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Product Release Pathways in Human and Plasmodium falciparum Phosphoribosyltransferase Tarak Karmakar, Sourav Roy, Hemalatha Balaram, Meher K. Prakash, and Sundaram Balasubramanian J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.6b00203 • Publication Date (Web): 12 Jul 2016 Downloaded from http://pubs.acs.org on July 15, 2016

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Journal of Chemical Information and Modeling

Product Release Pathways in Human and Plasmodium falciparum Phosphoribosyltransferase Tarak Karmakar1 , Sourav Roy2 , Hemalatha Balaram2 , Meher K. Prakash3∗ , and Sundaram Balasubramanian1∗ 1 3

Chemistry and Physics of Materials Unit, 2 Molecular Biology and Genetics Unit,

Theoretical Sciences Unit, Jawaharlal Nehru Centre for Advanced Scientific Research, Bangalore 560 064, India E-mail: [email protected],[email protected]

∗

To whom correspondence should be addressed

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Abstract Atomistic molecular dynamics simulations coupled with the metadynamics technique were carried out to delineate the product (PPi.2Mg and IMP) release mechanisms from the active site of both human (Hs) and Plasmodium falciparum (Pf) hypoxanthine-guanine-(xanthine) phosphoribosyltransferase (HG(X)PRT). An early movement of PPi.2Mg from its binding site has been observed. The swinging motion of Asp side chain (D134/D145) in the binding pocket facilitates the detachment of IMP which triggers the opening of the flexible loop II, the gateway to bulk solvent. In PfHGXPRT, PPi.2Mg and IMP are seen to get released via the same path in all the biased MD simulations. In HsHGPRT too, the product molecules follow similar routes from its active site; however, an alternate, but minor escape route for PPi.2Mg has been observed in the human enzyme. Tyr 104 and Phe 186 in HsHGPRT, and Tyr 116 and Phe 197 in PfHGXPRT are the key residues that mediate the release of IMP. While the motion of PPi.2Mg away from the reaction centre is guided by the negatively charged Asp and Glu, and few positively charged residues (Lys and Arg) which line the product release channels. Mutations on few key residues present in loop II of Trypanosoma cruzi (Tc) has been shown to reduce the catalytic efficiency of the enzyme. Herein, in silico mutation of corresponding residues in loop II of HsHGPRT and PfHGXPRT results in the partial opening of the flexible loop (loop II), thus exposing the active site to the bulk water, which offers a rationale for the reduced catalytic activity of these two enzymes. Investigations of the product release from these HsHGPRT and PfHGXPRT mutants delineate the role of these important residues in the enzymatic turnover.


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Introduction Plasmodium falciparum (Pf), a causative agent for malaria, lacks de novo purine metabolism and depends on salvage machinery for the generation of purine nucleotides. 1,2 Pf hypoxanthineguanine-xanthine phosphoribosyltransferase (PfHGXPRT) salvages nucleobases (hypoxanthine, xanthine and guanine) and converts them to their mononucleotides, inosine-5’-monophosphate (IMP), guanosine-5’-monophosphate (GMP), or xanthosine-5’-monophosphate (XMP), and inorganic pyrophosphate (PPi). PfHGXPRT and its human (Hs) homolog, HsHGPRT 3 exhibit 76% similarity and 44% identity in the sequence. 4 Understanding their behavior and identifying differences, if any, would aid ongoing efforts in the design of suitable inhibitors for the parasite enzyme to combat malaria. 5 Much of our knowledge about this class of enzymes has been gathered from its variants in different species like human (HsHGPRT), 3,6,7 Toxoplasma gondii, 8 Escherichia coli 9 etc. While the structures of PfHGXPRT complexed to transition state inhibitor 10 (PDB ID: 1CJB, see Figure 1(a)) and other ligands have been solved, 11 that of the ligand-free enzyme has not been experimentally determined yet. In contrast, HsHGPRT has been crystallized in both apo and various liganded states. 3,6,7,12 While PfHGXPRT exists as either a dimer (in Tris HCl) or as a tetramer (in phosphate), HsHGPRT exhibits a tetrameric structure in both the buffer conditions. 13 By comparing the apo and ligand complexed crystal structures, a large conformational change in loop II (residues 102 to 122) has been observed in HsHGPRT, 7 T. gondii, 8 as well as T. foetus HGXPRT upon ligand binding. 14 However, a knowledge of conformational changes of the enzyme during substrate inclusion and product release, and the role of active site loops or residues influencing this process are difficult to obtain through static structures alone. The activation mechanism of PfHGXPRT was investigated by us earlier, using both biochemical studies and molecular dynamics simulations. 15 Loop II (Figure 1(b), green colored loop) and IV (blue) together constitute a gate (Figure 1) whose dynamics enables substrate uptake and product release. The slow trans-cis conformational change of a non-X-Pro Leu76-Lys77 dipeptide explained the initial lag phase in 3 ACS Paragon Plus Environment


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Figure 1: (a) PfHGXPRT (one subunit of a tetramer) along with IMP, PPi and 2Mg2+ from 1CJB (ligands in the crystal structure are modified to generate the naturally occurring product molecules). IMP, PPi and 2Mg2+ are shown in ball and stick representation. Gate formed by loop II (green) and loop IV (blue) is shown by double-headed arrow. Schematic showing paths taken by the products to come out of their respective binding sites. (b) Residues (S115, Y116 and S121) in loop II (residues 112 to 133) that are mutated (in silico mutation) to investigate their participation in the product release are shown. A similar set in HsHGPRT is S103, Y104 and S109 (not shown). the activation of the enzyme. 15 Investigations on the ligand unbinding processes in pharmaceutically important enzymes provide several clues that further help in finding out suitable inhibitors for the specific target. 16–19 Thus, exploration of the last step of an enzymatic turnover, i.e., the release of the product molecules 20–22 from the active site of an enzyme is important as far as the druggability of the enzyme is concerned. 23–25 The product release step from HsHGPRT had been proposed to be the slowest (12 s−1 for PPi and 6 s−1 for IMP release) and thus rate limiting. 26 The order of release of the products (IMP and PPi.2Mg) is somewhat unclear. Giacomello et al. proposed a random release, 27 while Xu et al. 26 offered a sequential substrate binding and a partially random dissociation of the products. The kinetic mechanism of PfHGXPRT has recently been examined thoroughly; 28 the same was investigated earlier for homologous enzymes from other species. 26,29 The rate of dissociation of IMP (k−3 ) from a double mutant (W181S/F197W) of PfHGXPRT was determined to be 33 s−1 which cor-


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responds to a release time approximately of 30 milliseconds. 15 Despite the large number of investigations on the function of the parasite enzyme, several questions remain. How are the products (IMP and PPi.2Mg) released from their binding sites? What are the protein conformational changes that take place during the release of the product molecules? A flexible loop has been proposed to mediate the substrate uptake and product release processes, and it would be interesting to find out the roles of amino acid residues that are present in loop II in product release steps. Earlier mutational studies on few key residues present in loop II of Trypanosoma cruzi HPRT revealed their possible roles in enzymatic turnover. 30 In our study, in silico mutations were carried out on residues present in loop II of HsHGPRT and PfHGXPRT, similar to those performed experimentally on T. cruzi HPRT, to understand their role in product release. In this article, we investigate the release mechanisms of PPi.2Mg and IMP from the active sites of PfHGXPRT and HsHGPRT using molecular dynamics and metadynamics simulations and provide a comprehensive molecular level description of the specific events.

Computational details Unbiased equilibrium MD simulations MD simulations were performed at the all-atom explicit solvent level, starting from crystal structures 3OZF (PfHGXPRT) 11 and 1BZY (HsHGPRT) 3 collected from protein data bank (www.rcsb.org). The enzymes considered here are tetramers. The coordinates of the ligand molecules were obtained by overlaying one ligand-bound subunit of 1CJB (another crystal structure of PfHGXPRT containing a transition state inhibitor, immucillin-HP.PPi.2Mg2+ that is similar to IMP, PPi, and two Mg2+ ions) with the subunits of 3OZF. 11 On the other hand, the crystal structure of HsHGPRT, 1BZY is a tetramer with a transition state inhibitor. In both the cases, the C9 of the inhibitor was changed to N9 to generate the naturally occurring IMP. These IMP.PPi.2Mg-bound tetrameric systems were then simulated 5 ACS Paragon Plus Environment


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following a standard simulation protocol. (see ref. 38 for further details.) Point mutations such as K114A, S115G, Y116F and S121A for the PfHGXPRT and K102A, S103G, Y104F, and S109A for the HsHGPRT were generated (in all the subunits) using the mutagenesis module as implemented in PyMol software as follows. A rotamer was selected in such a manner that along with the backbone atoms (N, Cα , and C), there is an overlap between the Cβ of the residue to be mutated and the same atom in the new residue. Later on, energy minimization of the system provides proper orientation of the mutant residue that, however, was seen not to deviate much from that in the WT system. Each of the systems was solvated in water (∼38000-42000 water molecules per enzyme), and the net charge was neutralized by adding 8 and 16 Na+ ions for the HsHGPRT and PfHGXPRT enzymes, respectively. Each of the generated systems consisted of a total number of ∼127800-140500 atoms. Details about the simulated systems and durations of trajetories are listed in Table 1. The systems were minimized in two steps (with and without position restraints) using steepest-descent algorithm, followed by heating of the systems from 0 to 300 K within 300 ps time span in the NVT ensemble. A short run for 2 ns was performed to equilibrate each system in the NPT ensemble. Subsequently, each system was simulated for 100 ns (simulations of the WT systems were extended up to 200 ns) with a time step of 1 fs in NPT ensemble at 300 K temperature and 1 bar pressure using Nosè-Hoover thermostat 31 and Parinello-Rahman barostat. 32 LINCS algorithm 33 was used to constrain the covalent bonds involving the hydrogen atoms. A cut-off of 12 ˚ A was used for the van der Waals as well as for the electrostatic interactions. Particle-Mesh Ewald (PME) was employed to calculate the long-range electrostatic interactions. Gromacs-4.5.5 package 34,35 was used to run the unbiased simulations. Amber99sb-ildn force field 36,37 for the protein and Amber derived parameters for the ligand molecules were used. Details of the derivation of the RESP charges of the ligand molecules can be found in our earlier works. 15,38 Gromacs-4.5.5 patched with PLUMED-1.3 39 was used to perform the metadynamics simulations. Analyses were performed using Gromacs tools, a few home-grown Fortran codes and Tcl scripts. Tra-


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Table 1: Summary of the systems simulated without bias. PfHGXPRT Wild type (WT) K114A S115G Y116F S121A

HsHGPRT Wild type (WT) K102A S103G Y104F S109A

Trajectory length post-equilibration (ns) 200 100 100 100 100

jectories were visualized in VMD; 40 both VMD and PyMol 41 were utilised to produce the figures.

Metadynamics simulations The product release step of HsHGPRT is rather slow and rate limiting in the enzymatic turnover. 26 Unbiased MD simulations are limited in their ability to sample slow (rare) events involving high energy barriers. Several accelerated methods of simulations have been developed and employed in the literature, 42–49 out of which we have chosen to use the metadynamics 43,50 method. Metadynamics and its variants 43,51 have been found to be excellent techniques to study several chemical and biological problems. The detailed information about the development of these methods and their applications can be found in literature. 52–57 In metadynamics simulations, a history dependent bias potential is deposited along a few collective variables (CVs) which are physically relevant and difficult to sample. In this study, we have used two CVs relevant for the release of the two products from the binding pocket of a subunit (chain A) of the tetramer. While the bias was applied to only one subunit, the simulations were performed on the entire tetramer. The CV1 uses the configuration of PPi.2Mg bound to the protein as a reference (Figure 2(a)). CV1, at any instance in the trajectory, is defined as a distance in contact map (CMAP) space relative to this reference configuration (Figure 2(a)). CV2 is similarly defined based on the native contacts of IMP with the enzyme (Figure 2(b)). Both these CVs measure how the native contacts in the binding pocket are broken as the products are released. The definition of the CVs is inspired 7 ACS Paragon Plus Environment


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from the CMAP implementation first developed by Branduardi et al. 58 and has been used in several studies. 54–56 A recent technique, called funnel-type metadynamics developed by Limongelli et al. 59 has been found to be a promising method to study the binding/unbinding of ligands (substrate and/ products) and inhibitors in proteins. 59–61

Figure 2: Contacts made by (a) PPi.2Mg and (b) IMP with amino acid residues in the vicinity of their binding sites in PfHGXPRT. Table 2: Summary of the metadynamics simulations, trajectory length ∼10-50 ns, for each. Systems

Number of simulations

PfHGXPRT Wild type (WT) K114A S115G Y116F S121A HsHGPRT Wild type (WT) K102A S103G Y104F S109A

10 3 3 3 3 15 3 3 3 3

Metadynamics simulations were run by imposing bias on the two CVs simultaneously. 8 ACS Paragon Plus Environment

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A detailed description about the definition and choice of the collective variables has been provided in the SI. We randomly selected few structures from the equilibrated simulation trajectories and used them as initial configurations to carry out the metadynamics simulations. Clustering analysis was performed with the unbiased trajectories to find out the dominant conformations at equilibrium. The g cluster tool available with Gromacs package was used to perform the analysis. Ramachandran angles of all the residues in the initial structures chosen for metadynamics simulations and of the configurations obtained from the cluster analysis were calculated. Careful investigation of the Ramachandran plots (shown in Figure S1 of SI) indicates that the chosen configurations (for metadynamics runs) do not differ much from the dominant conformations obtained from the cluster analysis. Further details are provided in the SI. Multiple independent metadynamics simulations were performed for the WT as well as mutants by (i) taking different initial configurations, each drawn at arbitrary locations of a long equilibrium MD trajectory and (ii) also by varying the metadynamics parameters (different hills heights) to gather the statistics of the events. The biased simulations allow the observation of favored pathways for IMP and PPi.2Mg release from their corresponding binding sites. Comprehensive details of the collective variables (COLVARs) and metadynamics protocol are presented in SI. A summary of trajectories simulated is provided in Table 2.

Results The primary goal of the present study is to provide a qualitative description about the mechanism of release of the product molecules which would provide a wealth of structural and dynamical information about the important enzymes. Quantitative estimates of free energy barriers are computationally forbidding and are beyond the scope of the present work. In the first part, we discuss the outcomes of the metadynamics simulations of the WT



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HsHGPRT and PfHGXPRT systems, and in the next section, observations from the unbiased and biased simulations of the mutants are discussed. Release of the product molecules from the active site is captured by the time evolution of the two CVs. Two representative plots (one each for HsHGPRT and PfHGXPRT) of CV1 and CV2 vs metadynamics simulations time (ns) are presented in Figure 3. Similar plots of the other trajectories are provided in SI. As the values of CV1 and CV2 increase, the molecules PPi.2Mg and IMP respectively move away from their corresponding binding sites. The plateaus in the CV plots signify the final release of the product from the enzyme to the bulk solvent. Several key events that take place during the release of the product molecules are described in the subsequent paragraphs.

Figure 3: CVs (IMP: Blue & PPi.2Mg: Red) as a function of metadynamics simulation time obtained for (a) PfHGXPRT and (b) HsHGPRT systems. Similar plots obtained from different initial configurations are provided in Figures S2 and S3 of SI.

Early movement of PPi.2Mg and IMP Analyses of all the metadynamics trajectories allowed us to make the following observations, partly validated by experimental outcomes. In all the metadynamics simulations of both PfHGXPRT and HsHGPRT, a common event is the early movement (shown in Figure S4(a)) of PPi.2Mg complex within the active site. In the initial binding pose as seen in the crystal 10 ACS Paragon Plus Environment

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structure, the oxygen atoms of PPi interact with the positively charged residues e.g., R210, R112, K114, while, MgB interacts with D204 (Figure S4(a)). Within a few nanoseconds into the metadynamics trajectory, PPi.2Mg escapes its initial binding zone and stays near D204 and R210; we call this new position of PPi.2Mg as a ’secondary binding zone’(Figure S4(a)). Incidentally, a similar location of the PPi.Mg complex has recently been reported in the crystal structure of Mycobacterium tuberculosis HGPRT (Figure S4(b)). 62

Switching motion of Asp in the active site HsHGPRT was crystallized in its ligand-free state by Keough et al. 7 A comparison between this apo state of HsHGPRT with the ligand-bound (HPP.PRPP.Mg, IMP.PPi.Mg and GMPbound) states reveals subtle conformational changes in the enzyme during the catalytic reaction. Overlay of the apo and the ligand-bound structures of HsHGPRT indicated that major changes occur in the active site residues that bind the substrate in the reaction center. Out of those active site residues, E133 and D134 reorient themselves to bind the ribose ring via the formation of stable hydrogen bonds between the side chain of E133 and D134 and hydroxyl hydrogens of the sugar ring. The same purpose is served in PfHGXPRT by E144 and D145, during the ligand binding. In our simulations of product release, once PPi.2Mg and IMP move away from their respective binding sites, the side chain of D145 (in PfHGXPRT) and D134 (in HsHGPRT) switches from a ligand ’binding’ (binds -OH of ribose) to an ’unbinding’ pose (Figure 4). Thus, these observations are in good agreement with the reorientation of D134 side chain in HsHGPRT, originally proposed by Keough et al. solely from the inspection of the ligand-bound and apo crystal structures. 7 Once the side chain of D145 moves away from its binding pose, the detachment of IMP from the IMP.PPi.2Mg complex takes place, an event that allows the two ligand molecules (IMP and PPi.2Mg) to move independently. In a few instances, one of the Mg ions in PPi.2Mg was found to be coordinated with the ribose -OH of IMP for a long time before separation.



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Figure 4: Swinging motion of (a) Asp 145 in PfHGXPRT and (b) Asp 134 in HsHGPRT. Binding and unbinding poses are shown in green and orange colors, respectively.

Steps of product release Many events occur concurrently with the motion of PPi.2Mg and IMP from the active site (Figure 5). The opening of loop II was not biased in our simulations; however, the large displacement of IMP and PPi.2Mg from the active site was found to be concomitant to the widening of the gate via the opening of the more flexible loop II, thus exposing the reaction center to the solvent. Changes in the electrostatic environment upon solvation helps in the separation of IMP from the IMP.PPi.2Mg complex. As soon as IMP is detached from the complex, it starts moving away from its original binding site (Figure 5).


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Figure 5: Sequence of events that occur during product release from PfHGXPRT. Details of the individual steps and the interactions between ligand molecules and the protein residues are presented in latter sections

Details of the product release pathways Escape routes In the following, residue names and numbers are provided according to their identity in PfHGXPRT, while the corresponding ones in HsHGPRT are mentioned in parentheses. In metadynamics simulations of PfHGXPRT, IMP always follows path B (Figure 6(a)). PPi.2Mg initially moves towards path A but later, it too follows that same path as that of IMP. Similarly, in the case of the HsHGPRT, release of the two product molecules takes place largely via a similar path (path B) as that of PfHGXPRT (Figure 6(b)). In majority of the simulation trajectories of HsHGPRT, both the molecules come out of the pocket following path B. However, in few cases (3 out of 15 trajectories), PPi.2Mg follows an alternative route to egress from the active site of HsHGPRT (shown in Figure 6(c)). Surprisingly, in none of the ten metadynamics trajectories of PfHGXPRT, we observe this escape route for the 13 ACS Paragon Plus Environment


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Figure 6: Release of product molecules from the enzymes in different metadynamics trajectories. (a) PPi.2Mg and IMP via path B in case of PfHGXPRT (path A is shown by red arrows), (b) both the products via path B (major route) and (c) PPi.2Mg via path A (minor route) and IMP via path B, respectively in case of HsHGPRT. In the metadynamics simulation trajectory, the instantaneous positions of the PPi and IMP are shown in red and blue sticks, respectively. Release of the product molecules from subunit A of the PfHGXPRT and HsHGPRT tetramers is shown in Figure S5 of SI.


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PPi.2Mg complex. This rules out the possibility of an alternate release path for the complex in the parasite enzyme. Residues lining the escape routes, path A and B are found out by calculating the distance between the Cα atoms of residues and any atom of the ligands. The minimum of these distances between a residue and a ligand found in a simulation trajectory are listed in Tables S1-S8 of SI. Results are shown for two independent trajectories of the HsHGPRT system, where PPi.2Mg follows path A and IMP follows path B (Table S1-S4 of the SI). Similarly, residues present in the path B for the release of both the ligands from PfHGXPRT are listed in Tables S5-S8 of the SI. Details of the migration of two product molecules and the detailed interactions between the products and the residues are discussed in the subsequent sections.

PPi.2Mg release It is pertinent to examine the details of the release pathways of the IMP and PPi.2Mg in the two enzymes. The PPi.2Mg complex has both oxygen anionic centers and Mg cations. The former help the complex to stay near the positively charged residues while the MgA and MgB ions interact with the negatively charged regions generated by E144 (E133) and D145 (D134) residues (Figure 7). As the complex leaves the primary binding site in PfHGXPRT, two negatively charged residues, D204 and D207, located inside the active site interact with the MgB ion. PPi.2Mg complex stays near these two residues (Figure 7). The ionic interaction between Mg2+ and COO− restricts the motion of the highly charged PPi.2Mg complex. A similar set of residues in HsHGPRT are D193 and D196 that perform the similar job in holding PPi.2Mg for some time. On the other hand, positively charged residues like R112 (R100) and K114 (K102) can interact with the negatively charged oxygens on PPi. PPi.2Mg moves to and fro near the reaction center before it comes out of the pocket completely. The positions of the ligand molecules in different time frames are shown in red and blue sticks for PPi.2Mg and IMP, respectively (Figure 6). In HsHGPRT, K102 (present in loop II) interacts with PPi and pulls the complex from the active site towards itself leading to the 15 ACS Paragon Plus Environment


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Figure 7: Detailed steps in the release of PPi.2Mg from PfHGXPRT: a. Initial bound state of PPi.2Mg, b. detachment of PPi.2Mg and IMP, c. early movement of PPi.2Mg away from its binding site, d. further movement of the complex, e. movement of PPi.2Mg towards the entrance of the channel, & f. release of the complex to bulk water. Ligands are shown in ball & stick representation. Release of PPi.2Mg from the active site of HsHGPRT via the major (path B) and minor (path A) pathways are shown in Figure S6 & S7 of SI. alternative pathway, path A (Figure 6(c), for the PPi.2Mg release. These two exit channels are demarcated in Figure 6. 16 ACS Paragon Plus Environment

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IMP release The other ligand, IMP has three main interaction sites, (i) ribose ring hydroxyl groups forming strong hydrogen bonds with the carboxylate groups of E144 (E133) and D145 (D134) (Figure 8(a)), (ii) the nucleobase, which in the crystallographically bound state forms a πstack with the side chain of F197 (F186) (Figure 8(b)), and (iii) 5’-phosphate interacting with loop III residues (residues 146-152, shown in yellow color in Figure 1) via the formation of hydrogen bonds with the backbone -NH, side chain -OH of T150 (T139) (Figure 8(c)). Firstly, 5’-phosphate group of IMP comes out of the binding zone (residues 148 to 152) followed by the removal of its π-stacking interactions with F197. Later, IMP forms a new π-stack with Y116 present in loop II, which guides the ligand towards bulk water (Figure 8). A similar set of events take place in HsHGPRT where Y104 present in loop II guides the movement of IMP. Once these interactions are lost, IMP finds the only path (Figure 6(b)) to leave the protein environment. Although the release of PPi.2Mg from the active site of both the enzymes takes place via path B (major pathway), there is an alternate narrow escape route for the complex. A corresponding path was found to be unused in the parasite enzyme. In the simulation trajectories where PPi.2Mg follows the alternative pathway (path A), the residue K102 was found to interact with the PPi.2Mg complex and thus pull out it from the binding site. As a corresponding interaction is missing during the product release steps in PfHGXPRT, PPi.2Mg always exits via path B in the parasite enzyme. A pairwise sequential analysis (shown in Figure S8 of SI) of the HsHGPRT and PfHGXPRT shows that residues in loop II are largely similar. However, residues prior to K114 (K102) and after G123 (G111) differ in both the enzymes. This could lead to structural and dynamical differences (Figure S9 of SI) observed between the HsHGPRT and PfHGXPRT systems. Although sequential similarity (Figure S8 of SI) in region K114 (K102) to G123 (G111) exists between the two enzymes, K102 in Hs and K114 in Pf exhibit structural differences. The importance of K102 in the differential behavior of HsHGPRT over PfHGXPRT enzyme was further tested by mutating 17 ACS Paragon Plus Environment


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Figure 8: Detailed steps in the release of IMP from PfHGXPRT: a. Interactions of -OH of ribose sugar with E144 and D145, b. π-stack between IMP and F197, interactions between 6-oxo of IMP and K176, c. 5’-phosphate binding region, d. detachment of 5’-phosphate from its binding zone and opening of the flexible loop II, e. breaking of π-stack between the nucleobase of IMP and F197, f. formation of a new π-stack between nucleobase and Y116, and g. rattling of IMP inside the active site, h. IMP near the entrance of the pocket, and i. release of IMP to the bulk water. Release of IMP from the active site of HsHGPRT follows similar steps and hence is not shown. Ligands are shown in ball & stick representation. it to a hydrophobic Ala. Metadynamics simulations of this mutant (K102A) led to the release of both the products via path B, akin to the result for PfHGXPRT (Figure S10(a) of SI). A


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similar mutation on K114 of PfHGXPRT did not change the pathway for the release of the product molecules (Figure S10(b) of SI).

Product release from the mutants Experimental studies have shown that point mutation of residues S103, Y104 and S109 present in loop II reduces the catalytic efficiency of T. cruzi HPRT by 95%. 30 The corresponding residues are S115, Y116 and S121 in PfHGXPRT and S103, Y104 and S109 in HsHGPRT, respectively. We have earlier discussed (in ref. 38) the dynamics of the WT PfHGXPRT dimers and tetramers in their apo and ligand-bound states from classical unbiased simulations. Here, we briefly compare the results obtained from equilibrium simulations of the WT and the mutant of PfHGXPRT and HsHGPRT enzymes. Subtle structural changes are observed in equilibrium simulations of mutants as compared to the WT system. The RMSD fluctuations of the backbone atoms of all the enzymes (PfHGXPRT and HsHGPRT WT and mutants) show structural integrity over the entire simulation time span. However, in some cases, higher RMSDs of the mutants compared to that of the WT enzyme (Figure S11 for PfHGXPRT and Figure S16 for HsHGPRT of SI) indicate that mutation leads to lesser stability of the overall system. Furthermore, the RMSDs of the ligand molecules in all the subunits reveal that the motions are almost similar, except in some subunits of the mutants, they fluctuate to a greater extent (Figures S12 & S13 for PfHGXPRT and S17 & S18 for HsHGPRT). RMSDs of backbone atoms (Figure S14 and S19) of the residues present in loop II are higher for mutants than in WT systems. In some of the subunits of the mutant tetramers, the destabilization of loop II opens the gate within few nanoseconds of the trajectory (chain C of S115G and S121A, and chain D of Y116F), despite the presence of ligands in the active site. Consequently, the active site becomes solvent accessible (Figure S15 and S20), which could explain the reduced activity seen in experiments. 30 We now discuss the roles of these residues in product release. In the WT simulation 19 ACS Paragon Plus Environment


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trajectory, as PPi.2Mg comes out of its binding site, its oxygen atoms interact with the side chain -OH of S115 (and S103 in HsHGPRT) via hydrogen bonding (Figure S21(a) of SI). Mutation of S115 (S103) residue to G115 (G103) eliminates the possibility to form such hydrogen bonds. Hence, the only possible interactions are between the backbone -NH of G115 (G103) and oxygen atoms of PPi. Thus, in the S115G (S103G) mutant structure, PPi.2Mg moves away from loop II and goes towards D204 (D193) and D207 (D196) (Figure S22). These outcomes correlate with the critical role of S115 (S103) in the enzymatic activities of PfHGXPRT and HsHGPRT. The reduction in the catalytic efficiency of the mutants can be partially due to the difference in the interaction mechanisms established here. Another residue in loop II, Y116 (Y104), was proposed to stabilize the carbocation intermediate that is generated during the enzymatic reaction. It forms a π-stack with the nucleobase of the nucleotides (IMP). Additionally, the side chain hydroxyl hydrogen of Y116 forms hydrogen bonds with the 5’-phosphate oxygen atoms of IMP, thereby assisting its movement from the active site to bulk water (Figure S21(b)). Although the mutation of the Y116 (Y104) to Phe may not influence the stabilization of the cation-π interaction, the release of IMP however, can get affected by the change in the functional group from phenol to benzene. Release of the product molecules from the PfHGXPRT Y116F and HsHGPRT Y104F systems are depicted in Figure S23(a) and (b), respectively. During the early movement of IMP in its binding site, F116 (F104) forms π-stack with IMP but later, the release of IMP is guided by F197 in PfHGXPRT (Figure S24(a) & (b)). However, in HsHGPRT, F104 still assists the movement of IMP during its exit from the binding site (Figure S25(a) and (b). A 10 to 40 fold reduction in kcat of T. cruzi 30 HPRT was observed with Y116 (Y104)F mutantation and is possibly due to the decreased involvement of F104 in the substrate binding and stabilization of carbocation intermediate during the catalysis. Medrano et al. 30 also examined the participation of another residue S109 in T. cruzi HPRT in the closure of the active site by loop II. Higher RMSDs of backbone atoms of loop II (Figure S14 and S17 of SI) in the unbiased simulation of IMP.PPi.2Mg bound S121A


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(S109A) mutant, reassert its flexibility. The side chain -OH group of S121 (S109) forms hydrogen bonds with the 5’-phosphate of IMP while IMP moves out of the active site (Figure S21(d) of SI). Mutation of S121 (S109) to a hydrophobic Ala precludes its involvement in the product release process, and alters the release paths for the products (Figure S26). Thus, S121 (S109) not only participates in closing loop II but also in guiding the transport of the nucleotide (the movement of the 5’-phosphate) from its corresponding binding site to bulk water.

Discussion Several experiments have been carried out to investigate substrate binding and product release in HsHGPRT. 26,27 However, a detailed molecular level description and its dynamical characteristics were not explored until now. Specifically, the sequence of release of IMP and PPi.2Mg has been investigated experimentally and the latter has been suggested to get released first. 26 In our biased MD simulations, both the product molecules were allowed to traverse independently from the binding sites. In a majority (23 out of 25) of the simulations, IMP was seen to precede the release of PPi.2Mg. However, the sequence cannot be asserted, as that would require larger number of independent metadynamics simulations which is computationally forbidding; the same reasoning holds true for obtaining the free energy surface for the product release. In fact, IMP is closer to bulk water, while PPi.2Mg is located at the end of the channel (Figure 5). Further, IMP π-stacks weakly with F197 (F186) (Figure 2) and its 5’-phosphate interacts with the backbone -NH, -OH of T150 (T139); whereas PPi.2Mg is tightly held with the positively charged residues e.g., R212, R112, K114. These differences in interactions also indicate that IMP is more prone to move out of the pocket once bias was imposed on the products. The release of the product molecules have been studied from a single subunit of a tetramer, in our case subunit A of the enzymes. An additional metadynamics simulation on the release of the products from subunit C of



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PfHGXPRT provided similar results indicating that all the subunits of the tetramers would behave in a similar fashion. However, this needs further investigations which are beyond the scope of this work. We summarize our observations: (i) Products, IMP and PPi.2Mg follow chiefly a single release path (path B) to bulk water in both the human and parasite HG(X)PRT. However, on a few occasions, an alternative escape route for PPi.2Mg has been found in HsHGPRT. (ii) the minor escape channel for the release of PPi.2Mg in HsHGPRT can be ascribed to the orientation of K102 present in loop II of HsHGPRT. (iii) The release of IMP from its binding pocket is associated with the swinging motion of D145 and D134 in PfHGXPRT and HsHGPRT respectively. It would be interesting to investigate the release processes of the product molecules by means of biochemical experiments. Experimental mutations of specific residues as done here and studying their activity too can be explored. Investigations of the detailed steps involved in the catalytic turnover cycles i.e., substrate binding, catalytic reaction and product release is still a demanding task; the challenges lie in the computational complexities in handling each of these steps. On this note, we have previously identified key molecular events associated with the activation of PfHGXPRT. 15 Herein, our identification of the release pathways in the two enzymes and changes brought about by mutants of specific residues is hoped to spur further experiments on these vital enzymes.

Acknowledgement We acknowledge the DST, India for support. T. K. acknowledges UGC for senior research fellowship.


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Supporting Information The SI contains details of the metadynamics simulations, COLVAR plots from metadynamics simulations, comparison between the chosen initial conformations and that obtained from cluster analysis, figures showing secondary binding site of PPi.2Mg, release of the products from tetramers, figures showing details about the two release pathways for PPi.2Mg in HsHGPRT, distance between loop II residues and PPi obtained from unbiased simulations, sequence analysis, product release from the HsHGPRT K102A and PfHGXPRT K114A systems, results from the unbiased simulations of both the WT and mutants, interaction between the ligands and few residues lining the release pathways, product release in WT and mutants, tables containing details about the residues lining the product release pathways. This information is available free of charge via the Internet at http://pubs.acs.org.

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Plasmodium falciparum

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