Reaction Mechanism of Guanosine Triphosphate Hydrolysis by the

Apr 4, 2016 - ... of Guanosine Triphosphate Hydrolysis by the Vision-Related Protein ... In the Arl3–RP2 complex, the first two intermediates, corre...
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Reaction Mechanism of Guanosine Triphosphate Hydrolysis by the Vision-Related Protein Complex Arl3−RP2 Maria G. Khrenova,*,† Ekaterina D. Kots,†,‡ and Alexander V. Nemukhin†,‡ †

Chemistry Department, Lomonosov Moscow State University, Leninskie Gory 1/3, Moscow, 119991, Russian Federation N. M. Emanuel Institute of Biochemical Physics, Russian Academy of Sciences, Kosygina 4, Moscow, 119334, Russian Federation



S Supporting Information *

ABSTRACT: Complexes of small GTPases with GTPaseactivating proteins have been intensively studied with the main focus on the complex of H-Ras with p120GAP (Ras−GAP). The detailed mechanism of GTP hydrolysis is still unresolved. To clarify it, we calculated the energy profile of GTP hydrolysis in the active site of a recently characterized vision-related member of this family, the Arl3−RP2 complex. The mechanism suggested in this study retains the main features of GTP hydrolysis by the Ras−GAP complex, but the relative energies of the corresponding intermediates are different and an additional intermediate exists in the Arl3− RP2 complex compared with the Ras−GAP. These differences arise from small deviations in the catalytic arginine conformation of the active site. In the Arl3−RP2 complex, the first two intermediates, corresponding to the Pγ−Oβγ bond cleavage and the glutamine-assisted proton transfer, are almost isoenergetic with the ES complex. Numerical simulations of the kinetic curves demonstrate that the concentrations of these intermediates are comparable with that of ES during the reaction. The calculated IR spectra reveal specific vibrational bands, corresponding to these intermediates. These specific features of the Arl3−RP2 complex open the opportunity to identify spectroscopically two more reaction intermediates in GTP hydrolysis in addition to the ES and EP complexes.



INTRODUCTION Studies of guanosine triphosphate (GTP) hydrolysis to guanosine diphosphate (GDP) and inorganic phosphate (Pi) carried out by the hydrolase enzymes, GTPases, with and without the GTPase-activating proteins (GAPs), constitute an important research field.1,2 GTPases are responsible for signal transduction in living cells, and malfunction of these enzymes may lead to development of severe human diseases. In this respect, protein complexes of small GTPase Ras (p21Ras) and its activating partner GAP (p120GAP) have received considerable interest in the field of cancer research.3 Numerous experimental4−8 and theoretical9−23 studies have been aimed toward understanding the mechanism of the GAP· Ras·GTP → GAP·Ras·GDP·Pi transformation. Results of low temperature FTIR spectroscopy suggest ES breakdown with the same rate constant as formation of the reaction intermediate with Pi in the active site of the protein complex.5 Vibrational bands corresponding to the GAP·Ras·GDP·Pi complex are likely to be attributed to one reaction intermediate. Stop-flow measurements under single turnover conditions reveal the GTP/GDP ratio dynamics during the reaction.4 Kinetic curve analysis demonstrates that the rate constant of GDP and Pi formation in the active site of the protein is the same as that of ES breakdown, but in experiments, no information on the number and structures of GDP·Pi © XXXX American Chemical Society

intermediates can be extracted. Recently, the complete mechanism of GAP·Ras·GTP → GAP·Ras·GDP·Pi transformation was suggested.23 It can be divided into two principal stages: (1) formation of GDP and Pi from GTP accompanied by the catalytic glutamine tautomerization; (2) enzyme recovery, namely imide to amide transformation of the catalytic glutamine.23 According to the suggested mechanism23 and other recent papers,9,22 all reaction intermediates are higher in energy than the ES complex, and simulation of kinetic curves allows one to conclude that their concentrations are very low during the reaction. Combining data on the reaction mechanism together with the FTIR experiments, one can conclude that GAP·Ras·GDP·Pi intermediate observed in experiments corresponds to the enzyme−product complex. The Arl3−RP2 machinery has received much less attention although its significance for human health is very high as well. The protein Arl3 is a member of adenosine diphosphate ribosylation factor-like (Arf-like or Arl) GTPases of the Ras superfamily. In a complex with its GTPase-activating protein RP2 (retinitis pigmentosa 2), Arl3 is required for normal cytokinesis and cilia signaling and mediates the traffic of components between the inner and outer segments of the eye Received: April 2, 2016

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Figure 1. Computed energy profile of GTP hydrolysis in the Arl3−RP2 complex.

photoreceptor cells.24 It is known that certain mutations in RP2 abolish GTP hydrolysis acceleration, resulting in several eye diseases including hereditary blindness.25 Like all GTPases, Arl3 cycles between the active GTP-bound and inactive GDPbound forms, and the chemical transformations upon the reaction GTP + H2O → GDP + Pi regulate this cycle. Similarly to other GTPases, the intrinsic hydrolysis by Arl3 is very slow (k = 1.2 × 10−5 s−1), but upon binding to RP2, the reaction rate constant increases dramatically to 1.2 s−1.26 Comparison of the crystal structures of Arl3(Q71L)− GppNHp−RP2 (PDB ID: 3BH626) and Arl3−GDP−AlF4−− RP2 (PDB ID: 3BH726) to that of Ras−GDP−AlF3−GAP (PDB ID: 1WQ17) shows that the orientation of the important catalytic residue, the so-called arginine finger, in the Arl3−RP2 complex with respect to the phosphate groups of GTP slightly differs from that in Ras−GAP, but the glutamine residue (Gln71 in Arl3, and Gln61 in Ras) is located similarly in both active sites. Minor structural differences may result in tuning of the energy profile but conserving the main features of the reaction mechanism. Herein we present the computational characterization of the GTP hydrolysis by the complex of small GTPase Arl3 and the GTPase-activating protein RP2. The uniqueness and importance of this system is that it retains the main features of the reaction mechanism proposed for Ras−GAP,23 but it has stable intermediates that can be traced in spectroscopic experiments.

than 10 Å from Pγ were kept frozen. The QM part comprised a large fraction of the enzyme active site: the phosphate groups of GTP, the catalytic water molecule, the Arl3 side chains of Gly69, Gly70, Gln71, and Thr48, the backbones of Asp26 and Asn27, the RP2 side chains of Lys30, Thr31, and Arg118, and the magnesium ion Mg2+ and two water molecules from its coordination shell. The quantum part included 113 atoms, while almost 5000 atoms in total were considered in the QM/ MM scheme. For QM/MM calculations, we used the NWChem program package.29 Calculations of energies and forces in the QM subsystem were carried out using density functional theory with the PBE0 functional30 with the empirical dispersion correction D3.31 The cc-pVDZ basis set was applied. The MM subsystem was modeled with AMBER32 force field parameters. Structures corresponding to the ES and EP complexes and the reaction intermediates (I1−I5) were obtained in a series of unconstrained QM/MM minimizations following scans along the appropriate reaction coordinates. Vibrational analysis was performed to confirm that the located points corresponded to the true minima on the energy surface. Structures of the transition states (TS1−TS6) separating the minimum energy configurations were obtained as the points with single imaginary harmonic frequency. When the saddle points were located, we verified that the descent forward and backward correctly led to the respective minimum energy structures. Finally, we introduced corrections to the potential energy values due to zero-point energies and entropic contributions. For QM/MM MD simulations, the entire system after equilibration as mentioned above was considered with the same QM subsystem as in the QM/MM minimization. The energies and forces in the QM subsystem were estimated using the PBE functional with the empirical dispersion correction D3; the DZVP basis set and the Goedecker−Teter−Hutter pseudopotentials were applied.33 The multigrid approach34 with the cutoff of the finest grid level of 350 Ry was utilized. Two picosecond equilibration simulations were carried out before the 15 ps production run. QM/MM MD calculations were performed using the CP2K program35 under conditions of the NVT ensemble at 300 K. We used canonical sampling through a velocity rescaling (CSVR) thermostat with small relaxation times in equilibration runs and a Nosé−Hoover thermostat chain in production runs.



MODELS AND METHODS A molecular model for QM/MM-based simulations was constructed by motifs of the crystal structure PDB ID 3BH7.26 The AlF4− group bound to the Arl3−RP2·GDP complex was substituted with the PγO3 moiety to restore GTP, the hydrogen atoms were added by assuming positive charges for the side chains of Lys and Arg and negative charges for Glu and Asp, and the protonation state of His was manually suggested for every residue according to its local environment. The system was solvated in a rectangular water box 90 × 80 × 95 Å3 and neutralized. At preliminary stages, minimization with CHARMM force field27 was performed for the model system, keeping frozen the active site of the protein complex. Next, the MD trajectory of 500 ps length was run to adjust positions of the solvent water molecules, keeping frozen the protein groups and GTP with NAMD program package.28 Then the system was reduced to the size of approximately 20 Å from the phosphorus atom Pγ of GTP. In QM/MM minimization of the enzyme−substrate complex, all molecular groups located more B

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Figure 2. Equilibrium geometry configurations of the reaction intermediates in the first part (GTP·H2O·Gln → GDP·Pi·Gln*) of the GTP hydrolysis by the Arl3−RP2 complex. Here and in the next figures, carbon atoms are colored green, nitrogen blue, oxygen red, hydrogen white, and phosphorus ochre; distances are given in angstroms (Å).

Figure 3. Distribution of distances along the QM/MM MD trajectory in the I1 → I2 → I3 segment of the reaction pathway.



occurs similarly, with the planar structure of the PγO3− moiety at TS1 geometry configuration. Further decrease of the O(Wat)−Pγ distance leads to formation of the inorganic phosphate of the composition HPO42− and the protonated Gln71 side chain (denoted here as Gln+). The structure of this reaction intermediate is shown in the left bottom panel in Figure 2. A stationary point with the protonated form of catalytic glutamine cannot be located on the potential energy surface corresponding to GTP hydrolysis in the Ras−GAP complex. The energy barrier at the elementary step I1 → TS2 → I2 is very low, ∼0.1 kcal/mol. The next step is a proton transfer from the nitrogen atom of Gln+71 to HPO42−, forming the neutral imide form of Gln71 (denoted here as Gln*71) and the inorganic phosphate H2PO4−. The structure of this reaction intermediate I3 (illustrated in the right bottom panel in Figure 2) is similar to that in the GTP hydrolysis reaction in the Ras−GAP complex.23 Interestingly, its energy is 3.8 kcal/mol higher than that of the peculiar intermediate I2 with Gln+71. We carried out MD simulations with the QM/MM potentials to provide further support of the formation of the protonated form of the glutamine side chain Gln+71. We analyzed the

RESULTS

The computed energy profile for the GTP hydrolysis in the Arl3−RP2 complex is shown in Figure 1. The first segment in the pathway, GTP·H2O·Gln → GDP·Pi·Gln*, incorporates the elementary steps ES → TS1 → I1, I1 → TS2 → I2, I2 → TS3 → I3, resulting in formation of GDP, inorganic phosphate Pi of the composition H2PO4−, and the tautomeric imide form of the side chain of glutamine (Gln*). The corresponding structural changes are illustrated in Figure 2. In the ES complex (Figure 2), the catalytic water molecule is aligned by hydrogen bonds with the molecular groups of the Gln71 and Thr48 residues for a nucleophilic attack on the γphosphate of GTP. By gradually reducing the distance between the oxygen atom of Wat and the phosphorus atom Pγ and adjusting other geometry parameters in QM/MM minimizations, we could locate the first transition state TS1 and the first reaction intermediate I1 (Figure 2). In the I1 configuration, the Oβγ−Pγ bond in GTP is cleaved, but the water molecule Wat is not disrupted. This elementary step, ES → TS1 → I1, in the Ras−GAP-catalyzed hydrolysis of GTP has been modeled several times.12,22,23,36 In the Arl3−RP2 complex, this step C

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Figure 4. Equilibrium geometry configurations of the reaction intermediates at the stage of enzyme regeneration.

distribution of distances describing proton transfer between the side chain of Gln71, the catalytic water molecule, and the inorganic phosphate along the I1 → I2 → I3 segment of the reaction pathway (Figure 3). The left panel in Figure 3 suggests that the I2 intermediate is slightly more populated than I1 and that conformations corresponding to the I3 intermediate are rare. These population distributions are consistent with the relative energies of the intermediates presented in Figure 1. The right panel in Figure 3 shows the distribution of distances between the heavy atoms and the protons: N(Gln)···H···O(Pi) colored in red, and O(Gln)···H···O(Pi) colored in blue. We note that almost no overlap is seen between the red curves, which means that proton transitions from N(Gln71) to O(Pi) occur rarely (the I2⇄I3 transition). On the contrary, the overlap between the blue curves is considerable, indicating that the proton between O(Gln71) and O(Pi) migrates frequently (the I1⇄I2 transition). We can conclude that the I2 is a genuine reaction intermediate in the case of the Arl3−RP2 complex-catalyzed hydrolysis of GTP. The second segment of the reaction pathway denoted in Figure 1 as Gln* → Gln incorporates three elementary steps I3 → TS4 → I4, I4 → TS5 → I5, and I5 → TS6 → EP. These steps are required to restore the initial conformation of the Glu71 side chain in the amide form. Equilibrium geometry configurations of the reaction intermediates are shown in Figure 4. The first two steps correspond to O−H rotations in Pi around the P−O bonds. The energy barriers at these steps (the levels of TS4 and TS5 relative to the respective minimum energy points) are the highest for the entire pathway (Figure 1). The configuration of I5 is prepared for the concerted proton transfer between Pi and Gln71 which restores the amide isomer of glutamine. This process does not require considerable energy expense. To analyze the kinetic mechanism of the multistep reaction with the energy landscape illustrated in Figure 1, we consider six reversible stages along the pathway:

k1

k2

k3

k4

k5

k6

k −1

k −2

k −3

k −4

k −5

k −6

ES XooY I1 XooY I2 XooY I3 XooY I4 XooY I5 XooY EP

The rate constants of these elementary steps (k+n and k−n, n = 1−6) can be evaluated by using the traditional formulas of the transition state theory and the energy levels indicated in Figure 1. These values calculated for the temperature 298 K were introduced to the computer program KINET developed for numerical modeling of the kinetic properties of complex chemical reactions.37 The resulting kinetic curves are shown in Figure 5. The analytic fit of the curve corresponding to the EP

Figure 5. Kinetic curves for all reaction intermediates obtained by numerical solution of the set of differential equations.

accumulation allowed us to estimate the effective rate constant as 0.015 s−1 which is 2 orders of magnitude lower than the experimental value 1.2 s−1.26 The main contribution to this value comes from the energy barrier at the fourth stage. The potential energy surface of the O−H group rotation is rather complex; therefore, 2 kcal/mol error can be obtained, raising the difference with the experimental value. D

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corresponding to the coupled N−H and O−H vibrations of glutamine.

Consideration of all kinetic curves shown in Figure 5 demonstrates that pre-equilibrium conditions can be assumed for ES, I1, I2, and I3. Importantly, the concentrations of ES, I1, and I2 are comparable during the reaction with a constant ratio of [ES]:[I1]:[I2] = 1.8:1:1.4. This indicates that not only ES but also I1 and I2 can be detected in experiment. We calculated vibrational spectra for ES, I1, and I2, as these intermediates have high enough concentration to be observed (Figure 6). Though there are many intense spectral bands, we



DISCUSSION In this work, we characterize the GTP hydrolysis reaction in the vision-related protein complex Arl3−RP2, where Arl3 is a GTPase carrying out chemical reactions on the substrate and RP2 is the corresponding activating partner. Interestingly, this role of RP2 was revealed only recently;26 it was discovered that upon its binding to Arl3, GTP hydrolysis was accelerated dramatically as compared to the intrinsic reaction rate. Examination of the relevant crystal structures26 suggests that RP2 most likely performs similarly to the GTPase-activating protein GAP in the Ras−GAP complex. The QM/MM-based computer simulations presented in this paper prove that GTP hydrolysis in the Arl3−RP2 complex happens in a way similar to that with Ras−GAP,23 but there are certain differences that should be discussed. Figure 7 shows the superimposed calculated conformations of the molecular groups at the active sites in the Arl3−RP2 and the Ras−GAP systems at the respective enzyme−substrate geometry configurations.23 Starting from ES, the nucleophilic water molecule Wat performs an attack on the γ-phosphate group of GTP. The carbonyl oxygen atom of glutamine (Gln71 in Arl3, or Gln61 in Ras) assists in a proper alignment of this water molecule; the hydrogen atom of the amide group of Gln participates in the proton transfer required to form the inorganic phosphate. Figure 7 illustrates an apparent similarity in the “γ-phosphate−Wat−Gln” motif for both enzymes. Location of the arginine finger at the active site is different in these protein complexes. We show in Figure 8 the fragments of the hydrogen bond networks around the guanidinium group of the arginine side chains in the ES structures in the Arl3−RP2 and the Ras−GAP complexes. In the Arl3−RP2 complex (left panel), the bulky side chain of Gln116RP2 near the α-phosphate group of GTP occupies a considerable part of the cavity, shifting Arg118RP2 closer to the γ-phosphate group. In the Ras− GAP complex (right panel), the corresponding cavity is less tight, hosting a water molecule instead of glutamine; accordingly, Arg789GAP is shifted to the α-phosphate. The consequence of such arrangement of the arginine fingers as well as of other molecular groups at the active site is that the nucleophilic attack of the catalytic water molecule should be more efficient in the Arl3−RP2 complex. Also, the catalytic

Figure 6. Calculated IR spectra (middle panel) for ES (green), I1 (red), and I2 (blue) and difference IR spectra (lower panel) for I1−ES (red) and I2−ES (blue). In both difference spectra, negative peaks correspond to ES. The vibrational shapes of the IR bands marked with an asterisk in the difference spectra are shown in the upper panel.

focus on three vibrations responsible for the particular features of the stationary points. For the ES complex, the spectral band marked with the asterisk corresponds to the O−H bond vibrations in the catalytic water molecule. The spectral band corresponding to the C−N and C−O vibrations of the side chain of Gln71 coupled with the O−H vibrations of the catalytic water molecule is observed in I1. I2 is mainly characterized by the formation of the protonated amide group of Gln71; therefore, we choose the intense IR band

Figure 7. Superimposed calculated conformations of the molecular groups at the active sites in the Arl3−RP2 (balls and sticks) and the Ras−GAP (sticks) systems at the respective enzyme−substrate geometry configurations. The slash sign distinguishes the data referring to the Arl3−RP2 (first entries) and the Ras−GAP complexes (entries after the slash). E

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intermediates. This knowledge might be utilized to trace the reaction by the time-resolved FTIR technique.



ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.6b03363. Video file demonstrating chemical transformations at the active site of the Arl3−RP2 complex upon GTP hydrolysis (MPG)



Figure 8. Hydrogen bond networks near the arginine finger in the Arl3−RP2 (left panel) and the Ras−GAP (right panel) complexes.

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], tel: +74959392035. water molecule Wat is slightly closer to Pγ in the case of the Arl3−RP2 complex (Figure 7), facilitating the attack. This is observed in the first stage of the process, the energy barrier in the Ras−GAP complex is 6 kcal/mol whereas in Arl3−RP2 it decreases to 3 kcal/mol. On the other hand, the cavity in the Arl3−RP2 complex is less flexible, restricting internal rotations at the stage of enzyme recovery. These structural differences result in a ∼5 kcal/mol difference of the energy barrier corresponding to the first rotation of the OH group in Pi with the higher value corresponding to the Arl3−RP2 complex. This barrier is mainly responsible for the smaller value of the experimental rate constant in the Arl3−RP2 complex (1.2 s−1)26 compared with Ras−GAP (19 s−1).4 The role of the glutamine residue, Gln61 in Ras and Gln71 in Arl3, upon the GTP hydrolysis reactions should be emphasized. In both enzyme systems, the molecular groups of this residue participate directly in all stages of the process, the chemical transformations, and the enzyme regeneration. In the Arl3− RP2 complex we notice a reaction intermediate I2 which assumes the protonated form of glutamine (Figure 2). The relative energy of I2 (Figure 1) is fairly low, mainly due to stabilizing interactions of the positively charged Arg118RP2 with the negatively charged γ-phosphate group of the cleaved GTP. Both QM/MM and QM/MM MD calculations firmly demonstrate a proton attachment to the carbonyl oxygen of Gln at this segment of the pathway. The kinetic curves (Figure 5) suggest that concentrations of I1 and I2 should be comparable to those of ES during the reaction ([ES]:[I1]:[I2] = 1.8:1:1.4); thus, formation of I1 and I2 may be traced experimentally. We suggest the characteristic vibrational bands that can be used to detect ES, I1, and I2. Moreover, the Arl3−RP2 complex is more than 10 times slower than the Ras−GAP complex; thus, experimental evaluation can be obtained at temperatures closer to the standard kinetic experiment (298−303 K).

Notes

The authors declare no competing financial interest.



ACKNOWLEDGMENTS This study was supported by the Russian Foundation for Basic Research (15-33-20579). We acknowledge the use of supercomputer resources of Lomonosov Moscow State University38 and of the Joint Supercomputer Center of the Russian Academy of Sciences.



REFERENCES

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CONCLUSIONS The Arl3−RP2 complex is an important member of the complexes of small GTPases with GTPase-activating proteins. The calculated energy profile suggests the mechanism of the GTP hydrolysis to be basically similar to that in the Ras−GAP complex. Deviations in the local environment of the active sites of the protein complexes result in different conformations of the arginine finger. Also, in the Arl3−RP2 complex, the first two intermediates are almost isoenergetic with the ES complex and, thus, exist in concentrations comparable with that of ES during the reaction. Calculated IR spectra demonstrate the intense characteristic bands for these highly populated F

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DOI: 10.1021/acs.jpcb.6b03363 J. Phys. Chem. B XXXX, XXX, XXX−XXX