Umbrella Sampling and X-ray Crystallographic Analysis Unveil an Arg

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Umbrella Sampling and X-ray Crystallographic Analysis Unveil an Arg-Asp Gate Facilitating Inhibitor Binding Inside Phosphopantetheine Adenylyltransferase Allosteric Cleft Abhisek Mondal, Rakesh Chatterjee, and Saumen Datta J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.7b09543 • Publication Date (Web): 18 Jan 2018 Downloaded from http://pubs.acs.org on January 18, 2018

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The Journal of Physical Chemistry

Title: Umbrella Sampling and X-ray Crystallographic Analysis Unveil an Arg-Asp Gate Facilitating Inhibitor Binding Inside Phosphopantetheine Adenylyltransferase Allosteric Cleft

A. Mondal, R. Chatterjee and S. Datta*

Structural Biology and Bioinformatics Division, Council of Scientific and Industrial Research-Indian Institute of Chemical Biology, 4 Raja SC Mullick Road, Jadavpur, Kolkata, West Bengal, India. *

Corresponding Author: Email- [email protected] Telephone Number: +913324995896

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Abstract Phosphopantetheine adenylyltransferase (PPAT) is a rate limiting enzyme essential for biosynthesis of coenzyme A (CoA) which in turn is responsible to regulate the secretion of exotoxins via type III secretion system in Pseudomonas aeruginosa, causing severe health concerns ranging from nosocomial infections to respiratory failure. Acetyl Coenzyme A (AcCoA) is a newly reported inhibitor of PPAT, believed to regulate the cellular levels of CoA and thereby the pathogenesis. Very little is known so far regarding the mechanistic details of AcCoA binding inside PPAT binding cleft. Herein, we have used extensive umbrella sampling simulations to decipher mechanistic insight regarding the inhibitor accommodation inside the binding cavity. We found out that R90 and D94 residues act like a gate near the binding cavity to accommodate and stabilize the incoming ligand. Mutational models concerning these residues also show considerable difference in AcCoA binding thermodynamics. To substantiate our findings, we have solved the first crystal structure of apo-PPAT from P. aeruginosa which also found to agree with the simulation results. Collectively, these results describe the mechanistic details of accommodation of inhibitor molecule inside PPAT binding cavity and also offer valuable insight for regulating cellular levels of CoA/AcCoA and thus controlling the pathogenicity.


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Introduction: Pseudomonas aeruginosa is a frequent cause of nosocomial pneumonia, having mortality rate of ≤ 60%1. This gram-negative pathogen is a predominant cause of nosocomial bacteremia among immune compromised individuals2. Clinical reports show that chronic colonizing of these bacteria causes eventual respiratory failure and even death in cystic fibrosis patients2-3. World Health Organization published a report on Antimicrobial Resistance in 2014, saying increasing number of multidrug resistant bacteria continues to complicate medical treatment of infected individuals per year. P. aeruginosa infections in immune compromised patients primarily conserved to its own toxin repertoire, in spite of that increasing antibiotic resistance renders the treatment more difficult4-5. As suggested by Osterman and Begley, structural and mechanistic analysis of different conserve pathways, responsible for survival and proliferation of pathogens, would help us to provide new insights in order to develop novel drugs6-7. Pantothenate and Coenzyme A (CoA) biosynthetic pathway which is regulated by pantothenate kinase and phosphopantetheine adenylyltransferase (PPAT) fulfills these criteria. In major metabolic pathways CoA functions as an acyl carrier and also activates carbonyl groups in many reactions. It is also found to be involved in fatty acid biosynthesis, lipopolysaccharide biosynthesis, Krebs cycle6, 8. The biosynthetic precursor of CoA is pantothenate. However, the source of pantothenate varies in different organisms. It is produced de novo in some bacteria and other bacteria, like P. aeruginosa uses extracellular pantetheine for CoA biosynthesis in five different steps. Phosphopantetheine adenylyltransferase (PPAT) catalyzes the rate limiting step to reversibly transfer the adenyl group from adenosine tri-phosphate (ATP) to 4’phosphopanteheine (PhP) to produce 3’-dephosphocoenzyme A (dPCoA) and pyrophosphate (PPi). dPCoA-kinase further converts the dPCoA to produce CoA9. This second rate limiting step in CoA biosynthesis pathway is catalyzed by PPAT and also regulates the cellular CoA content, simultaneously with the catalytic protein pantothenate kinase10. Various virulence pathways are tightly regulated by metabolic pathways which act in a synchronized manner to ensure pathogenesis in correct environmental and cellular condition. Prior studies emphasized that CoA is utilized by Pyruvate Dehydrogenase Complex (PDC) to produce Acetyl-CoA which in turn regulates malate synthase (glcB) and isocitrate lyase (aceA) and thereby modulating the PcrG and PcrV (crucial exotoxin and chaperone for infection) secretion in Type III secretion system1112 . PPAT resembles the characteristic feature of alpha/beta phosphodiesterase superfamily in nucleotide binding mediated by 6 β Rossman fold. This family bears H/TXGH motifs13 which help to stabilize the pentacoordinate transition state during the transition from allosteric to substrate binding state and vice versa. PPAT is found to follow this mechanism and possesses a dinucleotide binding groove consistent with class I aminoacyl-tRNA synthetases which binds with ATP and undergoes 4’-phosphopantehteine mediated nucleophilic attack in an in-line displacement mechanism14-15. Many PPAT co-crystal structures, bound with different ligands, have been solved till date to understand the mechanism of interactions of various substrates or 3 ACS Paragon Plus Environment


ligands with this enzyme14-22. Recent X-ray crystallographic studies of PPAT from P. aeruginosa has reported a new allosteric molecule (AcCoA), which binds in the CoA binding groove and thereby inhibits the enzyme action. Structural characterization revealed that R90 and D94 residues play a crucial role in AcCoA binding in the PPAT allosteric site23. It is proposed earlier that these two residues show a gate or bridge like phenomena using ionic interactions that might allow the AcCoA binding23. Prior structural studies with apo form of M. tuberculosis showed that the ligand accommodation to the binding groove is solely mediated by movement of a loop region. Although in a more recent study, using a series of X-ray crystallographic structures, Chatterjee et al 23 has reported that in P. aeruginosa the mechanism of ligand accommodation happens in a completely different manner. The later study hypothesized that, unlike other structures from different organisms, in P. aeruginosa a ‘gate-like mechanism’ actively controls the entry and exit of ligands inside binding cleft. However, the exact mechanism or existence of such highly choreographed molecular movement still remains poorly understood. In this study, we have attempted to further comprehend the structural details and mechanisms involved in AcCoA binding. COM pulling and Umbrella Sampling simulation are used to decipher how these critical residues allow inhibitor binding from a mechanistic point of view. Additionally, we have also created PPATR90A/D94A double-mutants and performed molecular dynamic simulation to obtain the difference in dissociation free energy landscape. To further corroborate our results, we have solved the apo structure of PPAT for first time from P. aeruginosa using X-ray crystallographic analysis. Our results, clearly shows for the first time, the mechanistic details of the inhibitor binding in the PPAT groove mediated by a gatingmechanism of R90 and D94 residues using enhanced sampling of structures throughout reaction coordinate. The observations described herein are believed to be helpful for targeted drug designing that are able to alter most crucial interactions of the binding site.

Material and method: MD simulations The PPATWT-AcCoA structure used for this study was obtained from RCSB-PDB (PDB ID: 3X1J). This was the only structure reported of PPATWT co-crystallized with its inhibitor AcCoA. The structure had a trimeric asymmetric unit. We used a single chain with lower average Bfactor value for our further analysis. The molecule was parameterized using GROMOS96 43a1 force field and also applied to the system24. Automated Topology Builder web-server was used to build the topology of the acetyl-CoA25. Particle mesh Ewald (PME) algorithm was used for calculation of long-range electrostatics and short-range nonbonded interactions were cut off at 1.4 nm26-27. Periodic boundary condition was applied on the system in all directions. Before starting the pulling simulation, the crystal structure was well equilibrated. The structure was placed in a cubic box (5 nm) with simple point charge (SPC) water28. 100 mM NaCl was added to the system with neutralizing counterions (82 Na and 82 Cl ions). The energy was minimized with steepest descents minimization method and subjected to two phase of equilibration process, 4 ACS Paragon Plus Environment

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with position restraints applied to protein-ligand complex throughout. In the first phase, the structure was simulated for 20 ns with constant volume (NVT) ensemble. Separate temperature cooling bath was used for PPAT-AcCoA and water-ions, maintained at 310 K using Berendsen weak coupling method29. In phase two, another 20 ns of constant pressure (NPT) equilibration was performed. Production MD simulations were conducted for 50 ns after withdrawing all restraints. For production MD procedure, Noose-Hoover thermostat was used for maintaining temperature30-31 and Parrinello-Rahman barostat was used for isotopically regulation of pressure32. To perform all the simulations GROMACS-4.6.2 package was used33. After the equilibration of the structure, the residues interacting with the ligand were identified (depicts the active surface area of the binding cleft) (Figure 1b) and the center of mass (COM) of these residues was used to draw the pulling vector through the COM of ligand molecule towards outside of the cleft. Followed by, AcCoA was pulled along this vector using spring constant of 500 kJ mol-1 nm-2 and pull rate of 0.01 nm ps-1 to achieve complete separation. To check the method of pulling, slower pulling rate was also used which yielded nearly same force-vs-time curves. During the pulling simulation, snapshots were taken to generate staring configurations for umbrella sampling windows34-35. The unbinding vector was defined dynamically for each individual umbrella sampling simulations based on the COM of the ligand and COM of the binding cleft’s opening36-37. We analyzed the pulling trajectories and collected different windows for a robust potential of mean force (PMF) reconstruction with applied harmonic potential for the simulations. We used 35 different trajectories for reconstruction until the PMF appears convergent and total simulation time used was >350 ns. Error estimation was performed using bootstrap method38. Purification, crystallization and X-ray diffraction of apo-PPAT: The phosphopantetheine adenylyltransferase (PPATWT) was cloned and purified according to the previously reported methodologies23. Sparse matrix screening was performed with Crystal screens (Hampton research) using 16.6 mg/ml protein. Oily patches were appeared, after two months, when 2 µl of protein solution was added with 2 µl of Na-formate and incubated against Na-formate. To get diffraction size crystals a gradient of 3.25-3.3 M Na-formate was used and was optimized with or without 10 mM Caacetate. The crystals developed cracks when grown beyond 0.6 mm and had layered morphology. Additive screening of PPAT with 4% Polyvinyl Pyroliddone K-15 showed uneven shaped layered crystals and 1-5% PEG400 yielded showered crystals. Optimal sized crystals obtained in 3.25-3.3 M Na-formate with 10 mM Ca-acetate, were harvested in 9 well trays and were flash frozen in -180º C in cryo stream. All the diffraction data, for optimization purposes, were collected in our home source using Rigaku R-axis IV++ detector with 0.5º oscillation mode. A seed-stock was prepared using 2-3 single crystals and subjected to microseeding matrix screening. Crystals appeared using PEG 4000, 20% isopropanol and HEPES pH 7.0 condition. Gradual evaporation of alcohol made harvesting initial crystals very difficult. Finally, 16-18% 5 ACS Paragon Plus Environment


PEG 4000, 0.1 M HEPES pH 7.1, 5% isopropanol and 200 mM Na-acetate yielded diffraction quality crystals. 25-30% DMSO was used as cryo-protectant with the reservoir solution. Final data collection was performed in INDUS 2 Beamline PX21 using marCCD X-ray detector with 1º oscillation range at 93 K. Structure Solution: The oscillation files were integrated using HKL-2000 and iMosfilm39, followed by scaling was performed in Scalepack40. Crystals belong to the space group C121. PPAT-AcCoA structure from P. aeruginosa (3X1J) was used as a starting model for structure solution using molecular replacement method in MOLREP41 program. Subsequent model building was performed in Coot0.8.3 and refinement procedure was carried out using PHENIX.refine package42. MolProbity package was used for further stereochemical validation of the structure of PPAT43-44. Schrodinger and Chimera package was used for identifying interaction sites and molecular visualizations45.

RESULTS AND DISCUSSION: Interaction of inhibitor (AcCoA) with binding cavity PPAT is a very crucial enzyme responsible for catalyzing the penultimate rate limiting step in CoA biosynthesis pathway. The relation of this highly conserved metabolic pathway with regulation of bacterial virulence system (e.g. Type III secretion system) makes it an intriguing

Figure 1. Snapshot of Acetyl-CoA(represented in CPK) binding cavity inside PPAT of P. aeruginosa. a. Trimeric PPAT (representing one asymmetric unit) with bound Acetyl-CoA (PDB ID: 3X1J) with each protomer. b. 2D interaction diagram representing the major interactions between AcCoA and PPAT in the binding cleft. These interacting residues were used to calculate the COM of PPAT’s binding site.

subject of study. After all these years of study it came up very recently that Acetyl-CoA also can bind with the PPAT and downregulate the CoA biosynthesis. For our studies, we have obtained 6 ACS Paragon Plus Environment

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the only available crystal structure of PPAT bound with inhibitor Acetyl-CoA (Figure 1a) from RCSB-PDB. Later, we have chosen a single chain with lower average B-factor value for our further mechanistic studies. We performed 50 ns of NVT and NPT simulations on this structure using GROMACS-5.1.4 package and explored the interacting residues around the binding cavity of PPAT. Using Ligplot package46, we have identified all the interacting residues within 3.5 Å cut-off range of the bound inhibitor molecule (Figure 1b). In agreement with the prior report23 the equilibrated structure also shows residues like R90, R87, D94, K41, L73, R87, T129, H17 (neutral state) and T9 involved in electrostatic interactions within that distance cut off. As expected, the non-restrained molecular dynamics results yielded nearly same interaction diagram of allosteric site in accordance with the reported crystal structure (PDB ID: 3X1J). Thermodynamics of Acetyl-CoA binding: The prior study concerned with inhibitor binding also raised an intriguing hypothesis that possibly D94 and R90 residues are acting in a choreographic manner to perform the gating mechanism near binding cavity23. However, further clarification needed the crystal structure of apoPPAT from P. aeruginosa. In this study, we have used umbrella sampling (US) method to understand the energetics by which Acetyl-CoA gets Figure 2. PMF for the dissociation of Acetyl-CoA from the binding site into the binding cavity. of PPAT. The error associated with energy minima is ±0.14 kcal/mol. US gives a reliable way to The reaction coordinate here is represented in ζ (nm). comprehend systems behavior for certain time scales (say, transition phase) which might have otherwise remained inaccessible using both experimental and conventional molecular dynamics (MD) approach. US procedure had been used in earlier studies with reliable results from protein-protein, protein-ligand, lipid bilayerprotein systems46. The pulling force applied on the system perturbs the equilibrium of the system thus making it possible to calculate thermodynamic quantities from US trajectories without large errors. Further, we used weighted histogram analysis method (WHAM) for calculation of free 7 ACS Paragon Plus Environment


energy47 as it uses simulations performed on number of configurational windows generated from umbrella sampling simulation for calculation of ∆G.

We used COM pulling and US simulation to estimate the binding energetics as well as to understand the mechanism of inhibitor binding in the cavity of PPAT. After equilibrating the PPAT-AcCoA crystal structure (Figure S6), the residues involved in interaction with the ligand (within 3.5 Å cutoff range) were identified (Figure 1b). The unbinding vector was defined dynamically for each individual umbrella sampling simulation based on the COM of these interacting residues (representing the active surface area of the ligand binding cleft) through the COM of the ligand towards the opening of the cleft36-37 (Figure 3a). Starting configurations and velocities for the pulling simulations were taken at 15 ps intervals. The ∆G obtained from this US simulation (Figure 2) is in close agreement with the previously reported experimental observation23, which justifies the authenticity of our sampling procedure. Analysis of the snapshots collected from the trajectory showed that R90 and D94 residues together moved quite a lot to maintain the interaction with the ligand while in binding cavity (Figure 2-inset pictures). After the ligand got completely dissociated the displacement of R90 was found to be 12.5 Å and D94 residue was also found to be 4.8 Å away from the starting position (Video S11). Figure 4 clearly shows the difference in orientation of R90 and D94 as the ligand is moving out of its binding cleft. These observations are in good agreement with the hypothesis derived previously regarding the Figure 3. a. Overview of the vector used for movement of these residues. Therefore, it can now be center-of mass pulling along the egression of the binding cleft. b. View of PPAT binding said that these two residues do play very crucial role cleft after 0.5 nm displacement of AcCoA in insertion and accommodation of ligand inside the along the cleft opening. c. Changes in binding cavity. A large helix movement was also neighboring contacts (represented in black seen after AcCoA pulled out and possibly was to dotted lines) inside binding cleft as the ligand prevent entry of water molecules inside allosteric further egress 1 nm. cleft (discussed later) (Figure 4d).


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Figure 4. Snapshot of Acetyl-CoA at the entry gate of PPAT binding cavity. a. Shows the orientation of critical residues when Acetyl-CoA approaches at the gate. b. 2D interaction diagram of ligand acetyl-CoA with the PPAT while the ligand is residing near the gate of binding site. c. Superposed structures of apoPPAT(off white) with AcCoA-PPAT(sky blue) showing outward movement of R90 and D94 while binding cavity is empty. d. Superimposed structure of simulated-PPAT (after complete dissociation AcCoA, in off-white color) and apoPPAT crystal structure (sky blue) showing nearly same kind of outward movements of R90 and D94.

Thermodynamics of PPATR90A/D94A-AcCoA binding: Using PMF calculation and sampling over a large set of configurational windows, we have found evidences that led our belief towards the understanding of R90-D94 mediated gating mechanism. However, to completely rely on the results produced so far, we needed to check the free-energy profile of the same system, mutating those two critical residues. For this purpose, we took the structure prepared earlier after 100 ns of equilibration. The in-silico mutations intended were performed using Pymol package by replacing R90 and D94 residues to alanines (∆PPAT or PPATR90A/D94A). We used alanine mutations to eliminate any possible formation of ionic interaction of these two positions with ligand molecule. Thus, it would provide us a way to analyze either R90-D94 residues were acting as a gate or we just hit an artefactual mechanism.



The mutant was further equilibrated for 100 ns using both NVT and NPT method until the backbone RMSD reached certain stability. After performing thorough equilibration, the structure was subjected to US procedure. The COM of interacting residues around the binding site of ligand (within 3.5 Å cut-off distance) was used to calculate the force vector through the COM of Figure 5. PMF for AcCoA dissociation from PPATR90A/D94A binding ligand (same as used cleft. R90 and D94 residues are replaced with alanine double mutation. previously). COM Well equilibrated structure is used for PMF reconstruction. The error associated with energy minima is ±0.13 kcal/mol. The reaction pulling was performed with 500 kJ mol-1 nm-2 coordinate here is represented in ζ (nm). and pull rate of 0.01 nm -1 ps to achieve complete separation of ligand from PPATR90A/D94A. During this puling 36 different trajectories were recorded (Figure S9) and further umbrella sampling (US) simulations were performed utilizing over 360 ns of computation time for obtaining a convergent PMF∆PPAT. As evident very clearly from the figure that the PMF∆PPAT shows a considerable difference compared to PMFPPAT. PMF calculation shows that the ∆G of binding happens to be near -4.5 kcal/mol for PPATR90A/D94A-AcCoA. COM pulling started the dissociation around the ξ = -2.6 nm and it continued till the ligand got completely separated around ξ = 0.0 nm. The difference observed in PMF∆PPAT clearly suggests that due to the mutation of the R90 and D94 the ligand failed to maintain its position inside the binding cavity. Based on the results we must admit that the differences in ∆G between PPATWT and mutant are small (-2.5 kcal/mol). However, we would like to consider this significant as the ∆G obtained in case of PPATWT goes in accordance with previously reported experimental evidences23. We gave the same treatment for PPATR90A/D94A structure and thus we have a reason to believe that the ∆∆G, however small, but carries significant relevance towards the focus of this work. The dissociation of ligand from allosteric site in COM pulling simulation was also examined using different pulling rates (0.10 and 0.05 nm ps-1) to ensure the pulling rate (0.01 nm ps-1) used in this article did not induce any artifacts. Figure S7 shows that the ligand dissociation occurred in same way in different pulling rates, yielding same sequence of configurations. The required time to attain the maximum force and the magnitude of this force depends on the rate of pulling as the structural changes are happening at different rates. 10 ACS Paragon Plus Environment

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The PPATWT is found in hexameric state in nature. In our previous study23, we have showed that the AcCoA binds to all the protomers and the binding occurs mostly in independent or noncooperative manner. Structural analysis also showed that there is not much difference among individual AcCoA-bound protomers. However, compared to structures reported in other organisms, we found a unique region that possibly assists inhibitor binding/unbinding. We performed the simulation studies using a monomer of P. aeruginosa-PPAT-AcCoA as our primary concern was to decipher the unique ‘gating-mechanism’ of R90-D94 residues upon ligand movement. X-ray crystallographic analysis of apo-PPAT from P. aeruginosa: Based on the extensive simulations performed on both PPAT-AcCoA and PPATR90A/D94A -

Figure 6. Cartoon superposition with Cα traced superposition of the PPAT enzyme from various organisms. a-b. Cartoon representation and its corresponding Cα traced superposition. Showing clearly the loop movement (α3-β2) of M. tuberculosis-PPAT. c-d. 90˚ rotational view of a-b representation. Color code: Black – newly obtained structure of apo-PPAT(P. aeruginosa), PDB ID: 5X6F, red – 5TS2, green – 4RUK, blue – 3X1M, brown – 3X1K, pink – 3X1J, dark blue – 1TFU.

AcCoA structures, quite a vivid picture is formed regarding the mechanistic point of view of ligand accommodation. However, prior studies using X-ray crystallographic structures of PPAT from M. tuberculosis showed that the ligand accommodation is facilitated by movement of a loop region near the binding site. In a recent report Chatterjee et al.23 hypothesized that the mechanism employed by P. aeruginosa is completely different with that of reported earlier in M.



tuberculosis. Our results so far corroborate with the later statement clarifying the fact that instead of loop movement P. aeruginosa uses R90 and D94 residue’s gating mechanism (Figure 6). However, to establish such claim we needed to obtain the first crystal structure of full-length poform of PPAT from P. aeruginosa. Obtaining the crystal structure was very difficult as the purified protein always found to be in complex with either AcCoA or CoA. This experimental observation of strong binding also corroborates with the slow dissociation pattern of AcCoA from binding cavity upon application of constant force. The crystals of apo-PPAT always appeared in a layered manner, leading to interfere with data collection strategy (Figure S1, S2, S3). However, eventually we solved the crystal structure in C121 space group (Figure 7, Table S10).

Figure 7. Crystal structure of apo-PPAT from P. aeruginosa. The structure shows a hexameric asymmetric unit. The protomers of the asymmetric unit is shown in cartoon representation.

Analysis of the active site using PPAT-AcCoA and PPAT crystal structure revealed a striking similarity in movement of R90-D94 residues between the obtained results from US simulation and the crystal structure (Figure 4d). Figure 4c shows that in absence of bound AcCoA both R90 and D94 residues are shown to deviate 4.3 Å and 2.8 Å with side chain flip, respectively. E96 residue also found to be displaced by 3.2 Å in the PPAT crystal structure for avoiding any clash due to the movement of D94. However, a little difference in displacements is encountered between COM-pulling results and X-ray crystallographic structure. Solvent content analysis of the crystal structure revealed that in absence of any ligand, a glycerol and an isopropanol molecule are found to sit inside the binding cavity. This observation also indicates that R90 and D94 residue shows a lot of activity near binding cavity to provide ionic interactions and thereby maintaining stability of the binding cavity.


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Figure 8. Crystal structure superposition of apo forms of M. tuberculosis (sky blue) and P. aeruginosa (off white). a. Superposition cartoon diagram of apo structure from both the organism. The loop movement is very clear between α3 and β2. b-d. Cartoon superposition showing the deviations of critical residue near binding cavity of PPAT in P. aeruginosa, b. apoPPAT(off white) – anpnp-PPAT, c. apoPPAT – coA-PPAT(sea green), d. apoPPAT – dPCoA-PPAT(sea green).

To further illustrate the role of R90 and D94 gate, we used all the previously reported structures of PPAT (ligand bound) from P. aeruginosa and aligned the with apo-PPAT crystal structure. Figure 8b shows that D94 and R90 residue moved 6.9 Å and 3.7 Å, respectively to facilitate binding of AMP-PNP inside binding cavity. For providing binding interactions to dPCoA, D94 residue moved 2 Å towards binding cavity and R90 seems to move 2.1 Å closer to the ligand for providing stability (Figure 8d). Nearly similar observations were found in case of CoA binding also. The D94 residue is found to move 4.5 Å towards the binding pocket while R90 residue moved 2.1 Å towards the CoA to stabilize it (Figure 8c). Structural comparison with apo form of PPAT of M. tuberculosis made it evident that such gating mechanism is a characteristic phenomenon of P. aeruginosa for ligand accommodation in binding cavity. Figure 8a shows the loop movement in P. aeruginosa, with D94 and R90 residues, compared to M. tuberculosis in absence of any ligand. Indeed, Chatterjee et al23 argued about the existence of different kind of binding mechanism in P. aeruginosa which found to be regulating cellular level of CoA and AcCoA (Figure 9); affecting various pathogenic pathways like type III secretion system. 13 ACS Paragon Plus Environment


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However, our work here have provided the first demonstration regarding how this highly choreographed phenomena facilitate ligand accommodation and provide stability inside the binding cavity.

Figure 9. A schematic representation showing metabolic regulation of T3SS by cellular levels of AcetylCoA and CoA.

Conclusion: We have performed extensive molecular dynamic simulations using various approaches to provide detailed understanding of ligand binding inside PPAT binding cavity. We used explicit solvent MD simulations, COM pulling and umbrella sampling strategy to comprehend the mechanism of binding mediated by the key residues. In agreement with previous studies23, we find that the mode of ligand accommodation inside binding cavity is very different in P. aeruginosa compared to earlier report in M. tuberculosis. We have further deciphered the R90 and D94 residue mediated gating mechanism near the binding cavity that help maintain ligand accommodation and its stability by moving in and out from binding cleft. The PMF obtained from COM pulling MD simulations unraveled the thermodynamic details indicating the rapid movement of R90 and D94 residues to accommodate ligand molecule in binding cavity and providing stability to it. Expanding upon these findings, we obtained the first crystal structure of apo-PPAT from P. aeruginosa to further validate our prior theoretical observations. Corroborating with our extensive molecular dynamic studies, the X-ray crystallographic study also revealed that D94 and R90 residues indeed show a large movement near binding site and hence provide a gating mechanism for ligand accommodation and stability. Further validating these finding, we have shown considerable difference in binding free energy using PMF 14 ACS Paragon Plus Environment

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calculation of PPATR90A/D94A mutant. Thus, we propose that small molecular compounds, interfering with these particular residues or blocking the gating mechanism may provide a viable way for controlling the cellular level of CoA or Acetyl-CoA. This in turn could be helpful to down regulate the expression level of various pathogenic pathways like type III secretion system’s exotoxins and thereby avoiding the infection.

Supporting Information Supporting information contains figures, table and video files relevant to this manuscript.

Acknowledgements: We thank R.P. Thangavelu (Sr. Principal Scientist, CSIR CMMACS, Bangalore) for providing access to the Supercomputing facility. Financial support was provided by the Department of Science and Technology, Govt. of India (Grant no: SB/SO/BB – 36/2014) and Council of Scientific and Industrial Research, Govt. of India (Grant no: TREAT – BSC0113, UNSEEN – BSC0116). CSIR-SRF fellowship was provided to AM.

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TOC Graphic Title: Umbrella Sampling and X-ray Crystallographic Analysis Unveil an Arg-Asp Gate Facilitating Inhibitor Binding Inside Phosphopantetheine Adenylyltransferase Allosteric Cleft A. Mondal, R. Chatterjee and S. Datta*


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Figure 1. Snapshot of Acetyl-CoA(represented in CPK) binding cavity inside PPAT of P. aeruginosa. a. Trimeric PPAT (representing one asymmetric unit) with bound Acetyl-CoA (PDB ID: 3X1J) with each protomer. b. 2D interaction diagram representing the major interactions between AcCoA and PPAT in the binding cleft. These interacting residues were used to calculate the COM of PPAT’s binding site.

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Figure 2. PMF for the dissociation of Acetyl-CoA from the binding site of PPAT. The error associated with energy minima is ±0.14 kcal/mol. The reaction coordinate here is represented in ζ (nm). 38x29mm (600 x 600 DPI)


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Figure 3. a. Overview of the vector used for center-of mass pulling along the egression of the binding cleft. b. View of PPAT binding cleft after 0.5 nm displacement of AcCoA along the cleft opening. c. Changes in neighboring contacts (represented in black dotted lines) inside binding cleft as the ligand further egress 1 nm. 356x905mm (300 x 300 DPI)



Figure 4. Snapshot of Acetyl-CoA at the entry gate of PPAT binding cavity. a. Shows the orientation of critical residues when Acetyl-CoA approaches at the gate. b. 2D interaction diagram of ligand acetyl-CoA with the PPAT while the ligand is residing near the gate of binding site. c. Superposed structures of apoPPAT(off white) with AcCoA-PPAT(sky blue) showing outward movement of R90 and D94 while binding cavity is empty. d. Superimposed structure of simulated-PPAT (after complete dissociation AcCoA, in offwhite color) and apoPPAT crystal structure (sky blue) showing nearly same kind of outward movements of R90 and D94.


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Figure 5. PMF for AcCoA dissociation from PPATR90A/D94A binding cleft. R90 and D94 residues are replaced with alanine double mutation. Well equilibrated structure is used for PMF reconstruction. The error associated with energy minima is ±0.13 kcal/mol. The reaction coordinate here is represented in ζ (nm). 42x35mm (600 x 600 DPI)



Figure 6. Cartoon superposition with Cα traced superposition of the PPAT enzyme from various organisms. a-b. Cartoon representation and its corresponding Cα traced superposition. Showing clearly the loop movement (α3-β2) of M. tuberculosis-PPAT. c-d. 90˚ rotational view of a-b representation. Color code: Black – newly obtained structure of apo-PPAT(P. aeruginosa), PDB ID: 5X6F, red – 5TS2, green – 4RUK, blue – 3X1M, brown – 3X1K, pink – 3X1J, dark blue – 1TFU. 34x23mm (600 x 600 DPI)


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Figure 7. Crystal structure of apo-PPAT from P. aeruginosa. The structure shows a hexameric asymmetric unit. The protomers of the asymmetric unit is shown in cartoon representation.



Figure 8. Crystal structure superposition of apo forms of M. tuberculosis (sky blue) and P. aeruginosa (off white). a. Superposition cartoon diagram of apo structure from both the organism. The loop movement is very clear between α3 and β2. b-d. Cartoon superposition showing the deviations of critical residue near binding cavity of PPAT in P. aeruginosa, b. apoPPAT(off white) – anpnp-PPAT, c. apoPPAT – coA-PPAT(sea green), d. apoPPAT – dPCoA-PPAT(sea green). 37x27mm (600 x 600 DPI)


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Figure 9. A schematic representation showing metabolic regulation of T3SS by cellular levels of Acetyl-CoA and CoA. 118x81mm (600 x 600 DPI)



TOC Graphic 42x21mm (300 x 300 DPI)


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Umbrella Sampling and X-ray Crystallographic Analysis Unveil an Arg

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