to calculate reaction coordinates in large molecular systems in a stable and reasonably economic way. In the SPW approach the reaction coordinate is approximated by a discrete set of intermediates that interpolate between the known reactants and the known products. The existing NMR or X-ray crystal structure coordinates are used to generate the two end structures for normal and mutated Ras. The initial and final target structures of Ras protein are all in GTP-bound form, and the most conformational differences are located in GTP with a rmsd = 0.678 Å. In the target GTP structure, or the transient state, O3B is turned to the Mg2+ ion and becomes one of its ligands. The transient GTP structure is obtained by aligning the GDP in the 4Q21 [10], a RasD, with GTP in 5P21 [24], a RasT. In 4Q21, the O3B is already one of the Mg2+’s ligand but not in 5P21. GTP in 5P21 is replaced with the aligned GDP and a c phosphate is added to the GDP to form the transient structure. The PDB structures for mutants and their references are listed in Table 1.

Figure 2. In the pre-hydrolysis step, the bridge oxygen between b phosphate and c phosphate turns to Mg2+ and becomes one of its ligand. The red color represents the a, b and c phosphate of GTP. The green color represents residues 12, 13, 14, 15, 16, 17, 18 and 35. The four pictures are 1, 5, and 9 frames along the reaction pathway. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

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A total of 9 replicas, including the two end reference structures, RasT and the transient state, are generated for the Ras normal and seven mutants by linear interpolation between the two ends. Using wild type as an example, the atomic pictures of the reaction pathway around the b and c phosphate are shown in Figure 2, in which the three structures correspond to the 1, 5 and 9 frame. At the first frame, Mg2+ ligands are O2G from the c phosphate and O2B from b phosphate, together with two other water molecules and Ser17 and Thr35, forming an octahedron structure for both wild type and mutants. The main variation along the path is the bridge oxygen O3B turns and becomes a ligand of Mg2+, while the initial Mg2+ ligand O2B rotates away. c phosphate does not rotate, but is stretched to a little extent; the new Mg2+ ligands between O3B and O2G are not at a normal right angle, but a bent sharp one. The last frame is the transient state waiting for the hydrolysis cut by Mg2+ when it rearranges its ligands. Only electrostatic forces are used to represent the Mg2+–O dipolar bond (coordinate covalent bond). b and c phosphates sink into the cavity formed by residue 12–17, 35 and establish hydrogen bonds and salt bridges with Mg2+, G12, G13, V14, G15, K16. Q61 and G12, the hot mutation spots, are the caps that cover the pocket of GTP and constrain GTP in the cavity. The rotation of b phosphate needs to break these interactions and reform a network in this constrained space. These interactions provide a hindrance for the rotation. Figure 2 also shows the minimum path energy in the pre-hydrolysis steps for both wild type and seven mutants. The higher barrier of the potential energy indicates the harder the turning of GTP b phosphate. Table 1 lists the energy difference at transition structure (5th frame) between mutant and wild type. One can see the wild type has the lowest energy at the transition structure. All other mutants have higher energy barrier. The mutants Q61I and Q61K both have lower energy at the final 9th frame, i.e. the transient state. Free energy along the reaction path. In order to check if normal Ras has the lowest free energy at transient state, the umbrella sampling method [28] is improved and used to perform free energy calculations for the seven mutants and normal Ras. Umbrella sampling method is very efficient in sampling low probability events [29]. Figure 3 presents the free energy along the reaction pathway. The barrier in the potential energy disappeared because of the thermal motion of O3B and entropy contributions. From the Figure 3 one can find that the wild type has the steepest downhill

Table 2 The Free energy difference (in kcal/mol) from initial GTP-Ras to transient state for wild type and mutant Ras. Type

Wild

Q61K

Q61L

Q61G

Q61I

Q61V

G12V

G60A

DG T

3.96

2.29

1.45

1.06

1.08

1.48

1.77

2.20

process, and its final 9th frame (the transient state) has lower free energy than all of the mutants. The wild type has a gap of 3.96 kcal/mol between the last and the first step. That makes the turning of O3B steadily and mostly in one direction, so that the hydrolysis in the aid of Mg2+ in the next step becomes possible. The details of simulations are described in Section ‘‘Method’’. Table 2 shows the free energy gap between the initial and last steps for wild type and mutated Ras. In the experiment, mutations in codon 12, 13 or 61 all decrease the in vitro GTPase activity of Ras at least 10-fold less than normal Ras [30]. From Figure 3 and Table 2, one can find that, at the final frame, the mutant Q61K has the lowest free energy and a difference, 1.67 kcal/mol or 2.8 kT, higher than that of normal Ras, which gives about 16-fold decreased bioactivity for single Ras comparing to the experimental 10-fold transforming activity measured in the NIH 3T3 cell which is in vitro. At transient state, the mutantion’s free energy difference is 4.5 kcal/mol, which is 7.5 kT and gives about 1800 fold difference among mutations for single molecule comparing to experiment more than 1000 fold for in vivo. Note both transient and transition states refer to that of wild type. After the transient state, once the Mg2+ takes charge, it will be a downhill process, which will be shown in the following QM calculations. Analysis and explanation of the mutants’ malfunction. It seems that normal Ras has more freedom and space for the bridge oxgygen to move or turn. In order to check the structure correlation with free energy and understand the cause of the malfunction of mutants, the cavity size for c and b phosphate are calculated for the normal Ras and mutants using software package 3 V [31] after excluding the water and GTP molecules. Three angstrom is used to mimic the phosphate to find the solvent excluded volume and 9 Å is used to find the shell volume. The cavity volume is the difference between the shell volume and solvent excluded volume. In Figure 5, we show the cavity volume of GTP phosphate of wild type, Q61L and G12V at the transient state. The wires represent surrounding residues and the sticks represent GTP and water molecules. The transparent surface represents the Ras cavity for phosphates. Normal Ras clearly has greater cavity volume than

G60A G12V Q61V

-5 -6

3000

Q61I

-7

Q61L

-8

Wild

2500

Q61G

Cavity Volume (Å3)

Free Energy (kcal/mol)

-4

Q61K

-9

WILD -10

Ras-GTP

Transient

Q61G

2000

Q61V

1500

G12V 1000

Correlation Coefficient: −0.82

-11 1

2

3

4

5

6

7

8

9

Reaction Pathway Figure 3. Free energy along the reaction pathway in which the bridge oxygen between b and c phosphate turns into one of the ligand of Mg2+. The last frame is the transient state which will be hydrolyzed by Mg2+ in the next step. At transient state, the mutation’s free energy difference is 4.5 kcal/mol, which is about 7.5 kT and gives about 1800 fold difference among mutations in single molecule comparing to that in vivo which is more than 1000 fold.

Q61I

Q61K Q61L

500

G61A 0

0

1

2

3

4

5

6

7

Free Energy Gap Difference to Normal Ras (kcal/mol) Figure 4. The correlation plot between the b, c phosphate cavity volume of mutated or normal Ras and the free energy gap difference between mutant and normal Ras.

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Figure 5. At the transient state, the cavity volume of the normal Ras is clearly greater than that of mutants, as gives O3B enough room and freedom to rotate.

Figure 6. The energy barrier in prehydrolysis step need to be overcome with the help of Ras protein. Mg2+ will do the rest in the hydrolysis step. c and b phosphate are split when Mg2+ restoring the abnormal octahedron Mg2+ ligand complex. Mn2+ has the same function as Mg2+ does. It needs 2 kcal/mol less energy, or 100 fold faster to cross over the transition state, comparing to 4-fold faster in the additional help of a Ras protein in experiment [35]. GDP is represented by b phosphate only in the simplified model.

the mutants. In Figure 4, we show the correlation between the cavity volume and the free energy gap difference (DGmut–DGwild) for normal and mutated Ras. The normal Ras has the biggest cavity volume for turning or moving. On the contrary, G61A has the smallest phosphate cavity volume, correspondingly it has the highest free energy in Figure 4. G12V has the second least cavity volume, and correspondingly it has the second highest transient state free energy. The correlation coefficient between them is 0.82 for all mutants. The high correlation coefficient further confirms our hypothesis and the new hydrolysis mechanism. Quantum calculation of PAO bond breaking. In order to understand the bond association and breaking in both prehydrolysis and hydrolysis process, we performed the quantum calculations based on a simplified structure to save computer time, although the environmental residues could affect the hydrolysis. The simplified structure only includes b and c phosphate and the Mg2+ complex. For residues Thr17 and Thr35, only the oxygen atoms in their side chains are kept and become two water molecules

after adding hydrogen atoms. For prehydrolysis process, four structures were evenly chosen from the Chmin paths to perform the single point calculation. While for hydrolysis process, start from the transient state, both O3B and O2G are the ligands of Mg2+ and both form a bond with Pc which gives a twisted sharp angle for O2G–Mg2+–O3B, the single point energy scan is performed when the angle O2G–Mg2+–O3B extended from a sharp to a right one. All the atoms are frozen at their positions and only the O2G–Mg2+–O3B angle and Pc–O3B bond are allowed to change. The energy is calculated with tight option using the B3LYP density functional method and the 6-311++G⁄⁄ basis set [32] in GAUSSIAN98 [33] and shown in Table 3 and Figure 6. In Figure 6, during the hydrolysis process, last 6 frames are shown the increase of the O2G–Mg2+–O3B angle. While the energy is decreasing as the angle increases. In other words, it is a favorite process as the Pc–O3B bond is broken, c phosphate is hydrolyzed and the octahedron coordination complex structure is restored to normal (see Table 3).

Table 3 The QM energy (in kcal/mol) of the simplified GTP-Ion system are listed in details for Figure 6. Mn2+ has 2 kcal/mol less energy at the transition state, or 100 fold faster to cross over the transition state. Ras will dramatically lower the barrier in the case of both Mg2+ or Mn2+. However Mn2+ will still be 4.4-fold faster than that of Mg2+ [35]. QM energy

1

2

3

4

5

6

7

8

Mg2+ Mn2+

0.0 0.0

6.41 2.54

73.98 71.13

43.42 38.15

2.88 13.01

14.62 23.80

17.14 22.15

17.45 25.54

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Substitution of Mn2+ metal ions for Mg2+ can increase GTP hydrolysis by 4.4-fold [35]. In order to verify this fact, the Mn2+ ion is replaced with an Mn2+ ion in the simplified system. The GTP is taken from PDB 1JAI, which was used in the experiment [35]. Our simulation results, in Figure 6, show that Mn2+ gives lower free energy barrier about 2.9 kcal, or 4.6 kT, or 100 fold faster than that of Mg2+. Note we are using the simplified system comparing with the Ras protein in experiment, however the trend is clear and agree with the experiment. Besides the GDP with Mn2+ has lower energy and more energy will be released during hydrolysis than that of Mg2+. Charge shifting. Furthermore, one can better understand the bond breaking process by investigating the charge shifting from c phosphate to b phosphate as the Pc–O3B bond is hydrolyzed. The ESP charge is calculated to observe the charge shifting for each step. Table 4 lists the traditional estimated charge shifting [34] and the calculated charge shifting in the present quantum simulations. No symmetric constraint is applied. O3G and O3B are two ligands of Mg2+ and only these two have major charge shifting, other oxygen atoms do not change much. This is different from the traditional mechanism which requires the charge shifting for all the c and b oxygen atoms. The hydrolysis mechanism based on Mg2+ apparently requires less electron transfer energy and is easier for the hydrolysis to happen than caused by the nucleophilic water molecule. In the current work, we propose a new hydrolysis mechanism in which the magnesium ion takes the major responsibility for the GTPase-catalyzed reaction. The driving force of hydrolysis is due to the rearrangement of the ligands by Mg2+ back to the normal octahedron complex, and the bond between Pc and the bridge oxygen will be broken. All the energy and free energy calculations support our hypothesis for both normal Ras and seven typical cancerous mutants. Our mechanism can well explain the cause of the malfunction of the mutants, can confirm the fact that the replacement of Mn2+ will accelerate hydrolysis. This work could provide important clue to tackle with other cancerous mutants study involving both GTPases and ATPases, since they all have magnesium ion in their catalytic reaction.

PDB ID 5P21 [24] was used to represent the GTP-bound structure of wild type Ras. PDB ID 2UZI [26] was used for the GTP-bound form of the G12V mutant of Ras. PDB ID 1ZW6 [7], 2RGA, 2RGB, 2RGC and 2RGD [25] were used to represent the mutants Q61G, Q61I, Q61K, Q61V and Q61L, respectively. PDB ID 1XCM [27] was used to simulate G60A GTP-bound structure. The initial and target structures were minimized for 1000 steps with the steepest descent (SP) method and the value of the energy gradient of 0.01 kcal/mol Å was reached. Constant dielectric 1.0 was used because we use an explicit water shell surrounding the protein instead [22]. 2.2. Explicit water shell Structural water molecules in the aligned PDB structures of wild type and the seven mutants are collected. For each single Ras structure, either mutated or normal, the original water molecules are kept, the collected water molecules are added if there is no overlay. The water molecules between the initial and final structures are paired if the two water molecules from the two end structures are within the range of 3 Å. Unpaired water molecules will be removed. In addition, the numbers of water molecules are set to the same for all normal and mutants Ras. The rest are removed. This amounted to 223 water molecules for each mutant and wild type. 2.3. Minimum energy path simulations In our simulation, the initial and target structures are minimized for 1000 steps with the steepest descent (SP) method and the value of the energy gradient of 0.01 kcal/mol Å is reached. The initial paths are minimized with 1500 simulation steps with a spring constant of 200 kcal/mol. A final energy gradient of 0.001 kcal/mol Å is reached. There is no truncation of electrostatic and van der Waals interactions. Constant dielectric 1.0 was used because we use an explicit water shell surrounding the protein instead. 2.4. Umbrella sampling method simulations

2. Methods and materials: computational details

If P and G are simulated probability and free energy respectively, then they have the relation:

2.1. System preparation To investigate the structural changes that accompany GTPhydrolysis by wild type and single point mutants of Ras, existing NMR or X-ray crystal structure coordinates were used to generate initial paths. The initial and final target structures of Ras protein are all, in GTP-bound form, there are tiny differences (RMSD = 0.139 Å) between them before and after minimization. However the most conformational differences are located in GTP with a RMSD = 0.678 Å. In the target GTP structure, or the transient structure, O3B is turned to the Mg2+ ion and becomes one of its ligands. The transient structure was obtained by aligning the GDP in the 4Q21, a GDP-Ras crystal structure, with GTP in 5P21, a GTP-Ras crystal structure. In 4Q21, the O3B is already one of the Mg2+’s ligand. In 5P21, the GTP is replaced with the aligned GDP and then a c phosphate is added to the GDP to form the transient structure.

Table 4 The second row is the traditional estimated charge shifting from Ref. [34]. The third row is the charge shifting calculated according to our hydrolysis mechanism. Charge shifting

O1G

O2G

O3G

O1B

O2B

O3B

Traditional Mg2+ based

0.26 0.08

0.26 0.07

0.26 0.26

0.14 0.13

0.14 0.04

0.55 0.42

Pi ¼ eGi =kT ¼ eðF i þV i Þ=kT :

ð1Þ

Index i indicates each constrained window for umbrella sampling. Vi is the constraint or the biasing quadratic potential Vi = a(x–x0i)2, where a is the force constant, and x0i is the center of the constraint located on the nine structures along the reaction path. We assume approximately that Fi = bi(x–x0i) + ci, which is the piecewise linear free energy need to be found without constraint. bi and ci are parameters that need to be determined through simulation results. Then the simulated probability distribution at ith window can be written as

Pi ¼ eGi =kT ¼ eðaðxx0 i Þ

2

þbi ðxx0 i Þþci Þ=kT

:

ð2Þ

The x-value, xmi = x0i–bi/2a, at which the most probable distribution Pi(xmi) is, can be measured from the simulation results. Then Eq. (2) 2 becomes kTlnP i ðxmi Þ ¼ ci  bi =4a. Furthermore, we have 2 ci ¼ kTlnP i ðxmi Þ þ bi =4a and bi = 2a(xmi–x0i). After the run of 25 trajectories with different random initial velocities, ci becomes the same for each window. Thus the free energy Fi can be obtained by P integrating over the slope bi, F i ¼ i1 bj dj , dj is the reaction coordinate distance around the center of jth window or constraint. The improvement is that we use the probable distribution point to calculate the free energy since it is easier to be obtained and also gives

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-5 -6

Free Energy (kcal/mol)

on the SUNYSB Seawolf Supercomputer Cluster. We also thank Ron Elber for helping us about the usage of Moil package.

Avg1 Avg2 Avg3 Avg4

References

-7 -8 -9 -10 -11

1

2

3

4

5

6

7

8

9

Frames Figure 7. Avg1 represents the averaged results of umbrella constraint constant of 1.0, 2.0 and 3.0 kcal/mol Å2. Avg2 is of 2.0, 3.0 and 4.0 kcal/mol Å2, etc. The averaged error is within 0.5 kT through all the frames.

more accurate results than the traditional of umbrella sampling method in order to get the slope di for each window. Equilibration is performed for 5000 steps to heat the system to room temperature, 310 K, for each window in the initial path. The SHAKE algorithm is used to constrain the bonds involving hydrogen atoms with 0.0001 ps/step alongside the leapfrog integrator. The constraint applied for the purpose of umbrella sampling is a biasing quadratic potential [23]. For each window 8 ps, i.e. 80 000 steps are employed for the data collection; the constraint is applied to all the molecules excluding waters, although water molecules are kept in the dynamic simulations. The final free energy is the average of the productive results of biasing force constant of 2.0, 3.0 and 4.0 kcal/mol Å2. We also examine the reliability of the averaged results on different force constants. The averaged error is within 0.8 kcal through all the frames for different averaged constraint constant (in Figure 7). Acknowledgments We thank National Science Foundation for financial supports. Most of the simulations were performed on a Pacific Northwest National Laboratory supercomputer, and some were performed

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