Dynamics of the Active Sites of Dimeric Seryl tRNA

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Dynamics of the Active Sites of Dimeric Seryl tRNA Synthetase from Methanopyrus Kandleri Saheb Dutta, and Nilashis Nandi J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/jp511585w • Publication Date (Web): 20 Mar 2015 Downloaded from http://pubs.acs.org on March 23, 2015

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The Journal of Physical Chemistry

Dynamics of the Active Sites of Dimeric Seryl tRNA Synthetase from Methanopyrus Kandleri

Saheb Dutta and Nilashis Nandi ,1 ∗

Department of Chemistry University of Kalyani Kalyani, Nadia, West Bengal, 741235 India.

*To whom correspondence should be addressed. 1 Phone : +91 9433056943. Email: [email protected]

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Abstract Aminoacyl tRNA synthetases (aaRSs) carry out the first step of protein biosynthesis. Several aaRSs are multimeric and coordination between the dynamics of active sites present in each monomer is a prerequisite for the fast and accurate aminoacylation. However, important lacunae of understanding exist concerning the conformational dynamics of multimeric aaRSs. Questions remained unanswered pertaining to the dynamics of active site. Little is known concerning the conformational dynamics of the active sites in response to the substrate binding, reorganization of the catalytic residues around reactants, time dependent changes at the reaction center which are essential for facilitating the nucleophilic attack and interactions at the interface of neighboring monomers. In the present work, we carried out all atom molecular dynamics simulation of dimeric mkSerRS from Methanopyrus kandleri bound with tRNA using explicit solvent system. Two dimeric states of seryl tRNA synthetase (open, substrate bound and adenylate bound) and two monomeric states (open and substrate bound) are simulated with bound tRNA. Aim is to understand the conformational dynamics of mkSerRS during its reaction cycle. While the present results provide a clear dynamical perspective of the active sites of mk SerRS, corroborates with the results from the time averaged experimental data such as crystallographic and mutation analysis of methanogenic SerRS from M. kandleri and M. barkeri. It is observed from the present simulation, that the motif 2 loop gates the active site and its Glu351 and Arg360 stabilizes ATP in a bent state favorable for nucleophilic attack. The flexibility of the walls of the active site gradually reduces near reaction center which is a more organized region compared to the lid region. The motif 2 loop anchors Ser and ATP using Arg349 in a hydrogen bonded geometry crucial for nucleophilic attack and favorably influences the electrostatic potential at the reaction center. Synchronously, Arg366 of the β sheet at the base holds the syn oxygen of the attacking carboxylic group so that the attack by the anti oxygen is feasible. This residue also contributes to the reduction of the unfavorable electrostatic potential at the reaction center. Present simulation clearly shows the catalytic role of the residues at reaction center. A precise and stable geometry of hydrogen bonded network develops within active site which is essential for the development of an optimum transition state geometry. All loops move away from the platform of active site in the open or adenylate bound state and the network of hydrogen bond disappears. The serine binding site is most rigid among all three subsites. The Ser is held here in a highly organized geometry bound by Zn+2 and Cys residues. Present simulation further suggests that the helix-turn-helix motif connecting the monomers might have important role in coordinating the functional dynamics of the two active sites. The N terminal domain is involved in long range electrostatic interaction and specific hydrogen bond interaction (both direct and water mediated) with tRNA. Overall conformational fluctuation is less in N terminal compared to the catalytic domain due to the presence of motif 2 loop, loop f and serine ordering loop which change conformation in the later domain during the reaction cycle. The dynamic perspective of active site of mkSerRS with mobile loop acting as gate and dynamically silent β sheets performing as base has similarity with the perception of active site in various other enzymes.

Keywords: Aminoacyl tRNA synthetase (aaRS), dimeric aaRS, serRS, active site, dynamics


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I. Introduction Aminoacyl tRNA synthetases (aaRSs) carry out the first step of protein biosynthesis. In this reaction the tRNA is charged with its cognate amino acid (aa) by 3/ esterification (aminoacyl tRNA synthesis). The aaRSs mediate the important task of information transfer by translating genetic code into amino acid sequence in all kingdoms of life1-3. AaRSs perform noncannonical functions as well4,5. The structure, dynamics and function of aaRSs have drawn an enormous interest in recent times due to their vital role in life processes and for the possibility that the understanding of the enzymatic process may be harnessed to develop novel enzyme-mimetic system. Wealth of information about the structures of twenty aaRSs and the mechanism of aminoacylation in these enzymes are available from X-ray crystallographic analysis, NMR, mutation experiments, biochemical methods, kinetic analysis, classical molecular dynamics simulation, ab-initio calculations and theoretical analysis6-21. Twenty aaRSs are classified into two broad classes according to the structural organization of the aaRSs, participation of tRNA and conformation of adenosine triphosphate (ATP) during the course of the reaction, organization of the active site and molecular details of the reaction mechanism16-20. Aminoacylation reaction is a two step process. In the first step, aa and ATP binds with the aaRS and α-carboxylate oxygen of the aa makes nucleophilic attack at the α-phosphorous (αP) of ATP bound with Mg+2 ions (Mg-ATP). This step leads to the formation of aminoacyl adenylate (aaAMP) and pyrophosphate (PPi) is released. In the second step, 2/ or 3/ hydroxyl group of the terminal adenosine of the tRNA attacks the α-carbon of adenylate leading to formation of tRNAaa and release of adenosine monophosphate (AMP). It is known that participation of tRNA is obligatory for the first step in few class I aaRSs (GluRS, GlnRS, ArgRS and LysRSI) and none of the class II aaRSs requires the participation of tRNA for adenylation. Both steps occur within the active site of the aaRS which is extending from the ATP binding site to the binding site of aa. Despite the apparent simplicity of the two steps described, several lacunae of understanding exist about how the organization of the active site catalyzes the reaction. This question is challenging to address due to the idiosyncrasy of the active site organization of each of twenty aaRSs20. The residues constituting the walls of the active sites and their participation in the aminoacylation steps are different from one aaRS to the other. Even for a particular aaRS, the active site organization may differ in different species. It remains a question that how the


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microscopic organization of the active site in aaRS efficiently and accurately sequester the substrates and liberate product by dynamic reorganization. Both aaRS and tRNA are macromolecules with exotic dynamics and both undergo conformational transitions during aminoacylation. The conformational changes occur at the (long) length scale of the structural organization of the aaRS as well as at the nanodimension of the active site. The knowledge about the dynamics of conformational changes has pivotal importance in unraveling the molecular mechanism of aaRS catalysis. However, our understanding about the dynamics of the catalytic process in aaRSs is far from complete. The foregoing questions are complicated by the fact that several aaRSs are multimeric (Fig. 1a1k).22, 23 Dimeric aaRSs are for example, CysRS (from Rat liver and E.Coli), MetRS, TyrRS, TrpRS

in class I and HisRS, ProRS, SerRS, ThrRS, AspRS, AsnRS, LysRS in class II.

Tetrameric structures of PheRS and AlaRS (Eubacterial) are also known. Coordination between the dynamics of active sites present in each monomer is a prerequisite for the fast and efficient aminoacylation. Unfortunately, the dynamics of various important conformational states and transition between them during the reaction cycle in a multimeric aaRS are not amenable to time averaged experiments. Although molecular dynamics simulation of aaRSs revealed important aspects of conformational dynamics of aaRSs,

12-15

to the best of our knowledge, no study is

devoted to study of the dynamics of a dimeric aaRS.


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Fig. 1. Crystallographic structures of dimeric aaRSs (class I and class II) in surf representation available from protein data bank. (a) Class I CysRS (1LI5.pdb) from Escherichia coli. (b) Class I MetRS (2CSX.pdb) from Aquifex aeolicus. (c) Class I TyrRS (1H3F.pdb) from Thermus thermophilus. (d) Class I TrpRS (1MAW.pdb) from Geobacillus stearothermophilus. (e) Class II HisRS (1KMN.pdb) from Escherichia coli. (f) Class II ProRS (1H4Q.pdb) of species Thermus thermophilus. (g) Class II SerRS (1SER.pdb) from Thermus thermophilus. (h) Class II ThrRS (1EVK.pdb) of species Escherichia coli. (i) Class II AspRS (1IL2.pdb) of species Escherichia coli. (j) Class II AsnRS (11AS.pdb) from Escherichia coli. (k) Class II LysRS (4EX5.pdb) of species Burkholderia thailandesis. In each case, individual monomers are shown in surf representation with red and green color, respectively and tRNA is shown in tube representation with orange color. The images are prepared using VMD79.

In the present work, we investigated the dynamics of dimeric SerRS. SerRS is a member of the class II aaRSs and is homodimeric. The dimeric structure of SerRS from methanogenic Methanopyrus kandleri (mkSerRS) is interesting for the following reasons. SerRS is the only class II aaRS which exhibit cross dimer binding of tRNA24-26. The mode of binding of tRNASer across the dimer interface functionally correlates the two noncovalently bound monomers of SerRS. While one subunit accepts the CCA terminal of tRNASer in its catalytic domain, the variable arm of the same tRNASer is bound with the N terminal domain of the adjacent monomer. Thus the reaction is functionally dependent on both monomers. Cross dimer binding is also observed in class I aaRSs such as in TyrRS and TrpRS. The presence of a Zn+2 ion binding site is also a unique feature of

mk

SerRS (unusual among SerRSs from other species). The sequences and ACS Paragon Plus Environment

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crystallographic structures of SerRS from various species are studied in recent years24-42. The sequence, structure and recognition of tRNASer are also studied extensively26, 43-50. The structure of mkSerRS and its one of the active sites are shown in Fig. 2a-2b. The Stereo view of the active site with the ATP binding site located towards the observer is presented in the Fig. S.I of the supporting information.

(a)

(b) mk

Fig 2. (a) Structure of dimeric SerRS bound with tRNA (prepared from 3w3s.pdb). Details are given in computational methods. The catalytic domain and N terminal domain of monomer 1 (M1) are colored in red and pink respectively and these domains of monomer 2 (M2) are colored in green and yellow, respectively. The tRNA (colored in orange) is bound via anticodon binding region with N terminal domain of monomer 2 (M2) (b) views of the active site from the ATP binding site located towards the observer (in left) and view from the serine binding site (in right). The seven antiparallel β sheets are shown in grey, serine ordering loop in purple, motif 2 loop in red and the loop f in green color. The remaining structural elements are shown in light grey for clarity. The images are prepared using VMD79.

It is convenient to discuss the structure and dynamics of the active site of aaRSs according to the proximity and interaction between the residues in the walls of the active site and the substrates. These subsites are the ATP binding subsite (located near the opening of the active site and exposed towards the outer surface of enzyme), the reaction center which is the intervening region closest to the attacking oxygen and αP (crucial electronic rearrangements in transition state occurs here) and the amino acid binding subsite (located deeper within the active site) (Fig. 3a3c). Corresponding stereo image of the active site region is shown in S.II of the supporting information. The complementary interactions between the substrates and the active site residues are also responsible for effective binding of the substrates and conversion of the reactants to the product via transition state18, 20. Note that these subsites refer only the proximity of the different parts of substrates to easily understand the interaction and dynamics and nothing else. The stereo images of the three subsites are presented in the Fig. S.III of the supporting information. Seven antiparallel β sheets form the platform or base of the

mk

SerRS active site over which the

substrates reside. Surrounding walls of the active site are composed of various loops and helices. ACS Paragon Plus Environment

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Schematic representation of the organization of the active site residues (which are parts of the loops, helices and β sheets present in the active site wall) in the mkSerRS are shown in Fig. S.IV of the supporting information.

(a)

(b)

(c)

mk

Fig. 3. (a) The active site region of SerRS monomer bound with ATP and Ser (b) the active site is schematically shown in green (image prepared using CAVER55) and loops are shown in the same color scheme as shown in Fig. 2b, respectively (c) the three subsites of active site, namely, serine binding subsite, reaction center and ATP binding subsites are shown as slices of active site using space-filling model. All structures are based on 3w3s.pdb (details are given in computational methods). The images are prepared using VMD79.

The reaction scheme of aminoacylation for two active sites is discussed in literature which fits the experimental observations of TyrRS from Bacillus stearothermophilus.22 Based on mutation studies and kinetic analysis of HisRS, an alternating site catalysis model with initial priming followed by multiple turnovers is proposed for multimeric aaRSs23. Development of the understanding of the functional dynamics of aaRSs requires the study of important conformational states of the potential energy landscape of the aaRS-tRNA system in the context of the reaction. We studied the representative important conformational states that must appear during the reaction cycle. When each of the monomers of a dimeric aaRS contains one active site, the enzyme passes through various conformational states, for example, a state in which one active site in a monomer contains aa and ATP and an open active site is present in the other monomer. A subsequent state of the dimer is where adenylation is completed in the first active site and the second active site incorporated a second set of aa and ATP. In the present work, we carried out all atom simulations for four different states of

mk

SerRS with tRNA for 2

nanoseconds each. The first dimeric state contains aa and ATP within an active site in a monomer and an open active site in the other monomer. The second dimeric state contains an active site containing adenylate in a monomer and another active site containing aa and ATP in the neighboring monomer. Both states are bound with tRNA. As mentioned before, these two states are important conformational states that appear during reaction cycle. To better understand ACS Paragon Plus Environment

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the influence of dimerization, we further studied the dynamics of the active site of monomeric mk

SerRS with open active site as well as active site bound with substrates. In the next section we

present the computational procedure which is followed by results and discussion.

II. Computational Methods A dimeric aaRS (composed of two noncovalently bound monomers M1 and M2, respectively) bound with tRNA is a large macromolecular complex. The reaction starts with the priming step where the substrates (aa and ATP together abbreviated by ‘S’) bind to the active site of a monomer (say, M1). Once substrates (aa and ATP) are bound within the influence of the network of interaction of active site residues, the pre-transition state geometry suitable for nucleophilic attack is formed by the reorganization of the active site residues16-20. It is mentioned before that the tRNA binds across the dimer surface (a cross-dimer binding)24-26. Only one tRNA per dimer in SerRS-tRNASer complex in Thermus thermophilus is observed. Crystal structures of the SerRS-tRNASer complex shows the presence of only one tRNA bound across the two subunits25 while the solution studies showed that two tRNAs may bind cooperatively under some specific conditions26. While the anticodon arm or variable arm (depending on the particular tRNA) of cognate tRNA is bound to the N terminal of the second monomer with active site in the open state, denoted as M1(O), the 3/ end of the acceptor step approaches towards the active site of M1 for completing the aminoacyl transfer in M1 via cross dimer interaction. The state [M1(S).M2(O)]tRNA is denoted as dimeric state I in the present work. Once the substrates (a second set of aa and ATP) bind in the M2, the state is denoted as [M1(A).M2(S)]tRNA and is denoted as dimeric state II. In the present work we studied both dimeric state I with bound tRNA, [M1(S).M2(O)]tRNA and dimeric state II with bound tRNA, [M1(A).M2(S)]tRNA. Subsequently the 3/ end of acceptor stem of tRNA enter the active site of the M1(A) and the second step (charging of tRNA) takes place and tRNAaa leaves active site of M1 with completion of the cycle. The crystal structure of mkSerRS from M. kandleri in complex with tRNA from Aquifex aolicus is used as the starting structure of the present work (3w3s.pdb). SerRS is homodimeric in which each monomer contains 527 amino acids. The available crystal (3w3s.pdb) of SerRS from M. kandleri is monomeric. The homodimeric structure of mkSerRS is prepared from the monomer of mk

SerRS and comparing with the dimeric crystal structure of SerRS from methanogenic ACS Paragon Plus Environment

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Methanosarcina barkeri (2cja.pdb).30,31 Each monomer contains N terminal domain and C terminal domain connected via a linker. An active site is present in C terminal domain of each monomer. The tRNA is bound with the N terminal domain. It is known that49 that methanogenic SerRS accepts eukaryotic and bacterial tRNASer in addition to archaeal tRNASer. The crystal structure contains few selenomethionine residues which are mutated to methionine residues. The terminal adenosine base of 3/ end of tRNA was missing in the crystal and the missing adenosine residue is modeled from the CCA terminal of tRNAGlu (2cv1.pdb). Substrates (ATP and Ser) and two Mg+2 ions are docked at the active site using AutoDock Vina51. The geometry of ATP is taken from the geometry of ATP in SerRS from Methanosarcina barkeri (2cja.pdb).30,31 The dimeric state I (bound with tRNA) is solvated in a rectangular box of 186.8 × 122.0 × 136.7 Å3 containing 82854 TIP3P water molecules. The system is neutralized by adding 147 Na+ ions to achieve electro-neutrality. The dimeric state I solvated system contains 269271 atoms. The dimeric state II solvated system (bound with tRNA) is solvated in a rectangular box of 182.2 × 123.0 × 136.7 Å3 containing 80924 TIP3P water molecules. The system is neutralized by adding 147 Na+ ions to achieve electro-neutrality. The dimeric state II system contains 263528 atoms. The monomeric open state is solvated in a rectangular box of 99.8 × 120.2 × 86.6 Å3 containing 25835 TIP3P water molecules. The system is neutralized by adding 24 Na+ ions to achieve electro-neutrality. The monomeric open system contains 84534 atoms. The monomeric substrate bound state is solvated in a rectangular box of 99.8 × 120.2 × 87.1 Å3 containing 25659 TIP3P water molecules. The system is neutralized by adding 24 Na+ ions to achieve electro-neutrality. The system contains 85358 atoms. All simulations are performed using AMBER11 suite of programs52. First, all water molecules are energy minimized for 500 steps, while rest of the system was held constrained. This was followed by a 500 steps energy minimization of the overall system without any constraint. Subsequently, isothermal-isobaric (NPT) simulation was run for 150 ps with gradual relaxation of positional restraints over water molecules, amino acid side chains of protein, substrates and finally the backbone of protein. This step is followed by 2 ns simulation run for dimeric state I, dimeric state II, monomeric open state and monomeric substrate bound state using NPT condition without any restraint. The Generalized AMBER Force Field (GAFF)53 is used for substrates and AMBER (leaprc.ff99SB) force field52 is used for protein molecule. The parameters of Zn+2 are taken from standard literature54. The temperature is maintained at 300 K. ACS Paragon Plus Environment

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Langevin dynamics with collision frequency 1.0 ps-1 is used and pressure is maintained at 1 bar with a relaxation time of 2 ps. All simulations are carried out with a time step of 1 fs. The SHAKE algorithm is used to restrain the bonds linking the heavy atoms and hydrogen atoms. The electrostatic interactions are calculated using the Particle-mesh Ewald sum method. A nonbonded cutoff of 10 Å is used. The trajectories of 2 ns of dimeric states I, II and monomeric open and substrate bound states are used for analysis of the results. The dynamics of bottleneck radius (maximum radius of the narrowest region of the active site tunnel is the bottleneck radius) of the three subsites are computed from simulated trajectories at 50 ps intervals using CAVER.55 The bottleneck radius of the serine binding subsite is computed by starting from Cα of serine. The bottleneck radius of the reaction center subsite is computed starting from the mid point of the line joining carboxylic acid group of Ser and αP of ATP. The bottleneck radius of the ATP binding subsite is computed starting from αP of ATP. Following atoms are considered in computing the root mean square deviation (rmsd) of Zn+2 coordination sphere: (i) in substrate bound active sites Zn+2, γS of cys319, γS of cys478, ηO of Glu368 and N of amine group of Ser are used (ii) for adenylate bound states, Zn+2, γS of cys319, γS of cys478, ηO of Glu368 or N of amine group of adenylate are considered and (iii) Zn+2, γS of cys319, γS of cys478, ηO of Glu368 and oxygen of water molecule in open active site are used. The results are presented and discussed in the following section. Gaussian suit of programs56 are used for the calculation of electrostatic potential (ESP) using the structure of the active site from the simulated trajectory.

III. Results and discussions The results of four different simulations of dimeric (state I and state II) and monomeric (open and substrate bound) states of

mk

SerRS are presented and discussed in this section. While the

simulated structures are consistent with the crystallographic analysis, reveals the dynamics of the reaction in a clear way. We first present the observations about the time dependent changes of the active site from the fluctuation of the bottleneck radius of the active site computed from the MD trajectories. The bottleneck radius fluctuations of the three subsites of the active site are outcomes of the combined dynamical motions of α helices, β sheets and loops composing the active sites of

mk

SerRS. The analysis of the dynamics of these α helices, β sheets and loops and

important active site residues composing them are presented for three subsites in detail. This is followed by presentation of results about the dynamics of the base of the active site, dynamics of


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the helix turn helix (HTH) motif at dimer interface and dynamics of the N terminal domain. An overview of the results is presented followed by concluding remarks. III a. Dynamics of the ATP binding subsite ATP binding subsite is the gateway of the active site through which the substrates enter and products are released. The results of the analysis of the bottleneck radius fluctuation of the ATP binding subsite are presented in Fig. 4a-4c. Observed maximum value of the bottleneck radius in the open active site (in the M2(O) of the dimeric state I) is larger (4.4 Å) than the maximum value (3.4 Å) in the closed substrate bound active site (M1(S)). The average bottleneck radius of the open active site is also larger compared to the average radius of the substrate bound active site. The bottleneck radius of the ATP binding subsite decreases with time in the ATP bound state and exhibit maximum time dependent decrement among the three subsites. It is shown in the present work that the observed change is largely an outcome of the dynamics of the segment of the motif 2 loop near ATP which contributes to the open-close motion of the lid of the active site. This leads to the development of favorable interaction of the residues of the active site wall (motif 2 loop segment) with ATP. The average bottleneck radius of the active site containing adenylate (in dimeric state II) is larger compared to the substrate bound active site of the same dimer (Fig 4b). This is due to the loss of interaction of the active site wall with the β and γ phosphate of ATP in the adenylate containing active site after the release of PPi. In contrast, the bottleneck radius fluctuation of monomeric state does not exhibit effective closure of the lid of the active site upon substrate binding (Fig 4c).

(a)

(b)

(c)

Fig. 4. Fluctuation of the bottleneck radius of the ATP binding subsite for (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’.


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The wall of the ATP binding subsite is composed of motif 2 loop. Additionally, β4 of motif 2 as well as β7 and β8 of motif 3 are present at the walls of the subsite. Dynamics of the residues present in the loop and motifs contribute to the substrate binding and observed motion of the opening of the active site. The lid motion is noted from the movement of the motif 2 loop relative to the seven antiparallel β sheets at the base of the active site. The motif 2 loop moves closer to the base by changing its conformation in the ATP bound state. The rmsd of the motif 2 loop and its different segments are presented in S.V in the supporting information. The lid movement is presented as the time dependent separation between the Gly353 of motif 2 loop and Arg485 of β sheets at the base of the active site and is denoted as d (Gly353 − Arg 485) . Results are presented in Fig. 5a-5c, respectively for dimeric and monomeric states. The results are presented as δR = d (t ) − d min (t ) where d min (t ) which is the smallest separation observed in the adenylate bound active site. The motif 2 loop closes in substrate bound states in both dimeric state I (bound with ATP and aa) and dimeric state II (bound with adenylate in one active site and ATP, aa in the other). The loop opens up in the open active site. The closing (approach towards the substrates located over the base) and opening (movement away from the base of the active site) motions of the motif 2 loop is concluded from changes in δR . The loop movements are more clearly presented by superimposing the structures of the motif 2 loop at initial time (t=0) and at 2 ns (t=2) from the simulated trajectories of dimeric state I, dimeric state II and monomeric open and closed state, respectively (Fig. 5d-5f). The superimposed structures demonstrates that the loop is approaching towards the substrate located over the base of the active site in the closed state and moves away from the base in the open active site as a function of time.

(a)

(b)


(c)

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(d)

(e)

(f)

Fig. 5. Lid movement represented by δR (relative separation (Å) between motif 2 loop and the β-sheet at the base of the active site). For details see text. (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’ (d) superimposed structures of motif 2 loop at initial time (t=0) and at 2 ns (t=2) from trajectories of dimeric state I, both monomers are shown (e) superimposed structures of motif 2 loop at initial time (t=0) and at 2 ns (t=2) from trajectories of dimeric state II, both monomers are shown (f) comparison of the superimposed structures of motif 2 loop at initial time (t=0) and at 2 ns (t=2) from trajectories of superimposed open (red) and closed (green) monomeric states. All images are prepared using VMD79.

Videos of the time dependent movement of the motif 2 loop with respect to Arg485 are presented in the supporting information (section S.VI) for dimeric state I for monomer 1 (video S.VI.1.avi for substrate bound active site), monomer 2 (video S.VI.2.avi for open active site) and for dimeric state II for monomer 1 (video S.VI.3 for adenylate bound substrate bound active site) as well as monomer 2 (video S.VI.4 for substrate bound substrate bound active site). The closing and opening motions of the motif 2 loop are clearly visible from the changes in separations in the respective videos. Thus, the dimeric state of

mk

SerRS bound with tRNASer influence the

flexibility and movement of motif 2 loop necessary for serylation. This result is in agreement with the mutational study of the motif 2 loop in yeast SerRS37. The possible role of the flexible loops as lid over the active site which stabilizes upon substrate binding was proposed in SerRS from E.Coli.27 Our results corroborate the proposition and present a dynamic view of the same. The observed structural dynamics of the ATP binding subsite leads to the development of interactions of the residues of the subsite and ATP and stabilize the ATP in the bent conformation. The bent conformation of ATP is favored in class II aaRSs over extended conformation for nucleophilic attack19. The closure of the lid allows the development of hydrogen bonding interaction between loop residues (for example, Glu351 and Arg360) and the ATP. The rmsd of Glu351 is shown in Fig. 6a-6c. The side chain of Glu351 of the motif 2 loop ACS Paragon Plus Environment

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develops hydrogen bonds with the amine group of adenine ring of ATP in the substrate bound active sites (in dimeric state I, dimeric state II and monomeric states) and exhibit smaller fluctuation compared to the open or adenylate bound states. The percentages of hydrogen bond occupancies of Glu351 in the trajectories over the course of the simulation are presented in the S.VII.a-S.VII.c of the supporting information. The superimposed structures of Glu351 at initial and final time for different simulated states are shown in supporting information (Fig. S.VIII.aS.VIII.c, respectively). The superimposed structures clearly show the interactions developed during the course of the simulation as discussed.

(a)

(b)

(c)

Fig. 6. Rmsd (in Å) of Glu351 of motif 2 loop for (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’.

Side chain of Arg360 of the motif 2 loop interacts with both βP and γP oxygens of ATP. The fluctuation of the rmsd of the residue indicates the mobile nature of the residue (Fig. 7a-7c). Arg360 side chain is labile and flips its two NH2 protons to bind with the two βP and γP oxygens and the conformational changes are reflected in the fluctuation of the rmsd of Arg360. The superimposed structures of Arg360 at initial time (t=0) and at 2 ns (t=2) from the trajectories of dimeric state I, dimeric state II and monomeric open and closed states are shown in supporting information (Fig. S.IXa-S.IXc). The interactions between Arg360 and ATP are noted in the superimposed structures during the course of the simulation.


14

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(a)

(b)

(c)

Fig. 7. Rmsd (Å) of Arg360 of motif 2 loop for (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in red). M2(O) indicates monomer 2 with open active site (shown in black). (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’.

The side chain of Arg 485 of motif 3 forms hydrogen bond with γP oxygen of ATP in substrate bound. The motif 3 is located at the base of the active site near ATP binding site. The adenine ring of ATP is stacked between phenyl ring of Phe364 and positively charged guanidinium group of Arg485. Low rmsd values are observed compared to the open active sites in both cases (Fig. 8a-8c).

(a)

(b)

(c)

Fig. 8. Rmsd (Å) of Arg485 for (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’.

The presence of π- π interaction between the Phe364 side chain and adenosine ring as well as cation-π interaction between Arg485 and adenosine ring observed in both ATP and seryladenylate bound states are responsible for the smaller fluctuation of the side chain. The corresponding geometries of adenine ring of ATP, stacked between phenyl ring of Phe364 and positively charged guanidinium group of Arg485 involved in π- π and cation- π interactions are ACS Paragon Plus Environment

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Page 16 of 60

shown in Fig. S.X.a-S.X.c, respectively in the supporting information. The percentage of hydrogen bond occupancies of Arg485 in the trajectories are shown in Fig. S.XI.a-S.XI.c of the supporting information. The interactions between ATP and Glu as well as Arg residues (conserved in class II aaRSs) are known from experimental data23 and the present simulation corroborates the same. Videos of the time dependent variations of the hydrogen bond geometry between Arg485 and oxygen attached with γP in monomer 1 in substrate bound active site of dimeric state I are presented in the video S.XII.1.avi and the time dependent variation of the hydrogen bond geometry between Arg485 and oxygen attached with γP in monomer 2 in substrate bound active site of dimeric state II are presented in the video S.XII.2.avi, respectively in the supporting information (section S.XII). The βP and γP segments of ATP molecule exhibit conformational fluctuation which is the result of coupled movement with the nearby residues. Once the adenylate formation is complete, the interaction between the ATP (in the βP and γP) with the active site wall is lost and active site wall recede. This is observed from the highest magnitude of the bottleneck radius in adenylate bound active site in dimeric state II (maximum 4.8 Å radius) as shown in Fig. 4b. III b. Dynamics of the reaction center The reaction center subsite is the region around the carboxylic group of Ser and αP of ATP. This is the region where the most significant reorganization of the electronic structures of the substrates during the nucleophilic attack takes place. The subsite is composed of parts of motif 2 loop, serine ordering loop, loop f (a loop spanning the residues Leu253 to Met269 which is topologically equivalent to flipping loop and shown in green color in Fig. 2b), β4 of motif 2, β6, β7 and β8 of motif 3, H11 and its adjacent loop. The fluctuation of the bottleneck radius of the reaction center is presented in Fig. 9a-9c. Present simulation shows that the bottleneck radius of the reaction center subsite is smallest in the substrate bound active site. As the active site residues clamp on the substrates (Ser and ATP), the subsite shrinks. The radius of open and adenylate bound active sites are larger due to missing interactions between the subsite and nearby segments of the substrate. The bottleneck radii of the reaction center in both the substrate bound active site of dimeric state I and II show least fluctuation. This result shows that the region is well ordered and dynamically more rigid compared to the lid of the active site (ATP binding subsite). In contrast, the bottleneck radius fluctuation of the reaction center in the monomeric


16

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simulation showed that substrate bound state is less compact compared to the open state indicating the lesser efficiency of the monomeric state in forming an organized reaction center.

(a)

(b)

(c)

Fig. 9. Fluctuation of bottleneck radius (Å) of the reaction center for (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’.

The present simulation shows that the αP region exhibit less conformational fluctuation compared to the βP and γP segments of ATP. The organized structure of the reaction center around αP is a prerequisite of the effective nucleophilic attack. Development of a network of hydrogen bonds between active site residues and substrates, as the reaction center subsite clamps upon the substrate, is responsible for this. The motif 2 loop plays a significant catalytic role for reaction, in addition to stabilizing ATP near the ATP binding subsite via its residues. This will be elaborated later in this subsection. The rmsd of the loop f in the substrate bound active site, M1(S), of the dimeric state I indicates that the loop f is less labile compared to the monomer with open active site, M2(O), as shown in Fig. 10a-10c. Same feature is also noted in the substrate bound state in the dimeric state II where the rmsd of the loop f in adenylate bound active site is higher compared to the substrate bound active site. The motions of the loop f observed by superimposing the structures of loop f at initial time (t=0) and at 2 ns (t=2) from simulated trajectories of dimeric state I, dimeric state II and monomeric open and closed state, respectively are presented in the supporting information (Fig. S.XIII.a-S.XIII.c). The role of flipping loop in mediation of the open-close conformational change is discussed32 in the case of methanogenic SerRS and present data corroborates the same. The result shows that the loop f interacts with substrates and remain in closed conformation in the substrate bound state and opens up in both in the open state and adenylate bound active sites.


17


(a)

(b)

Page 18 of 60

(c)

Fig. 10. Rmsd (Å) of loop f for (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’.

The amine groups of the side chain of Arg349 of motif 2 loop forms hydrogen bond with the oxygen of the carboxylic group of Ser and oxygen atom attached with the αP of ATP in the substrate bound state (Fig.11a-11c). The percentage of hydrogen bond occupancies of Arg349 in the trajectories are shown in the Fig. S.XIV.a - S.XIV.g, respectively of the supporting information. The hydrogen bonds with Arg349 are necessary for anchoring both substrates. This residue anchors both the Ser and ATP in the reactive conformation and facilitates the formation and stabilization of the transition state geometry. Time dependent variation of the hydrogen bond geometry of Arg349 with both substrates (Ser and ATP) via a pair of hydrogen bonds depicting the anchoring action of the residue in monomer 1 in substrate bound active site of dimeric state I and monomer 2 in dimeric state II in videos S.XV.1.avi and S.XV.2.avi, respectively in the supporting information. The anchoring action of the Arg349 using a pair of hydrogen bonds is clearly visible from the videos of the substrate bound states. The result points out important catalytic role of the residue. In contrast, the higher conformational flexibility of the Arg349 is noted in open active site of dimeric state I (Fig. 11a). Similar observation is made in substrate bound state in dimeric state II (Fig. 11b). Hydrogen bonding with Arg349 is absent in adenylate state and the rmsd of the residue is higher than the active site in the substrate bound state.


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(a)

(b)

(c)

Fig. 11. Rmsd (Å) of Arg349 of motif 2 loop for (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’.

In the case of

mk

SerRS, the invariant Arg349 of motif 2 loop and Arg366 have similar role as

played by Arg259 and Arg 113 in the case of HisRS16. We studied the ESP around the reaction center using quantum mechanical method (at the HF/6-31G(d,p) level of theory) based on the simulated structures. The electrostatic potential (ESP) around the αP is negative and is unfavorable for the nucleophilic attack (by the electron density of the carboxylic group of His) when the Arg residues and Mg2+ ions are absent. The results show that Arg349 and Arg366 changes the ESP to a more favorable value at the reaction center and thereby facilitate the ease of nucleophilic attack at αP. The computed ESP contours are shown in the Fig S.XVI.a and Fig. S.XVI.b of supporting information. Similar roles of Arg residues are observed in the cases of HisRS.16 Detailed quantum mechanical analysis of transition state and computation of electrostatic potential in HisRS showed that Arg residues (Arg113 and Arg259) near the reaction center reduces the unfavorable electrostatic potential and facilitates the nucleophilic attack. The change in the hydrogen bonding pattern from the reactant to the product via a transition state further demonstrated that the Arg 259 anchors the carboxylic acid group of His and the oxygen atom attached with αP in the aminoacylation reaction pathway in HisRS.16 The present dynamical study of SerRS shows similar anchoring role of Arg349. It was suggested from crystallographic studies of SerRS from T. Thermophilus that the conserved Arg residue from motif 2 loop plays a key role in positioning the αP and charge neutralization, forming hydrogen bond with both carboxylic oxygen and αP and assembling the Ser and ATP by a synergistic binding.29 However, the dynamic perspective of the anchoring role of Arg residue as an important catalytic residues and dynamics of the reorganization of the substrates is presented here for the first time. ACS Paragon Plus Environment

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Recent studies established that attack by the syn oxygen carboxylic acid group of aa is favorable for class I aaRSs whereas attack by anti oxygen is favorable for class II which is a class specific difference between the reaction mechanism in class I and class II aaRSs17,19. SerRS being a class II aaRS, side chain of Arg366 holds the syn oxygen of carboxylic group of Ser by hydrogen bond and anti oxygen attacks to the αP of ATP. Arg366 residue of β4 of motif 2 located at the base of the reaction center subsite is important for controlling the mode of nucleophilic attack by the carboxylic group of amino acid. The rmsd of Arg366 provides an indication of hydrogen bond formation as in open state rmsd is slightly higher (Fig. 12a-12c). The percentages of hydrogen bond occupancies are shown in Fig. S.XVII.a-Fig.S.XVII.f, respectively of the supporting information. Time dependent variation of the hydrogen bond geometry between syn oxygen of carboxylate group of Ser and Arg366 via a pair of hydrogen bonds in monomer 1 in substrate bound active site of dimeric state I and monomer 2 in dimeric state II in videos S.XVIII.1.avi and S.XVIII.2.avi, respectively in the supporting information. The videos clearly show the hydrogen bond formation with the syn oxygen of carboxylate group of Ser favoring the anti attack, a class specific feature of class II synthetases17.

(a)

(b)

(c)

Fig.12. Rmsd (Å) of Arg366 in (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’.

III c. Dynamics of the serine binding subsite The serine binding subsite is composed of residues from serine ordering loop, loop f , H11 and adjacent loop, β4 of motif 2, β6, β7 and β8 of motif 3 among other residues. The bottleneck radius fluctuation is of the serine binding subsite is least among the three subsites (Fig. 13a-13c). ACS Paragon Plus Environment

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Bottleneck radius fluctuations and loop movements computed from trajectories show that the serine binding subsite is the most rigid region of the active site.

(a)

(b)

(c)

Fig.13. Variation of bottleneck radius (Å) in the serine binding site for (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’.

Our results show that the side chains of Cys319, Glu368 and Cys478 form a tetrahedral coordination geometry around the Zn+2 ion. The fourth coordination site of Zn+2 ion is occupied by a water molecule in the open active site. The water molecule is replaced by the substrate Ser in the substrate bound active site and the Zn+2 ion interacts with the lone pair of nitrogen of the amino group of Ser. The significantly low rmsd of the coordination sphere around Zn+2 ion indicates a rigid region (Fig. 14a-14c). The individual rmsd values of each ion and residues of the coordination sphere such as Zn+2 ion, Cys319, Glu368, Cys478 and water molecule are presented in the Fig.S.XIX.a-Fig.S.XIX.i of the supporting information. The geometry of the coordination sphere from the present simulation of

mk

SerRS agree nicely with the conclusions

made from the crystal structure of SerRS from Methanosarcina barkeri.30,31

(a)

(b)

(c)

+2

Fig. 14. The rmsd (Å) of coordination sphere around Zn in (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’.


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The rmsd of serine ordering loop in the substrate bound active site of the dimeric state I is significantly lower than the open active site of the same dimer (Fig 15a-15c). The residues present in loop interact with Ser. The serine ordering loop places serine in a reactive position and the concomitant organization of the loop lowers the rmsd relative to open or adenylate bound state. The interaction is lost in the adenylate bound active site and rmsd rises compared to the serine bound active site as observed in the dimeric state II. This is not observed in the simulation of the monomer.

(a)

(b)

(c)

Fig. 15. The Rmsd (Å) of serine ordering loop for (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’.

The motions of the serine ordering loop observed by superimposing the structures of the loop at initial time (t=0) and at 2 ns (t=2) from simulated trajectories of dimeric state I, dimeric state II and monomeric open and closed state, respectively are presented in the supporting information (Fig. S.XX.a-S.XX.c). The organization of the loop is noted in the superimposed figures of substrate bound states. The ordered state of the serine ordering loop in the serine bound active sites is evident from the time dependent separation of the loop residues (Phe409 and Leu411) and the β-sheets at the base of the active site. For example, the separation between the Phe409 or Leu411 with Gly480 of the β sheet at the base of the active site shows that the respective separations are increasing in the open active site of dimeric state I or in the adenylate bound state in dimeric state II while the separation changes little in the Ser bound states in both dimeric states indicating the organized structure of the subsite in the substrate bound states. This result is in nice agreement with structural analysis of serine ordering loop in SerRS from methanogenic species.32 The opening-up of the loop in the open active site and adenylate bound active site is ACS Paragon Plus Environment

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due to the loss of interaction between serine and loop residues. The observed rigidity of the Ser binding subsite (located deeper within the aaRS) is effective for better placement of the Ser in a reactive geometry. The rmsd rises in the adenylate state indicating that the adenylate moved towards the opening of the cavity and the integrity of the tetracoordination is gradually lost. This might be suggestive of the functional requirement that adenylate needed to be closer to the opening of the cavity to interact with the incoming tRNA. The Zn+2 ion and Cys dependent Ser recognition in mkSerRS might be related with the early origin of the methanogenic Methanopyrus kandleri belonging to the base of Euryarchaeota universal tree57. III d. Base of the active site Seven antiparallel β strands, B1, B3 to B8, respectively27 form the base of the active site. The schematic representations of these strands for

mk

SerRS are given in the S.XXI in the supporting

information. These strands act together as a cradle for the substrates and form the base of the cavity. One end of each of the seven antiparallel β strands is located near serine ordering loop (SOL end) and another end is in proximity of motif 2 loop (M2L end). The time dependent variations of the average of the cosine of the angle between adjacent antiparallel β sheets (cosβ) are measured. Definition of cosβ used to describe the time dependent mutual orientation of seven antiparallel β sheets is presented in the section S.XXI of the supporting information and the organization of seven antiparallel β strands are depicted in detail in Fig. S.XXI.a and Fig. S.XXI.b, respectively. The results presented in Fig. 16a-16c show that the set of β sheets do not show any mutual movement and retain the organized structure. The β sheets are dynamically silent in open, substrate bound and adenylate bound states. The result shows that the base of the active site is practically immobile compared to the dynamics of the loops over the time scale of all simulations performed.

(a)

(b)

(c)

Fig. 16. Time dependent variation of the average of the cosine of the mutual orientations of the successive antiparallel β sheets, cosβ, in the active site with time for (a) dimeric state I. M1(S) indicates monomer 1 bound with


23


Page 24 of 60

substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’. In all cases, the cos(β) values are near the serine ordering loop (SOL end) and near motif 2 loop (M2L end) are presented.

The apparent slow motion of β sheets composing the active site is also noted in the active site of ribonuclease binase where the dynamically silent nature of the antiparallel β sheets is demonstrated by NMR spectroscopy58. It will be interesting to see if structurally similar antiparallel β sheets present in the active sites of in other proteins play similar role. III e. Dynamics of the helix turn helix (HTH) motif at dimer interface The pair of idiosyncratic helix turn helix (HTH) motifs at the interface acts as a hinge between catalytic domain and N terminal domain. This motif provides a covalent connectivity between motif 2 loop and loop f and the loops are shown to have important role in the reaction as presented in this work. The results of the present simulations show that HTH motifs of two neighboring monomers are held by noncovalent interaction (salt bridges) and constitute an important part of dimer interface in the present simulation (Fig. 17a). This result agrees fairly well with the crystallographic proposition that HTH motif reduces the degrees of freedom of two domains of a monomer. The structure of noncovalently bound HTH motifs of two monomers of mk

SerRS is shown in Fig. S.XXII.a of the supporting information. The structure is consistent with

the crystallographic, mutational and kinetic analysis of SerRS from M. barkeri59. The present results show that the pair of HTH (one from each monomer) forms an integrated structure (Fig. 17b-17c). This result also corroborates the suggestion that HTH motif contribute to the interdomain interaction based on the crystal structure of SerRS from M. barkeri. 30,31,59,60

(a)

(b)

(c)

Fig. 17 (a) The structure of nonbonded HTH motifs from two monomers at the dimeric interface of mkSerRS. Motif 2 loop and loop f of each monomer bound with the HTH motif are shown (b) the separations between the HTH motifs in dimeric state I and (c) in dimeric state II. Separations between various pairs of residues are measured and shown in the legend.


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The HTH motif of each monomer is flanked by loop f and motif 2 loop and acts as a continuity of the primary structure of the two loops. Stereo image of HTH motif connected with the motif 2 loop and loop f of the respective monomer are shown for dimeric state I in the supporting information (Fig. S.XXII.b). The present work showed that the motif 2 loop and loop f changes their conformation in going from open to closed state (we refer to Fig. 5, Fig. 10 and related discussions). The HTH motif connected with these two loops reorganizes its own conformation. The pair of the neighboring HTH motifs of the two monomers being a coupled structure, the reorganization is also expected to be correlated with the dynamics of motif 2 loop and loop f of the adjacent active site. This might have influence on the coordination of the reaction in two active sites. It is pointed out in literature that class II aaRSs such as HisRS, AlaRS, ProRS and ThrRS exhibit slower overall catalytic cycle than the rate of aminoacyl transfer which is suggestive of coupling across dimeric interface.23 However, the detailed nature of the coupling was not pointed out. Considering the coupled dynamics of two neighboring HTH motifs of adjacent monomers and the covalent linkage between labile motif 2 loop and loop f of each monomer with the HTH motif, this structural region might be one of the responsible factors controlling the coupling. III f. Dynamics of the N terminal domain So far we discussed the dynamics and structure of catalytic domain which is linked with the N terminal domain via a connecting loop. SerRS from methanogenic species (from M. Kandleri or M. barkeri) contains a unique N terminal tRNA binding domain which is composed of antiparallel β sheets capped by helical bundle and is different from the coiled-coil tRNA binding domain in bacterial-type SerRS30. The N terminal domain of variable arm of tRNA

Ser

mk

SerRS recognizes the long

and the acceptor end approaches the catalytic domain for tRNA

charging. The recognition takes place between conserved helices H2 and H3 of N terminal domain of SerRS and long variable arm of tRNASer. The binding of the tRNA is driven by long range electrostatic interaction.61 We computed the electrostatic potential of N terminal domain and the results are presented in the supporting information (Fig. S.XXIII). The positive electrostatic potential of N terminal domain is complementary to the negatively charged tRNA and is responsible for recognized via long range interaction. Information about the residues participating in interaction between long variable arm of tRNA and SerRS from M. barkeri is ACS Paragon Plus Environment

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known. The corresponding residues of the N terminal of

mk

Page 26 of 60

SerRS involved in interactions with

tRNASer are available by sequence comparison62. The residues are mentioned in supporting information (Table S.XXIV). Hydrogen bond analysis shows that Arg40 and Lys82 interact with the residues of long variable arm of tRNA via hydrogen bonding through the 2 ns trajectory of both dimeric state I and dimeric state II. The percentage occupancy of the hydrogen bond between

mk

SerRS and tRNASer of the relevant residues are presented in the supporting

information (Fig. S.XXIV.a –S.XXIV.f). Water mediated hydrogen bond between Arg79 and C47p of long variable arm of tRNA is noted as shown in the supporting information (Fig. S.XXV.a-S.XXV.d, respectively). It is interesting to note that N terminal domain exhibits lesser overall conformational fluctuation compared to the catalytic domain. The β-sheets and α helices of both domains exhibit low rmsd. However, the loops present in catalytic domain (motif 2 loop, loop f and serine ordering loop) change their conformation during the time scale of simulation and rmsd increases when the rmsd values are included in computation. In contrast, the inclusion of loops in N terminal loops does not increase the rmsd of the N terminal domain exhibiting the dynamical difference between the two domains. The results are shown in Fig. 18a-18d, respectively.

(a)

(b)

(c) (d) Fig. 18. (A) The rmsd of catalytic domain including only the β-sheets and α helices (B) the rmsd of catalytic domain including the β-sheets, α helices and the connecting loops between them (C) the rmsd of catalytic domain including β-sheets, α helices, connecting loops, motif 2 loop, loop f, serine ordering loop (D) the rmsd of N terminal domain including the β-sheets and α helices (E) the rmsd of N terminal domain including the β-sheets, α helices and the connecting loops between them.


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III g. Overview of the active site dynamics Here we summarize the principal findings of the present simulation from which an overview of the dynamics of active site in dimeric

mk

SerRS emerges. The active site tunnel has a rigid base

and the walls are composed of labile loops such as motif 2 loop, loop f and serine ordering loop. The relatively rigid base of the active site is composed of seven antiparallel β sheets which are dynamically silent compared to the walls. The part of the motif 2 loop near ATP binding site acts as a lid of the entrance of the active site and controls the substrate access and egress. The residue in this part of the loop such as Arg360 interacts with βP and γP region of ATP by hydrogen bonds and Glu351 interacts with adenosine base. The interaction helps retaining the bent conformation of ATP which is favorable for the reaction compared to the extended conformation. The movement of the loop is such that the hydrogen bonds with substrates which are important for catalysis are retained over nanosecond time scale. The flexibility of the walls of the active site gradually reduces near reaction center where the segments of motif 2 loop as well as loop f exhibit lesser fluctuation. Arg349 is an important catalytic residue of motif 2 loop in the reaction center subsite, which anchors both substrates (Ser and ATP) via hydrogen bonding. The anchoring act by Arg349 is very important for the feasibility of the nucleophilic attack. Further, Arg366 of the β sheet at the base of the active site holds the syn oxygen so that the attack by the anti oxygen is feasible. The functional reason for making a compact reaction center subsite is to create the optimum transition state geometry and to reduce the unfavorable electrostatic potential to facilitate the catalytic process. The reduction of ESP by the catalytic Arg residues (Arg349 and Arg366) is observed from present simulated structures in closed states. All loops move away from the platform of active site in the open or adenylate bound state and the network of hydrogen bonding is absent therein. A precise and stable geometry of hydrogen bonded network develops within active site which is essential for the reaction. All loops move away from the base of the active site in open state. The Ser binding site is the most rigid among all three subsites. The Ser is held here in a highly organized geometry bound by Zn+2 and Cys residues. The loop organizations are not well-organized for catalysis in the monomeric closed state, although some important hydrogen bonds are noted to be developed. Present simulation further suggests that the HTH motif connecting the monomers might have important role in controlling the functional dynamics in the two active sites. The result is consistent with the experimental observation of


27


Gruic-Sovulj et al37 that dimeric

mk

Page 28 of 60

SerRS enzyme complexed with one molecule of tRNASer is

more specific and more effective in catalyzing seryl adenylate formation than the apoenzyme. The N terminal domain is involved in long range electrostatic interaction and specific hydrogen bond interaction (both direct and water mediated) with tRNA which are important for binding of tRNA with SerRS. Overall conformational fluctuation is less in N terminal compared to the catalytic domain due to the presence of motif 2 loop, loop f and serine ordering loop which change conformation in the later domain during the reaction cycle.

IV. Concluding remarks Questions are raised in the introduction that how the active site of dimeric enzyme influences the positioning of the reactants in geometry suitable for reaction, organizes the catalytic residues around reactants and influences the electrostatic potential around the substrates to facilitate nucleophilic attack. In presenting a dynamical perspective of the active sites of the dimeric mk

SerRS bound with tRNA, the results provide answers to the foregoing questions. Classical

molecular dynamics simulations shows that the active site tunnel of each monomer in dimeric mk

SerRS in has a dynamically silent base composed of seven antiparallel β sheets and relatively

more flexible walls made up of motif 2 loop, loop f and serine ordering loop. The role of flexible loops in protein catalysis is well known.63-72 The role of conformational changes in the loop in controlling the functional dynamics of various proteins are known such as motility protein B in bacterial flagellar motor, cytochorme c peroxidase, triosephosphate isomerase, protein tyrosine phosphatase,

phosphoenolpyruvate

carboxykinase,

basillus

stearothermophiluslactate

dehydrogenase among others. The study of the dynamics of active site using molecular dynamics simulation is an important area of research since the pioneering works of Karplus and coworkers on the ribonuclease active site.73 The present work is expected to contribute to the understanding of active site dynamics of the aaRSs, an important class of enzyme for which the studies of the dynamics are rather limited. Another aspect of the present study is related to the study of homomeric structures which have fundamental importance in biology.74 A large number of aaRSs are multimers and the study of dynamics of dimeric enzyme is more relevant in understanding the catalytic activities rather than monomeric structures. The present study of dimeric SerRS is expected to contribute the understanding of functional importance of multimeric structure of various biomolecules. Another important aspect of the study of active site as carried out in this work is that the principles learned from architectonics of active sites can be ACS Paragon Plus Environment

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utilized in synthesizing useful biomimetic functional materials (enzyme mimetics). Novel functional nanomaterials can be developed once the molecular understanding of the correlation between the enzyme action and the structural organization is known. Numerous nanomaterials have been designed through the process of self-assembly and molecular recognition. The knowledge gained from the present study about the nanodimensional space of active site is useful in understanding the enzyme nanostructures and nanostructures capable of incorporation of enzymes75-78. The present study of SerRS active site organization and dynamics is expected to be useful in these directions.

Acknowledgements This work is supported by a SERB project grant (to N.N.) from DST, India. S.D. thanks University of Kalyani for a research scholarship. Partial support by UGC SAP DRS II program of the Chemistry department is acknowledged. This paper is dedicated in honoring Professor Biman Bagchi.

Supporting Information available The following figures, videos and Table are presented in the supporting information. Fig.S.I: Stereo view of the close-up of the structure of the active site of dimeric with tRNA. Fig.S.II: Stereo image of the active site region of

mk

mk

SerRS bound

SerRS monomer bound with

ATP and Ser. Fig.S.III: Stereo images of (a) ATP binding site (b) reaction center (c) serine binding site of mkSerRS. Fig.S.IV: Schematic representation of the active site residues present in the active site wall and interacting with Ser and ATP in the mkSerRS. Fig.S.V: Rmsd of motif 2 loop and its various fragments. S.VI.1.avi: Changes of the motif 2 loop with respect to Arg485 in monomer 1 in substrate bound active site of dimeric state I. S.VI.2.avi: Changes of the motif 2 loop with respect to Arg485 in monomer 2 with open active site of dimeric state I. S.VI.3.avi: Changes of the motif 2 loop with respect to Arg485 in monomer 1 in adenylate bound active site of dimeric state II. S.VI.4.avi: Changes of the motif 2 loop with respect to Arg485 in monomer 2 with substrate bound active site of dimeric state II. Fig.S.VII: The percentage of hydrogen bond occupancy of Glu351 in the simulation. Fig.S.VIII: superimposed structures of Glu351 of motif 2 loop at initial time and at 2 ns for dimeric state I, dimeric state II and monomeric states. ACS Paragon Plus Environment

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Fig.S.IX: (a) uperimposed structures of Arg360 of motif 2 loop at initial time and at 2 ns of dimeric state I. Fig.S.X: Demonstration of π- π interaction between the Phe364 side chain and adenosine ring as well as cation-π interaction between Arg485 and adenosine ring. Fig.S.XI: The percentage of hydrogen bond occupancy of Arg485 in the simulations. S.XII.1.avi: Time dependent variation of the hydrogen bond geometry between Arg485 and oxygen attached with γP in monomer 1 in substrate bound active site of dimeric state I. S.XII.2.avi: Time dependent variation of the hydrogen bond geometry between Arg485 and oxygen attached with γP in monomer 2 in substrate bound active site of dimeric state II. Fig.S.XIII: Superimposed structures of loop f at initial time and at 2 ns of (a) dimeric state I (b) dimeric state II and (c) monomeric open and closed state, respectively. Fig.S.XIV: The percentage of hydrogen bond occupancy of Arg349 in the trajectory. S.XV.1.avi: Time dependent variation of the hydrogen bond geometry of Arg349 with both substrates via a pair of hydrogen bonds depicting the anchoring action of the residue in monomer 1 in substrate bound active site of dimeric state I. S.XV.2.avi: Time dependent variation of the hydrogen bond geometry of Arg349 with both substrates via a pair of hydrogen bonds depicting the anchoring action of the residue in monomer 2 in substrate bound active site of dimeric state II. Fig.S.XVI: The ESP contour at the reaction center of the

mk

SerRS using HF/6-31G(d,p) level of theory. Fig.S.XVII: The percentage of

hydrogen bond occupancy of Arg366 in the trajectory. S.XVIII.1.avi: Time dependent variation of the hydrogen bond geometry between syn oxygen of carboxylate group of Ser and Arg366 via a pair of hydrogen bonds in monomer 1 in substrate bound active site of dimeric state I. S.XVIII.2.avi: Time dependent variation of the hydrogen bond geometry between syn oxygen of carboxylate group of Ser and Arg366 via a pair of hydrogen bonds in monomer 2 in substrate bound active site of dimeric state II. Fig. S.XIX: Rmsd of various residues in the Zn+2 coordination site. Fig. S.XX: Superimposed structures of loop at initial time and at 2 ns from simulated trajectories of (a) dimeric state I (b) dimeric state II and (c) monomeric open and closed state, respectively. Fig.S.XXI.a: Schematic representation of seven antiparallel β sheets at the base of active site. Fig.S.XXI.b: Catalytic core of mkSerRS showing arrangement of β sheets for two ends. Fig.S.XXII.a: HTH motifs of two monomers at the dimer interface obtained from simulation. Fig.S.XXII.b: Stereo image of HTH motif connected with the motif 2 loop and loop f of the respective monomer. Fig.S.XXIII: (a) Electrostatic potential on molecular surface of Nterminal domain of

mk

SerRS

(b) electrostatic potential surface of tRNA (c) the ribbon

representation of N-terminal domain in the background of ESP of mkSerRS (d) long variable arm ACS Paragon Plus Environment

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of tRNA recognized by the N-terminal domain. Table S.XXIV: The residues observed to be involved in tRNA recognition via direct hydrogen bonding and water mediated hydrogen bond between residues of N terminal domain and bases of long variable arm in mkSerRS. Fig.S.XXIV: The percentage of occupancy of hydrogen bonds between residues of N terminal domain and bases of long variable arm in

mk

SerRS. Fig. S.XXV: The images of water mediated hydrogen

bonds between residues of N terminal domain and bases of long variable arm in water mediated H-bond between Arg79 of N-terminal domain of

mk

mk

SerRS. The

SerRS and C47p of long

variable arm of tRNA of dimeric state II. This information is available free of charge via the Internet at http://pubs.acs.org.


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Figure legends Fig. 1. Crystallographic structures of dimeric aaRSs (class I and class II) in surf representation available from protein data bank. (a) Class I CysRS (1LI5.pdb) from Escherichia coli. (b) Class I MetRS (2CSX.pdb) from Aquifex aeolicus. (c) Class I TyrRS (1H3F.pdb) from Thermus thermophilus. (d) Class I TrpRS (1MAW.pdb) from Geobacillus stearothermophilus. (e) Class II HisRS (1KMN.pdb) from Escherichia coli. (f) Class II ProRS (1H4Q.pdb) of species Thermus thermophilus. (g) Class II SerRS (1SER.pdb) from Thermus thermophilus. (h) Class II ThrRS (1EVK.pdb) of species Escherichia coli. (i) Class II AspRS (1IL2.pdb) of species Escherichia coli. (j) Class II AsnRS (11AS.pdb) from Escherichia coli. (k) Class II LysRS (4EX5.pdb) of species Burkholderia thailandesis. In each case, individual monomers are shown in surf representation with red and green color, respectively and tRNA is shown in tube representation with orange color. The images are prepared using VMD79. Fig 2. (a) Structure of dimeric mkSerRS bound with tRNA (prepared from 3w3s.pdb). Details are given in computational methods. The catalytic domain and N terminal domain of monomer 1 (M1) are colored in red and pink respectively and these domains of monomer 2 (M2) are colored in green and yellow, respectively. The tRNA (colored in orange) is bound via anticodon binding region with N terminal domain of monomer 2 (M2) (b) views of the active site from the ATP binding site located towards the observer (in left) and view from the serine binding site (in right). The seven antiparallel β sheets are shown in grey, serine ordering loop in purple, motif 2 loop in red and the loop f in green color. The remaining structural elements are shown in light grey for clarity. The images are prepared using VMD79. Fig. 3. (a) The active site region of mkSerRS monomer bound with ATP and Ser (b) the active site is schematically shown in green (image prepared using CAVER55) and loops are shown in the same color scheme as shown in Fig. 2b, respectively (c) the three subsites of active site, namely, serine binding subsite, reaction center and ATP binding subsites are shown as slices of active site using space-filling model. All structures are based on 3w3s.pdb (details are given in computational methods). The images are prepared using VMD79. Fig. 4. Fluctuation of the bottleneck radius of the ATP binding subsite for (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’. Fig. 5. Lid movement represented by δR (relative separation (Å) between motif 2 loop and the βsheet at the base of the active site). For details see text. (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’ (d) superimposed structures of motif 2 loop at initial time (t=0) and at 2 ns (t=2) from trajectories of dimeric state I, both monomers are shown (e) superimposed structures of motif 2 loop at initial time (t=0) and at 2 ns (t=2) from trajectories of dimeric state II, both monomers are shown (f) comparison of the superimposed structures of motif 2 loop at ACS Paragon Plus Environment

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initial time (t=0) and at 2 ns (t=2) from trajectories of superimposed open (red) and closed (green) monomeric states. All images are prepared using VMD79. Fig. 6. Rmsd (in Å) of Glu351 of motif 2 loop for (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’. Fig. 7. Rmsd (Å) of Arg360 of motif 2 loop for (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in red). M2(O) indicates monomer 2 with open active site (shown in black). (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’. Fig. 8. Rmsd (Å) of Arg485 for (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’. Fig. 9. Fluctuation of bottleneck radius (Å) of the reaction center for (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’. Fig. 10. Rmsd (Å) of loop f for (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’. Fig. 11. Rmsd (Å) of Arg349 of motif 2 loop for (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’. Fig.12. Rmsd (Å) of Arg366 in (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) ACS Paragon Plus Environment

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dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’. Fig.13. Variation of bottleneck radius (Å) in the serine binding site for (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’. Fig. 14. The rmsd (Å) of coordination sphere around Zn+2 in (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’. Fig. 15. The Rmsd (Å) of serine ordering loop for (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’. Fig. 16. Time dependent variation of the average of the cosine of the mutual orientations of the successive antiparallel β sheets, cosβ, in the active site with time for (a) dimeric state I. M1(S) indicates monomer 1 bound with substrate (shown in black), M2(O) indicates monomer 2 with open active site (shown in red) (b) dimeric state II. M1(A) indicates monomer 1 bound with adenylate (shown in blue). M2(S) indicates monomer 2 with substrate bound active site (shown in black) (c) monomeric state. The substrate bound state (shown in black) is denoted by ‘S’ and open state (shown in red) is denoted by ‘O’. In all cases, the cos(β) values are near the serine ordering loop (SOL end) and near motif 2 loop (M2L end) are presented. Fig. 17. (a) The structure of nonbonded HTH motifs from two monomers at the dimeric interface of mkSerRS. Motif 2 loop and loop f of each monomer bound with the HTH motif are shown (b) the separations between the HTH motifs in dimeric state I and (c) in dimeric state II. Separations between various pairs of residues are measured and shown in the legend. Fig. 18. (A) The rmsd of catalytic domain including only the β-sheets and α helices (B) the rmsd of catalytic domain including the β-sheets, α helices and the connecting loops between them (C) the rmsd of catalytic domain including β-sheets, α helices, connecting loops, motif 2 loop, loop f, serine ordering loop (D) the rmsd of N terminal domain including the β-sheets and α helices (E) the rmsd of N terminal domain including the β-sheets, α helices and the connecting loops between them.


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Fig. 2. ACS Paragon Plus Environment

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Dynamics of the Active Sites of Dimeric Seryl tRNA

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