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Elucidating the GTP Hydrolysis Mechanism in FeoB – a Hydrophobic Amino Acid Substituted GTPase. Neha Vithani, Sahil Batra, Balaji Prakash, and Nisanth N. Nair ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.6b03365 • Publication Date (Web): 23 Dec 2016 Downloaded from http://pubs.acs.org on December 27, 2016

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Elucidating the GTP Hydrolysis Mechanism in FeoB – a Hydrophobic Amino Acid Substituted GTPase. Neha Vithani1, Sahil Batra1, Balaji Prakash2* and Nisanth N. Nair3* 1

Department of Biological Sciences and Bioengineering, Indian Institute of Technology, Kanpur 208016, India

2

Department of Molecular Nutrition, CSIR-Central Food Technological Research Institute, Mysore, 570020, India

3

Department of Chemistry, Indian Institute of Technology, Kanpur 208016, India

* Authors for correspondence Email: [email protected], [email protected] ABSTRACT: Employing hybrid QM/MM molecular dynamics simulations and experimental mutational studies, we investigate the GTP hydrolysis mechanism in a HAS (Hydrophobic Amino-acid Substituted)-GTPase, FeoB. We identify glutamates, Glu66 and Glu67, acting as bases and find that proton transfer occurs from the attacking water to either glutamates through a water chain. However, GTP hydrolysis does not abolish despite mutating these glutamates; instead an alternative substrate-assisted hydrolysis becomes active with the same rate. Thus, mutational studies would misinterpret the role of glutamates. We trace the origin of the alternative mechanism to a structural feature conserved across all HASGTPases, distinct from the Ras-like GTPases.

KEYWORDS: Metadynamics, QM/MM, Metaphosphate, HAS-GTPase, Substrate-assisted-catalysis, Mutational studies, Proton transfer, Iron transporter. The enzymes belonging to the GTPase superfamily regulate a wide range of cellular events such as protein biosynthesis, cell division, cell signaling and transportation processes across all kingdoms of life. GTPases render this regulation by switching between their GTP (guanosine tri-phosphate) bound ‘on’ and GDP (guanosine diphosphate) bound ‘off’ states.1-3 The switching between the ‘on’ and ‘off’ states occurs through GTP binding and hydrolysis, usually accelerated by a GTPase Activating Protein (GAP)4 For the extensively studied Ras and EF-Tu GTPases, existing literature suggests a substrate-assisted-catalysis (SAC), wherein the γ phosphate of the substrate GTP acts as the final proton acceptor for the activation of the lytic water.5-9 The process is facilitated by a conserved glutamine (Gln61 in Ras) or a histidine (His85 in EF-Tu) (Fig. S1A, S1B).1,5-17 Gln/His is believed to orient the lytic water17 and assist proton transfer during GTP hydrolysis.5,8-16, 18-20 Owing to such key role(s), their substitution with a hydrophobic residue abolishes GTPase activity.10,17,21 Interestingly, a group of GTPases termed HAS (Hydrophobic Amino-acid Substituted) GTPases, naturally possess a hydrophobic residue in lieu of the polar residue (Gln or His) and yet efficiently hydrolyze GTP (Fig. S1C).22 Structural and biochemical studies on a few of the HASGTPases (such as hGBP1, MnmE and dynamin) show that the hydrophobic residue varies among the different subclasses.23 Unlike Gln61 in Ras, side chain of the hydrophobic residue, in HAS-GTPases, is retracted out of the active

site pocket. The role of these hydrophobic residues and the detailed mechanism of GTP hydrolysis in HASGTPases remains a puzzle. Here, employing a combination of molecular simulations and experimental mutational studies, we attempt to identify the catalytically important residues and the mechanism for GTP hydrolysis in a bacterial HAS-GTPase, FeoB. We further find an extrapolation of the newly identified mechanism to other HAS-GTPases. FeoB, ferrous iron transport protein B, is a Fe2+ iron transporter required for bacterial growth in acidic and anaerobic environment; and a virulence factor in pathogenic bacteria.24-27 GTP hydrolysis by FeoB is imperative for its biological function.28,29 Previously, attempts to understand the GTP hydrolysis mechanism were made based on the structures of FeoB from Streptococcus thermophilus (FeoBSt) determined in complex with GDP, GMPPNP (a non-hydrolyzable GTP analogue) and GDP.AlF4- (a transition-state analogue). FeoB has a hydrophobic residue (Ile57) at the position equivalent to Gln61 of Ras (Fig. S1C). Unlike Ras like GTPases that use an Arg residue supplied by a GTPase activating protein to accelerate GTPase activity, some of the HAS-GTPases like FeoB use a K+ ion at their active site to satisfy this role.23,24 Experimental studies suggested that the lytic water in FeoB would be stabilized only via the protein backbone interactions. None of side chains of the active site residues were found to participate in GTP hydrolysis.24 With this, the mechanism for proton transfer and activation of

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Table 1. Metadynamics simulations for the wild type (WT) and the mutants of the FeoB. Mechanism labels are according to figure 1. An error of less than 2 kcal mol-1 is expected for all the free energy estimates. System

Sim1

WT

a

C

17

WT

b

C

17

Sim4

WT

c

A

33

Sim5

E66A

C

20

Sim6

E67A

C

19

Sim7, Sim8

E66A.E67A

B

18

Sim2

Figure 1. Probable mechanisms for GTP hydrolysis in FeoB. (A) Mechanism A: A direct proton transfer mechanism. Oxygen of the γ phosphate of GTP directly abstracts proton from the lytic water. (B) Mechanism B: Proton transfer from lytic water to GTP γ phosphate via additional water molecule. (C) Mechanism C: Proton transfer from lytic water to Glu66/Glu67 via a chain of water molecules.

lytic water for GTP hydrolysis in FeoB remains speculative.24 To address this, we employed hybrid quantum mechanical/molecular mechanical (QM/MM)30 techniques combined with metadynamics31 for modeling GTP hydrolysis in the wild type and mutants of FeoB. This combination was successfully applied to study various enzymatic reactions.32 QM/MM technique has been successfully used for understanding the reaction mechanisms for GTP and ATP hydrolysis in several cases.8,33-36 Here, we also carried out experimental mutational studies to validate our computational findings. The details of the simulations and the experimental procedures are provided in the Supporting Information. In brief, the substrate GTP, a Mg2+ ion, a K+ ion, the reactive water molecules along with the side chains of important active site residues were treated by plane-wave density functional theory using PBE functional (see also Table S3 for the error estimation).37 The rest of the solvated enzyme was treated by AMBER38 molecular mechanics (MM) force-field. Employing metadynamics, certain coordinates relevant to a reaction (termed collective coordinates, CCs) were enhance-sampled. This allows the system to accelerate barrier-crossing events in a selfguided manner. Free energy surface of the reaction constructed along the selected CCs elucidate the reaction mechanism and free energy barrier for the reaction (see Supporting Information section 2.4). An error of less than 2 kcal mol-1 is expected for the free energy estimates (see Supporting Information section 2.4.3., section 2.4.5.). For the experimental study, the constructs of wild type and mutants (E66A, E67A and a double mutant E66A.E67A) of FeoB from Streptococcus thermophilus were expressed and purified. Their rate constants kcat for GTP hydrolysis were determined by colorimetric phosphate detection method (see Supporting Information section 3). As a first step, we carried out metadynamics simulation of GTP hydrolysis reaction in the wild type FeoB. We enabled enhanced sampling of Oβ-Pγ bond breaking and PγOw bond formation by selecting appropriate collective

‡

Simulation

-1

Mechanism (ΔF ) kcal mol observed

ΔF‡ is the Free energy barrier for the reaction. a: Glu66 and Glu67 treated by QM; b: Glu66 treated by MM, Glu67 treated by QM; c: Glu66 and Glu67 treated by MM.

Table 2. Experimentally measured kcat for the wild type (WT) and mutants of the FeoB -1

Enzyme construct

kcat (min )

WT

3.21 ± 0.12

E66A

1.32 ± 0.02

E67A

0.89 ± 0.03

E66A.E67A

0.70 ± 0.02

coordinates in metadynamics simulation (Table S1). The main objective was to simulate the formation of an intermediate structure. Surprisingly, this simulation (Sim1) resulted in the final product (GDP and HPO4-) formation via a metaphosphate intermediate ‘I’ (see Supporting Information section 2.4.4.1.), followed by a nucleophilic attack by the water. Once the Pγ-Ow bond was formed, the lytic water molecule spontaneously deprotonated (see Supporting Information section 2.4.7.) in our simulations, and its proton was transferred to Glu66 through a water chain of three water molecules (Fig. 1C & Fig. 2). Spontaneous proton transfer from lytic water following the transition state (TS) has also been demonstrated for similar systems.5,39,40 With the dissociation of the Oβ-Pγ bond, W1 and W2 water molecules flip their positions in a coordinated manner (Fig. 2, S17). The overall barrier for the hydrolysis reaction is 17 kcal mol-1 (Table 1), and the intermediate ‘I’ is short-lived with a lifetime of nearly 48 ps. The TS and the ‘I’ state structures are further characterized by the transition path sampling simulations (see Supporting Information section 2.4.4). These simulations further confirm that the observed results are not the artifacts of the chosen CVs. A similar transient intermediate was observed during GTP hydrolysis studied in the aqueous solution.41 A proton transfer mechanism via a chain of water molecules on a glutamate was also reported for ATP hydrolysis by Kinesin36 and a very recent study on GTP hydrolysis by hGBP142. Based on structural studies, MnmE, another HAS-GTPase, was also proposed to follow such a proton transfer, via a chain of water molecules, to a conserved glutamate residue.43 The water chain observed in our simulation was extending up to two highly con-

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Figure 2. Snapshots representing (A) the reactant ‘R’ (B) the intermediate ‘I’ state; and (C) the product ‘P’ during Sim1. Atom colors: C(gray); N(blue); O(red); H(white); P(tan); 2+ + Mg (dark green); K (pink); the four active site water molecules are colored differently. Blue dotted lines show the Hbond interactions. Oβ-Pγ and Pγ-Ow distances are labeled in the figures. (For distance distributions, see Supporting Information; Figure S13) (D) Reconstructed free energy surface for Sim1. The collective variables CV1 and CV2 corresponding to CC1 and CC2 for Sim1 are defined in the Supporting Information. The position of TS is shown by cyan colored dot and the ‘I’ state is enclosed by cyan colored square box.

served glutamates-Glu66 and Glu67 (Fig. 2B). Such a water chain was also seen in the crystal structure of FeoB[GDP.AlF4-].26 Both, in our simulation and also in the crystal structure of FeoB[GDP.AlF4-], the water molecules in this chain have H-bonding interactions with a) the residues Thr35, Gly56, Glu66 and Glu67, and b) between the water molecules themselves. Especially, the terminal water molecule interacts with both Glu66 and Glu67 (Fig. 2B) prompting that both of these residues could act as the base. To test this further, we carried out a simulation (Sim2) where Glu66 was treated by MM (and thus cannot accept a proton), while retaining Glu67 in the QM region. In this simulation, we found that the hydrolysis occurs by the same mechanism; and proton transfer occurs to Glu67 (Fig. S5). The free energy barrier for this reaction is also identical to the case where Glu66 acts as the base (Table 1). From these simulations, we conclude that both Glu66 and Glu67 are equally likely to act as a base to facilitate the phosphate hydrolysis reaction. As we find that there is no barrier for the proton transfer event while going from the TS to the product (Fig. 2D), we conclude that Glu-catalyzed reaction is the preferred reaction route in wild type FeoB. To verify alternative pathways, especially substrate-assisted mechanisms, we performed additional computations where we explicitly included sampling of collective coordinates that would accelerate these events. Further, we screened the proton transfer pathways to Glu66 and Glu67 by treating both these residues using MM (so that they cannot accept proton), without which we always observed spontaneous

Figure 3. Snapshots representing the (A) reactant ‘R’ state from Sim7 (B) intermediate ‘I’ state from Sim7 (C) the transition structure formed in Sim8 and (D) the product ‘P’ state in Sim8 for E66A.EE67 mutant. Color scheme is according to figure 2.

proton transfer to these residues irrespective of the collective coordinates sampled in our simulation. In the first of such simulations, we explicitly sampled the auxiliary water-assisted proton transfer i.e. Mechanism B (Sim3). We observed the formation of intermediate ‘I’, with water chain extending up to Glu residues (Fig. S6C). Subsequently, the water molecules arrange in a manner to assist an auxiliary water-assisted mechanism. However, such configurations which could promote proton transfer were unstable (Fig. S6D). Proton transfer was not observed even after applying a biasing potential of 26 kcal mol-1 (Fig. S6). Thus, we rule out the preference for such auxiliary water-assisted mechanism (i.e. Mechanism B) over Mechanism C in wild type FeoB. We performed another simulation (Sim4) where we sampled the direct proton transfer from W1 to γ-phosphate (Fig. 1A). Interestingly, we observed that the proton transfer occurs crossing a free energy barrier of 33 kcal mol-1 (Fig. S7). Such a high energy barrier does not concur with the experimentally measured reaction rate in the present study (~ 3.3 min-1) (see Table 2) as well as the previously reported (~ 2.6 min-1).24 Our computations thus demonstrate that hydrolysis in wild type FeoB GTPase occurs by a Glu-catalyzed mechanism, wherein either Glu66 or Glu67 acts as a base. This proton transfer mechanism is also in line with prior experimental studies21 where the mutation of Glu66 (E66A) and Glu67 (E67A) marginally affected GTP hydrolysis rates (with only ~2 fold and ~3.5 fold decrease in kcat observed for E66A and E67A, respectively).24 To further confirm this effect, we performed experimental kinetics studies on these two FeoB mutants. In accordance with the previous reports, in our experiments too, E66A and E67A mutants showed only a ~3 fold and ~4 fold decrease in GTP hydrolysis rates, respectively (Table 2). Based on these, it can be argued that in E66A, Glu67 would facilitate the proton transfer and in the E67A mutant, Glu66 would play a similar role; thereby significant hydrolysis may en-

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Figure 4. The distribution of backbone dihedrals Ψ and Φ of Gly56 in GTPases is shown on the Ramachandran map of glycine residue. (A) The regions enclosed by light blue colored lines are the (sterically) allowed regions for the backbone dihedrals of Gly; whereas, those enclosed by the blue colored lines are generously allowed (more favorable) regions for Gly backbone dihedrals. The black colored circles represent the backbone dihedrals for the Gly in HAS-GTPases. The red colored circles correspond to those of the Ras, EF-Tu and related GTPases. The backbone dihedral Ψ for Gly from (B) FeoB (Ψ2); and (C) Ras (Ψ1) is shown along with the lytic water at the active site. The atoms defining the dihedral Ψ are labeled 1 to 4.

sue in both the mutants. To validate this, we carried out two independent metadynamics simulations (Sim5 & Sim6) for the mutants E66A and E67A (see Supporting Information section 2.4). For the single mutants, water chain formation and proton transfer from the lytic water to the glutamate occurred in a similar manner as observed in wild type FeoB (Fig. S8, S9). The free energy barrier for both mutants was only about 2-3 kcal mol-1 higher than that of the wild type (Table 1). This result qualitatively agrees with our experimental kinetics data too (Table 2). Since the glutamates, Glu66 and Glu67 can individually act as a base during GTP hydrolysis, in the double mutant E66A.E67A FeoB, GTP hydrolysis was expected to be completely abolished. Since, kinetics data is not available for the E67A.E66A double mutant, we generated this mutant and measured the rate of GTP hydrolysis experimentally. Surprisingly, the GTPase activity for the double mutant exhibited only a 4.5 fold decrease in activity (kcat) compared to the wild type FeoB (Table 2). To decipher how GTP is hydrolyzed in this double mutant, we performed another simulation (Sim7), in which both the glutamates were mutated to alanines. The reaction proceeded with Oβ-Pγ bond breaking, followed by an intermediate formation (Fig. 3). This is similar to the observation in Sim1, except that the water chain is absent in Sim7. The absence of an extended water chain is expected since Glu66 and Glu67 that stabilize the terminal water of the water chain do not exist in the double mutant. The alanine residues replacing the glutamates alter the way active site water molecules arrange in the intermediate state (Fig. 3B, Fig. S18). Interestingly, this altered arrangement of water molecules opens up a possibility for Mechanism B to be employed: proton transfer from the lytic water occurs on the γ phosphate of GTP via an auxil-

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iary water (Wa) molecule (Fig. 1B, 3C). To probe this possibility, we performed Sim8 (Fig. S10), starting from the intermediate formed in Sim7. Of great importance, we observed the hydrolysis reaction along Mechanism B in this simulation, and the overall free energy barrier (combining Sim7 and Sim8) was computed to be 18 kcal mol-1 (Table 1, Fig. S10). Such altered arrangement of water i.e. Mechanism B, was not observed in the wild type FeoB due to the way W3 and W4 –terminal waters of the chain - were stabilized by glutamates (see Supporting Information section 2.4.6 for detailed discussion). Thus, Glu66 and Glu67 influence the configuration of W2, W3 and W4, which in turn allows the employment of a different mechanism in the wild type and the mutants. Interestingly, the auxiliary water-assisted proton transfer observed in Mechanism B resembles the one proposed for Ras GTPase (Fig. 1B).6,8 In Ras, the lytic water (W1) and the auxiliary water (Wa) are shown to be stabilized by the Gln61 side chain.8 In FeoB that has a hydrophobic residue in place of the Gln61, W1 and Wa are stabilized by active site water molecules, W2 and W3. H-bond interaction provided by the backbone carbonyl of Gly56 arranges W2 and W3 in a manner that mimics the active state configuration of Gln61 of Ras (Fig. S23). It is worth noting that the residue equivalent to Gly56 is conserved across all GTPases. This then raises a question, ‘why is the Gly residue not activating auxiliary-water based hydrolysis in Q61L mutant of Ras?’ We find a striking difference in the conformation adopted by the Gly in Ras and the Gly in FeoB. In Ras, the backbone dihedral Ψ of the Gly has a value close to 0 (termed ‘Ψ1’) (Fig. 4C). Whereas, in FeoB the same Gly residue has a dihedral angle Ψ close to 180, termed ‘Ψ2’ (Fig. 4B). We found that only Ψ2 could allow the backbone carbonyl of Gly to aid in precisely orienting the chain of water molecules. On the other hand, Ψ1 (in both Ras and EF-Tu) results in the backbone carbonyl pointing away from the active site. An extensive structural bioinformatics analysis (see Supporting Information section 4) elucidates that in Ras, EF-Tu and related GTPases, the backbone dihedral for the Gly is invariably close to 0, i.e. Ψ1. Whereas, in HAS-GTPases, this dihedral is close to 180 (Ψ2) (Fig. 4A). The conserved conformation (Ψ2) of Gly56 across all HAS-GTPases suggests that all HASGTPases may be capable of employing SAC in the absence of canonical catalytic residue (equivalent to Glu66, Glu77 of FeoB). Indeed, this appears plausible as the catalytic mutants of a few HAS-GTPases continue to hydrolyze GTP at reasonable rates (Table S5). A recent theoretical work on hGBP1, a HAS-GTPase also shows Glu-catalyzed GTP and GDP hydrolysis.41 We predict, based on our structural bioinformatics analysis, that mutation of catalytic Glu in hGBP1 should not abolish its activity due to Ψ2 conformation of Gly backbone. This is also consistent with the experimental data (Table S5). The catalytic mechanisms revealed for FeoB in the present study provide a broader perspective on GTP hydrolysis reactions. The understanding obtained for the Gluassisted GTP hydrolysis in FeoB, by proton transfer via a ‘water-chain’, can be extrapolated to other HAS-GTPases

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having a conserved, catalytically indispensible Glu residue such as in MnmE. GTPases are involved in the temporal regulation of biological processes. In this context, SAC is of significance as it likely operates in several HASGTPases, and does not allow the complete shut down of GTP hydrolysis despite the mutation of the catalytic residues. HAS-GTPases may be acting as slow timers and SAC appears to be a means to achieve this temporal regulation. In summary, this study provides another example of non-canonical GTP hydrolysis mechanisms and underscores the importance of structural plasticity to generate diverse mechanistic strategies to efficiently hydrolyze GTP.

AUTHOR INFORMATION Corresponding Author

*[email protected], [email protected] Author Contributions All authors have given approval to the final version of the manuscript.

ASSOCIATED CONTENT Supporting Information. Details of QM/MM metadynamics simulations, benchmark studies and experimental procedure. This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENT All the QM/MM calculations were performed using the High Performance Computing (HPC) facility at IIT Kanpur. N.V. and S.B. acknowledges the Ministry of Human Resource Development, India, for financial assistance. We thank Shalini Awasthi for providing calculations on the error analysis in metadynamics. We thank Abhishek Acharya for critical comments and help with the manuscript.

ABBREVIATIONS GAP, GTPase Activating Protein; HAS, Hydrophobic Aminoacid Substituted.

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