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Modulation of the Conformational Dynamics of Apo Adenylate Kinase through a #-cation Interaction Ritaban Halder, Rabindra Nath Manna, Sandipan Chakraborty, and Biman Jana J. Phys. Chem. B, Just Accepted Manuscript • Publication Date (Web): 23 May 2017 Downloaded from http://pubs.acs.org on May 29, 2017

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The Journal of Physical Chemistry

Modulation of the Conformational Dynamics of Apo Adenylate Kinase through a π-cation Interaction

Ritaban Halder, Rabindra Nath Manna, Sandipan Chakraborty and Biman Jana*

Department of Physical Chemistry, Indian Association for the Cultivation of Science, Jadavpur, Kolkata-700032

*Corresponding author: Dr. Biman Jana: Department of Physical chemistry, IACS, Kolkata-700032, India. Phone: +91 33 2473 4971; Fax: +91 33 2473 2805; E-mail: [email protected] 1

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Abstract

Large scale conformational transition from open to closed state of adenylate kinase (ADK) is essential for its catalytic cycle. Apo-ADK undergoes conformational transition in such a way that closely resembles open to closed conformational transition. Here, equilibrium simulations, free energy simulations, QM/MM calculations in combination with several bioinformatics approaches have been used to explore the molecular origin of this conformational transition in apo-ADK. Apart from its conventional open state, E. coli apo-ADK adopts conformations that resemble a closed-like intermediate, the “half-open-half-closed” (HOHC) state and a π-cationic interaction can account for the stability of this HOHC state. Energetics and electronic properties of this πcationic interaction have been explored using QM/MM calculations. Upon rescinding the πcationic interaction, the conformational landscape of the apo-ADK changes completely. The apoADK population is shifted completely towards the open state. This π-cationic interaction is highly conserved in bacterial ADK, the cationic guanidinium moiety of a conserved ARG interacts with the delocalised π-electron cloud of either PHE or TYR. This study fascinatingly demonstrates modulation a principal protein dynamics by a conserved specific π-cationic interaction across different organisms.

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Introduction Adenylate kinase (ADK) is an important metabolic monitor that involves in cellular energy homoeostasis and signaling.1 The enzyme catalyzes the phosphoryl transfer reaction: Mg+2ATP + AMP ↔ Mg+2ADP + ADP Structurally, the protein is composed of three domains: LID, CORE and NMP.1 During the chemical reaction, ligand binding induces large scale allosteric motions of LID and NMP domains with respect to CORE domain (Figure S1, ESI).2,3 This collective dynamics between several domains is a prerequisite to facilitate the chemical reaction.4,5 Wolf-watz et al. showed that dynamics of the protein significantly influences the enzymatic turnover of ADK.4 Particularly, opening of the nucleotide binding LID has been found to be the rate-limiting step, evident from NMR relaxation data.4 Substrate binding induces conformational changes of the active site residues in such a way that facilitate enzymatic reaction. ATP binds at the interface between the CORE and LID domains while AMP binds at the space between the CORE and NMP domains. Substantial movement of the LID and NMP domain towards the CORE domain occurs during the open to closed transition of ADK.6 Many computational studies using all-atom simulations7-10 and various coarse-grained models11-16 have been applied to explore the origin of the co-operative movement of LID and NMP domain and its functional significance. Recent well-tempered metadynamics simulation of adenylate kinase using an all-atom model (ff99sb amber force field) in explicit TIP3P water model reveals that the LID domain is more dynamic than the NMP domain.17 Free energy calculation using all-atom model (ff99sb amber force field) of ADK reveals that the LID domain motion access several conformations ranging from open to closed state, however, the closed and open states of NMP domain are separated by a free-energy 3



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barrier of 9.9 kJ/mol.17 Notably, free energy calculation using coarse-grained model also revealed similar barrier height for the conformational transition of the NMP domain from open to closed form.18 Exploration of the sequence of events of this ligand induced conformational transition and its coherence with the catalytic turnover of ADK has been studied by both experimental and simulation techniques.14,17,19,20 It has been demonstrated that ATP/AMP binding induces a dynamic equilibrium between the open and closed state of their respective ligand binding domain of ADK and binding of both ATP and AMP is required for complete closure of ADK which is highly co-operative in nature.6 Although, the large-scale ligand induced structural dynamics of ADK has been studied extensively, the role of this structural dynamics on the processivity of the enzyme is still debatable.4,21-24 Using reaction funnel model, Min et al. demonstrated that slow conformational motion in a collective protein coordinate expedites the catalytic reaction along the intrinsic reaction coordinates.22 NMR relaxation data showed that the hyper-thermophilic ADK shows slower catalytic activity due to slow lid-opening rate.4 However, Pisliakov et al., showed that conformational dynamics of ADK does not contribute significantly to catalysis using a renormalization approach which convert the energetics and dynamics of the protein into an effective 2-dimensional system.23 Mechanism of ligand recognition by ADK is another subject of interest. Early concepts about these motions are thought to be induced fit in nature which is primarily motivated from the crystal structures of the apo (open state) and ligand-bound (closed state) ADK.3,25 Schulz et al., solved three high resolution crystal structures of ADK, one in ligand free form and the other two are ADK bound with one and two ligands, respectively.25 The ligand considered was a substrate mimicking inhibitor. Based on these structures they proposed the induced fit model for substrate

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recognition. Binding of AMP induces conformational changes in the NMP binding domain while ATP binding induces conformational changes in both LID and NMP binding domain.25 However, NMR and single molecule experiments revealed that the protein is highly dynamic even in the apo form.19,26 Hanson et al. demonstrated that apo-ADK is in equilibrium between two distinct states and the equilibrium favours the closed like state due to the closure of the LID domain, evident from high resolution FRET study.26 Henzler-Wildman et al. showed that apoADK can undergo conformational transition in such a way that closely resembles open to closed conformational transition.19 Free ADK can adopt conformations that are not open rather resembles the closed conformation27,28 but it is not completely closed, termed as “half-open-halfclosed” (HOHC) conformation.7,15,19 Appearance of such state implicates functional relevance.8,29,30 This state perhaps facilitates ligand binding due to the reduction of the energy barrier of the conformational transition compared to the transition from fully open to close state. Currently, a conformational selection hypothesis has been proposed to explain the ligand recognition mechanism of ADK.8,28,31 Many salt bridge interactions at the different domain interfaces of ADK, like the salt bridges LYS136-ASP118 at the LID-CORE interface and the LYS57-GLU170 and ASP33-ARG156 at the CORE-NMP and LID-NMP interfaces, respectively, have been shown to exhibit profound influence on the protein conformational dynamics.32 However, despite many experimental and theoretical studies, a systematic study of the molecular origin of this conformational dynamics of apo-ADK in terms of its intermediate is yet to be determined. Most importantly, natural selection of this conformational dynamics of ADK has not been explored earlier.

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Here, our primary objective is to explore in details the conformational dynamics of the wild-type as well as different mutants of E. coli ADK to identify crucial interactions that dictates the functional dynamics of the apo ADK using long equilibrium simulations, free energy simulations in combination with QM/MM calculations. In addition, we have also explored the conservedness of the interaction in bacterial ADK protein sequences. Materials and Methods Equilibrium Simulation methodology Open state ADK structure was obtained from protein data bank (PDB ID: 4AKE). Crystallographic waters and all heteroatoms were removed. All the simulations were carried out using GROMACS 4.5.533-35 using OPLS/AA force field.36,37 Open state ADK was then subjected to a preliminary short energy minimization in vacuo using the steepest descent algorithm. This minimized structure was then used to generate different ADK mutants using the mutagenesis toolkit implemented in VMD.38 Position of each mutation is summarized in Table S1 (ESI). All the mutants were then energy minimized in vacuo using the steepest descent algorithm. Each minimized protein was then solvated with SPC/E39 explicit water model in a cubic box with periodic boundary condition. The box dimension was chosen such that all the protein atoms were at a distance equal to or greater than 10 Å from the box edges. The simulated system was then made charge neutral by adding appropriate number of Na+ ion. The solvated system was then subjected to 1000 steps of energy minimization using steepest descent algorithm which is followed by 5000 steps of minimization using conjugate gradient algorithm. After that each system was subjected to 5 ns position restrained dynamics in NPT ensemble where the protein was restrained by adding restraining forces while the water molecules were allowed to move 6


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freely. Temperature was maintained at 300 K by using Nose-Hoover thermostat40,41 and pressure was maintained by Parrinello-Rahman barostat.42 Electrostatic interactions were calculated using particle mesh Ewald summation method using a grid spacing of 0.12 nm and interpolation order of 4. Van der Waals interactions were truncated at 10 Å. The trajectories were stored at every 1 ps. Analyses were carried out with the trajectory analysis tools implemented in GROMACS. Details of the timescales for different simulations are summarized in Table S1 (ESI). Free energy simulation using umbrella sampling We have carried out umbrella sampling simulation43 in order to explore the thermodynamic stability of different states of wild type as well as several ADK mutants. For this purpose, two dimensional free energy surfaces were constructed by taking distance between different domains of ADK as the order parameter. LID-CORE and NMP-CORE distances were considered as order parameters. In our multidimensional free energy studies, we varied the LID-CORE distances from 1.9 to 3.4 nm and also the NMP-CORE distances from 1.8 to 2.5 nm with an interval of 0.1 nm. At each window umbrella force constant of 500 kJ mol-1 nm-2 was used to keep the domains at the desired position. In each umbrella window, 1ns equilibration was carried out followed by 2ns production run. Weighted histogram analysis44 method available in GROMACS was used to construct the potential of mean force profile. Sufficient overlap among all the windows was confirmed by histogram analysis. Quantum Mechanics/Molecular Mechanics (QM/MM) optimization The structure of wild type ADK in HOHC state obtained from the 2-D free-energy simulation was used as the initial guess structure for the QM/MM optimization methodology.

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Before QM/MM calculations, each system comprising of protein, water and ions were optimized classically using 5000 steps of conjugate gradient. Then the optimized system was used to perform QM/MM calculation.45 All the QM/MM optimization calculations were performed using the micro–macro iteration scheme46 implemented in the fDYNAMO library.47 For wild-type ADK, the phenyl side chain of PHE137 and the guanidinium moiety of ARG119 were considered in the QM part while rest of the system was considered in MM part. Based on previously reported good performance in model studies of the π-cation systems, the M06-2X functional48 in combination with the split valence 6-31+G(d,p) basis set was used to optimize the QM region in our QM/MM calculations, whereas rest of the system was treated with the OPLSAA force-field. In QM/MM calculations, we considered two different mutants of ADK. In one case the PHE137 was mutated with TYR residue and in other case the aromatic side chain of PHE137 was mutated with cyclohexane. Cyclohexane parameters were developed according to the OPLS force-field defined atomic groups. The partial atomic charges were calculated using AM1-BCC charge model.49 Both the mutants were solvated with SPC/E water and made charge neutral. Then each of the system was energy minimized using conjugate gradient algorithm which is followed by the QM/MM calculation using the similar protocol mentioned above. Herein, π-cation interactions energy (E) was calculated using the equation (1) E = Etotal – [Ecation + Eπ]

(1)

Where, Etotal is the total MM perturbed QM energy of the system where both the cation and π system were considered in the QM system, Ecation is the MM perturbed QM energy of the system where only cationic moiety was considered in the QM system. Eπ is the MM perturbed QM energy of the system where the aromatic residue was considered in the QM system. 8


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Multiple sequence alignment and phylogeny Bacterial ADK sequences were obtained from NCBI (www.ncbi.nlm.nih.gov ) protein sequence database. Redundant sequences were removed by using the CD-HIT program50 using a sequence identity cut-off of 0.95. Multiple sequence alignment (MSA) was carried out using MUSCLE program.51 The MSA was then used to draw the phylogenetic tree using PhyML52 web-interface and the generated tree was rendered using TreeDyn program.53 Modeling and simulation of Streptococcus pneumonia ADK Streptococcus pneumoniae ADK sequence was obtained from NCBI protein sequence database and sequence alignment with E. coli ADK revealed a 45.77 % sequence identity. Thus the structure of E. coli ADK (PDB ID: 4AKE) in open form was used as a template to model Streptococcus pneumoniae ADK. It is noteworthy that the crystal structure of the Streptococcus pneumonia ADK is available in PDB, but there is a missing region in the structure at the LIDCORE interface participating in the π-cation interaction. The open state structure was then subjected to a preliminary short energy minimization in vacuo using the steepest descent algorithm. The minimized protein was then solvated by SPC/E explicit water model in a cubic box with periodic boundary condition. The box dimension was chosen such that all the protein atoms were at a distance equal to or greater than 10 Å from the box edges. The simulated system was then made charge neutral by adding appropriate number of Na+ ion. The solvated system was then subjected to 1000 steps of energy minimization using steepest descent algorithm followed by 1 ns position restrained dynamics in NPT ensemble. All the details of the barostat and thermostat used were similar to the wild-type E. coli ADK. It is noteworthy that the open form of Streptococcus pneumoniae ADK is more open compared to E. coli ADK. Therefore, to 9



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achieve this initial more open form, we performed 30 ns equilibration run. During this period the modeled Streptococcus pneumoniae ADK became more open with a LID-CORE distance of 3.25 nm which is similar to the crystal structure of the Streptococcus pneumoniae ADK. After this long equilibration, production simulation was carried out for 100 ns in NPT ensemble.

Results and Discussions Conformational dynamics of the wild-type and different mutants of E. coli ADK have been probed using equilibrium simulations, free energy simulations in combination with QM/MM calculations. Comparisons of the crystal structures of open and closed ADK show that the LIDCORE distance varies from 3.1 to 1.9 nm while the NMP-CORE distance changes from 2.25 to 1.85 nm on going from open to closed state.7 Thus LID-CORE distance variation undergoes more dramatic changes during the conformational transition, in accordance with recent simulation studies.17,27 Equilibrium simulation of wild-type ADK at 300 K shows that the LID domain is more flexible compare to the NMP domain which also resembles well with the recent meta-dynamics study17 which shows that the LID motion is barrier free while the NMP domain has relatively high free-energy barrier between the open and closed states.17 Interestingly, although we have started the simulation with the ADK in open state and during the simulation many different conformations of the protein with widely varied LID opening are visited which are primarily grouped into two different conformational states (Figure 1A). It is noteworthy that we have considered residues 1-29, 60-121 and 160-214 as CORE domain; residues 30-59 as NMP domain and residues 122-159 as LID domain, as considered by Hongfeng et al.,54 and the distances are referred in term of the center of mass (COM) distance between different domains. 10


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Figure 1: Conformational sub-states of apo wild-type ADK during equilibrium molecular dynamics simulation. A: 2-D scatter plot of LID-CORE distances with RMSD from the open state of ADK. Two different states are shown within circles and labelled. Representative conformations from each state are shown in 1B. LID, CORE and NMP domain is shown in green, yellow and blue colour, respectively.

Open state conformation represents conformation where LID-CORE distance varies from 3-3.6 nm. Remarkably, majority of the protein during the simulation time-scale adopts conformation where the LID domain comes close to the CORE domain, resembling a closed-like state but not completely closed, as evident from the crystal structure of the closed ADK. These conformations are refereed as “Half-Open-Half-Closed” (HOHC) state.7 During the simulation these conformations scanned a wide region of LID-CORE distances from 2.8 to 2.2 nm. It has been 11



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reported from the experiments of kerns et al.,5 that ligand free ADK can adopt conformations which are intermediate between open and closed state and these states are catalytically competent.5 A representative conformation of the open and HOHC state of ADK is shown in Figure 1B. It is evident from the figure that in the HOHC state LID (green) domain is considerably closer to the CORE domain compared to the open state. This conformational transition is a consequence of the hinge bending motion along the LID-CORE interface and this motion is believed to be crucial for catalysis.27,55 It is noteworthy that there are long standing discussions on the nature of inter-domain motions observed in ADK. Several studies demonstrate that the conformation transition of ADK is mediated through the order-disorder transition of the inter-domain region using different coarse-grained (CG) models.11-13 This local folding transition is commonly referred as cracking. However, the predicted degree of cracking in ADK varies greatly in different studies. Microscopically mixed multi-basin Hamiltonian predicts higher degree of cracking in ADK,12 while the macroscopic mixing model proposed a lower degree of cracking during the conformational transition of ADK.14 However, Snow et al., predicted a hinge bending motion during the conformational transition of ADK using an united atom model representation of the protein using GROMOS96 force field.56 Critical residues that play important role in this LID-CORE bending motion have been identified by residue-residue contact map analysis (Figure 2A & 2B). New contacts in case of the HOHC conformation of ADK, involving residues ~134-138 from the LID domain and residues ~118119 from the CORE domains, appear in the contact map (indicated by arrow in the contact map) which is absent in open state of ADK.

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Figure 2: Contact map of open (A) and HOHC (B) conformation of ADK. Specific contacts are zoomed in inset. To further specify the residues involved in that critical interactions, we have performed GLY scanning around that region. Interestingly, when residue 137 from the LID domain or residue 119 from the CORE domain has been mutated to GLY, during the simulation time scale ADK remains in the open state (Figure 3A). Whereas GLY mutation at 118 and 136 position does not change the intrinsic dynamics of ADK, both the open and HOHC states are evident from the equilibrium simulation.

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Figure 3: Conformational dynamics of wild-type and ADK mutants. A: Histogram representation of the LID-CORE distances of different ADK mutants obtained from MD simulations are shown. B: Orientations of PHE137 and ARG119 in open and HOHC state are shown. Residues are shown as stick whereas the protein backbone is rendered in ribbon mode. C: 2-D free energy surfaces of wild type and F137G and R119G mutant ADK are shown as contour plot and colored according to the free energy gradient.

We have performed several additional GLY scanning simulations around the 137 and 119 positions and found that apart from mutation of 119th and 137th residues, mutations in all other 14


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positions do not alter the dynamics of the apo-ADK; the HOHC state is clearly evident from the simulation (Figure S2, ESI). Close inspection of the trajectory reveals that in open conformation, PHE 137 and ARG 119 are wide apart and whenever ADK visits the HOHC state these two residues come close to each other and form a stable stacked orientation (Figure 3B). However, results obtained from an equilibrium simulation are always limited and depends on the simulation timescale as the large scale collective domain dynamics are generally long timescale process.19 Therefore, free-energy calculations are essential to understand the thermodynamic stability of different sub-states appear in the equilibrium simulation. We have performed a 2-D free-energy calculation of wild-type and its different mutants using LID-CORE and NMP-CORE distances as two collective variables (Figure 3C). In case of wild-type ADK, it is evident that the HOHC state is the stable state where the LID-CORE distances are in the range of 2.4-2.6 nm and the open state where both the LID and NMP domains are widely apart from the CORE domain is around 1.5 kJ/mol higher compared to the HOHC state. Thus apo ADK is expected to be in equilibrium between the HOHC and the open state with little bias towards the HOHC state which complements well with the recent FRET study of apo-ADK.26 Also, very recently using single molecule force spectroscopy, Pelz et al., also observe a dominant energetic minimum where both the lids of the ADK are half-closed.57 Another interesting observation is that when the LID domain is in open conformation, NMP domain is always in open form. NMP closing is only possible when the LID domain approaches closely to the CORE domain. Interestingly, upon mutation of either 137 or 119 residue with GLY, the open state becomes the thermodynamically stable state and the HOHC state population decreases significantly (Figure 3C) which complement our equilibrium simulation data. Thus both the equilibrium simulation and free energy data reinforce the fact that the interaction between PHE137 and ARG119 is 15



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crucial for stabilizing the HOHC conformational state of ADK. Critical insight into the structure of the HOHC conformation corresponds to the minima in the 2-D free energy plot of wild-type ADK reveals that the guanidinium moiety of ARG119 is stacked with the benzene ring of PHE137 indicating a π-cation type of interaction (Figure 3B). However, due the electronic nature of this kind of interaction, it is difficult to conclude from classical simulation using molecular mechanical force-field. We need a quantum description of the system to shed light into the nature of the interaction. We have performed QM/MM optimization of the wild-type ADK HOHC conformation. In QM/MM optimized HOHC conformation, the mean distance between guanidinium moiety and the benzene ring further reduces to 3.7 Å with highly favorable interaction energy of -34.3 kJ/mol (Table 1). Table 1: Predicted interaction energies (E) obtained from QM/MM calculations using the M062X/6-31+G(d,p)/OPLS-AA level of theory for wild type and mutant ADK. System

E (kJ/mol)

Distance (Å)

PHE137-ARG119

-34.3

3.70

CYC137-ARG119

1.4

4.53

TYR137-ARG119

-25.4

3.88

Electron density mapped on electrostatic surface potential analysis reveals that it is π-cationic in nature with a distributed electronic cloud from the benzene to guanidinium moiety. The Cationic charges are primarily distributed over the guanidinium group while benzene becomes the 16


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electronegative center (Figure 4A). To understand the effect of π-electron cloud on this interaction, the phenyl ring of PHE137 is replaced by cyclohexane ring (CYC). Noticeably the πelectronic cloud is absent on the cyclohexane moiety which in turn reduces the negative charge density on the cyclohexane moiety (Figure 4B). Due to the electrostatic repulsion, the distance between the ARG and CYC moiety increases to 4.53 Å which concomitantly decreases the electron density overlap between ARG and CYC. Therefore the interaction energy (1.4 kJ/mol) becomes unfavourable (Table 1).

Figure 4: Role of benzene ring π-electron conjugation on stabilizing the PHE137-ARG119 interaction obtained from QM/MM calculation and 2-D free energy simulation of F137A ADK mutant. A: Defining the QM system in QM/MM calculation. QM portion is represented in vdW mode while the protein is rendered in ribbon mode (MM part), water box (MM) is not shown due 17



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to the sake of clarity. Electron density mapped on the electrostatic surface potential of the QM system is shown within the square box. Red to blue color indicates a negative to positive electrostatic potential. B: Electron density mapped on the electrostatic potential plot of the CYC137-ARG119 system. C: 2-D free energy of the PHE137ALA (F137A) ADK.

QM/MM calculations clearly depict that the interaction is essentially π-cationic in nature and upon removal of the electron density overlap between the benzene ring and the cationic center destabilizes the interaction. Now to understand the effect of this interaction on the dynamics of ADK, we have performed both equilibrium simulation and 2-D free energy analysis of a mutant ADK where the aromatic PHE group was mutated to non-aromatic ALA. For PHE137ALA mutant ADK, the open state is the thermodynamically stable state (Figure 4C). Thus the PHE137-ARG119 interaction which is essentially π-cationic in nature stabilizes the HOHC state of ADK. We have done a series of mutation where PHE137 has been mutated by other amino acids and found that ADK visits HOHC state only when PHE is mutated with other aromatic amino acid while mutations of PHE with non-aromatic amino acids confine the ADK dynamics strictly within the open state (Figure S3, ESI). Previously, π-cationic interactions have been shown to play seminal role in stabilizing protein structure in other related systems.58-61 We have then tried to shed insight on the fact that the π-cation interaction present in the E. coli ADK is conserved across different species or not. Multiple sequence alignments of bacterial ADK protein sequences reveal an interesting feature (Figure 5A). The CORE ARG residue is found to be absolutely conserved in all bacterial organisms and its interaction counterpart, the LID PHE, is also highly conserved (Figure 5A). 18


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Figure 5: Evolutionary conserved π-cation interaction across different bacterial ADK. A: Multiple sequence alignment of bacterial ADK. Only LID-CORE interface residues are shown for clarity. B: 2-D free energy of PHE137TYR mutant of E. coli ADK is shown. C: Electron density mapped on the electrostatic potential of the TYR-ARG system is shown. D: 2-D scatter plot of LID-CORE distances with RMSD obtained from open state of Streptococcus pneumoniae ADK is shown.

Interestingly, in some organisms PHE is replaced by only TYR which is also an aromatic amino acid. Upon mutation of the PHE to TYR, the π-cation interactions are preserved, evident from 19



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favourable interaction energy and a close stacking interaction between the phenolic group of TYR and the guanidinium moiety of ARG with a calculated distance of 3.88 Å (Table 1). 2-D free energy surface of PHE137TYR mutant ADK reveals that like the wild-type, the HOHC state is the thermodynamically stable state (Figure 5B). Interestingly, the open state of ADK is also a stable minima separated from the HOHC state with a barrier of 4 kJ/mol. QM/MM calculations reveal distributed π-electronic cloud from the benzene to guanidinium moiety and cationic charges are primarily distributed over the guanidinium group while benzene ring of TYR is the electronegative center (Figure 5C). Therefore, the interaction is indeed a π-cation interaction. This result suggests that the stability of the HOHC state of ADK through a π-cation interaction is highly conserved across all bacterial species. Stabilization of a protein conformation through an evolutionary conserved interaction were also demonstrated in prion protein and other related protein system.59-61 Even recently, directly coupled evolutionary residue pairs have been used to successfully explore the conformational landscape of diverse protein family.62 One interesting question still remain unanswered is that the π-cation interaction is necessary and sufficient to provide stability of the HOHC state? To probe this question we have used phylogenetic analysis to all bacterial ADK sequences to identify distant homologs of E. coli ADK. ADK sequences are primarily distributed into two clades, E. coli together with Vibrio, Shigella, Enterobacter, Klebsiella and Salmonella sp. form one clade whereas all Streptococcus sp. are grouped together in a separate clade, evident from the phylogenetic tree (Figure S4, ESI). Thus Streptococcus sp. is distantly related with E. coli ADK. We chose Streptococcus pneumoniae ADK as a representative distant homolog of E. coli ADK. Interestingly, in Streptococcus pneumoniae ADK, the π-cation interaction between PHE-ARG is conserved but the other LID, CORE and NMP residues varied greatly. Sequence alignment reveals a 45.77% 20


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sequence identity between Streptococcus pneumoniae and E. coli ADK sequence. We have modelled the Streptococcus pneumonia ADK in open form using E. coli ADK structure as template and performed equilibrium simulation to explore the conformational dynamics of the protein. Streptococcus pneumoniae ADK also visited both the open and HOHC state during simulation timescale (Figure 5D) similar to E. coli ADK and the whenever the HOHC state appear in the simulation the π-cation interaction is evident which is absent in the open state (Figure 6).

Figure 6: Orientations of PHE and ARG in open and HOHC state of Streptococcus pneumoniae ADK obtained from MD simulation are shown.

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Conclusions Using multi-disciplinary approaches comprising of equilibrium simulations, free energy simulations, QM/MM calculations in combination with multiple-sequence alignment and phylogeny, we are able to shed new insight into the collective dynamics of apo-ADK. Both open and HOHC state appear during the simulation starting from open state of ADK. The HOHC state is thermodynamically more stable than the open state in apo-ADK, evident from free energy calculation. Single molecule experiments also confirms that the equilibrium favours a closed like structure for ADK even in absence of ligand.26 Stability of the HOHC state is primarily dictated by a π-cationic interaction involved between PHE137-ARG119 in E. coli ADK. Astonishingly, this interaction is conserved across bacterial species in ADK protein family. Even, distant E. coli ADK homolog with greatly divergent sequence but with the conserved π-cation interaction at the similar position adopts HOHC state in apo form. Formoso et al., also recently demonstrated that the interaction between PHE137 and ARG119 is one of the important interaction stabilizing the closed state of ADK.17 Hayward et al. previously demonstrated that in presence of the ligand, ARG119 interacts with the adenine part of AMP region.63 Thus our results implicate that the dynamics of apo ADK parallel to the ligand induced conformational transition of the protein. In the HOHC state, PHE137 and ARG119 adopt conformations that can easily facilitates formation of necessary interactions to stabilize the ligand within the binding site of ADK. Thus the HOHC state observed during the dynamics of the apo-ADK has functional implication. This study exhibits modulation of a functional dynamics in ADK protein through a conserved π-cation interaction across different organisms.

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Supporting Information Details of the materials and methodology and additional supplementary figures are provided as supporting information (ESI). This material is available free of charge via the Internet at http://pubs.acs.org.

Notes The authors declare no competing financial interests.

Acknowledgment Authors gratefully acknowledge the central supercomputing facility at Indian Association for the Cultivation of Science, Kolkata for providing the computational resources. Ritaban Halder is thankful to CSIR for providing the fellowship.

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