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Molecular dynamics of local protein motions in lactate dehydrogenase
THEO CHEM ELSEVIER
Journal of Molecular Structure (Tbecchem) 368 (1996) 205-212
Molecular dynamics of local protein motions in lactate dehydrogenase’ Charles Letner, Gerald Alter* Wright State University, School of Medicine, Department of Biochemistry and Molecular Biology Dayton, OH 45435, USA
Received 1 November 1995; accepted 29 March 1996
Abstract The conformation and dynamics of an active-site loop in Bacillus stearothermophilus lactate dehydrogenaae has been examined. A series of molecular dynarniea calculations were performed in which only this loop and neighboring amino acid residues were allowed to move. Though the remainder of the protein was held rigid, it was still included in energy calculations.
In this initial study, solvent water was not included. Several interesting features were observed. Two triads of electrostatically interacting amino acid side chains were identified.Their functional significancewas assessed in simulationsby changing the
charge properties of each triad’s amino acid side chains. Resultsindicate one triad reduces the loop’s internal flexibility while the other mediates both loop closing and the precise positioning of the catalytically important arginine 92 side chain. These results link loop motions with the catalytic function of lactate dehydrogenase. Keywords:
Active site; Lactate dehydrogenase; Loop; Molecular dynamics; Protein dynamics
1. Introduction
The various isozymes of lactate dehydrogenases (LDH) all share a common feature, a flexible peptide loop that is adjacent to their active site. During a catalytic cycle this loop goes through a large conformational change as it moves from an open to a closed conformation. In the closed conformation the loop contributes catalytically important amino acid side chains to the active site. In the open conformation these side chains move away from the active site. Fig. 1 demonstrates the magnitude of this change obtained from crystallography data. Comparing the * Ccrresponding author. ’ Presented at the 2nd Electronic Computational Chemistry Conference, November 1995. This issue along witb any supplimentary material can be accessed from the THBOCHBM HomePage at URL:http://www.elsevier.co.rrJ/lccste/thecchem
open, apo, conformation (green or lighter ribbon) to the closed, holo, conformation (blue or darker ribbon) a considerable change in conformation is apparent upon ligand binding. The crystal data for malate dehydrogenase (MDH), a structurally homologous protein, give results consistent with this observation [1,2]. Kinetic data from Bacillus stearothermophihs LDH (bsLDH) indicate that the rate limiting step for catalysis is loop closure [3,4]. The link between catalysis and loop residues resides with Arg-92 of bsLDH. This residue, a loop residue, has been proposed by [5] to decrease the pKa of His-179 as the loop moves from the open to the closed conformation bringing Arg-92 closer to His179. This histidine acts as an acid catalyst in the utilization of pyruvate and a base catalyst in the utilization of lactate. A more recent mechanism proposes that Arg-92 acts to polarize the carbonyl of the
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Fig. 1. Superposition of the apo and holo crystal structures of dfLDH. The green (lighter) ribbon corresponds to the dogfish LDH (dfLDH) backbone conformation in the absence of ligands ([l], PDB code 6LDH). The blue (darker) ribbon represents the dfLDH backbone conformation when the enzyme is crystallized in the presence of oxamate (a substrate analog) and NADH ([l], PDB code 1LDM). The oxamate and NADH from the ternary complex of oxamate, NADH, and dfLDH are represented as space-filling models.
substrate [6]. That the loop can influence catalysis is further supported by kinetic experiments in the presence of Ficoll 70. In these experiments, MDH and LDH kinetics are altered in crowded solutions resulting from high concentrations of Ficoll 70 [7]. Holbrook [4] used a mutant bsLDH and stop flow fluorescence to examine loop motions in crowded solutions. These experiments indicated that loop motions are altered by solution crowding (Holbrook personal communication). From these results we have proposed that the loop is the structural feature responsible for alterations of catalysis seen in crowded solutions. To further establish a mechanism for the influence of loop motions on catalysis, we have undertaken a theoretical study of loop motion in bsLDH. This enzyme was chosen since a body of kinetic information including stop flow experiments examining loop motions have been reported [3]. Further, the consequences of specific mutations in bsLDH have been reported and a bsLDH clone is available. This offers the possibility for verification of computational results presented in this paper by mutation experiments. A complete molecular dynamics simulation study of the loop’s role in bsLDH catalysis requires simulations performed in the presence as well as the
absence of active site ligands while the protein is adequately hydrated. As a first step towards achieving this goal, we have determined how the molecular model of the protein may be simplified so that computing resources can be conserved. We have also searched for probable electrostatic interactions important for the loop’s conformation and dynamics. We report results of these initial studies, performed using in vacua simulations, in this communication. Results from these molecular dynamics simulations suggest probable charge/charge interactions that may have importance in loop conformation and in the positioning of Arg-92. We take advantage of the unique features of this conference to present movies generated from these simulations.
2. Methods The crystal structure for bsLDH ([8], PDB code 1LDB) was obtained from the Brookhaven Protein Database 19,101.The loop is not defined in this structure and was modeled using the loop search routine of SYBYL [ll]. The details are described in another manuscript currently in preparation. Molecular dynamics simulations were calculated with the MINMD module of AMBER 4.0 [12] with the allatom force field [13]. To pre-condition the modeled structure, energy minimization was conducted for 2 000 steps of steepest descent and 8 000 steps of conjugate gradient minimization. A distance dependent dielectric function was used with the dielectric constant set to 1.0. The non-bonded cutoff was set to 8 A and was updated every 25 steps. The resulting structure was used as the input for molecular dynamics. Molecular dynamics simulations were run at a temperature of 50 K. As the loop closes early in the simulation (within 5 ps) and remains closed for the rest of the simulation, it was not possible to increase the temperature to a more physiologically relevant temperature. This is probably owing to the unphysiological conditions of the simulation, particularly the absence if water. Under these conditions, it is likely that the polar/charged loop is more stable in the polar/ charged active site than in a vacuum, which would surround it when it is in an open conformation. In initial 100 ps simulations the loop is seen to close in 5 ps and remain in that general conformation for the
C. Letner, G. Alter/Journalof MolecularStructure(Theochem)368 (1996) 205-212
remaining 95 ps. The simulations presented here were run for 10000 steps with a 0.002 ps time step for a total simulation time of 20 ps. SHAKE was applied to all carbon-hydrogen bonds so that time step of 0.002 ps could be used. As in Fe minimization, the non-bonded cutoff was set to 8 A, the non-bonded list was updated every 25 steps, and a distance dependent dielectric function with the dielectric constant set to 1.0 was used. For the truncated model, the BELLY command of MINMD was used to allow only the atoms of residues 74-118,175184, and 216-236 to move. In this way the atoms of the stationary residues still contribute to the force felt by each atom allowed to move. However, the force on each stationary atom is not calculated. Model verification was done by comparisons of the truncated model to the dfLDH crystal data and full 20 ps simulations allowing all atoms to move. The results of both comparisons demonstrate that the truncated model gave conformations consistent with the open and closed dfLDH and motions consistent with the full bsLDH simulation. Mutants were modeled using SYBYL by altering the native residue of the energy minimized structure to the desired mutant. The side chain torsional angles of the mutant residue were adjusted to match the torsional angles of the native residue. The structures obtained were used in molecular dynamics simulations without further alteration or minimization. Simulations in which all residues are allowed to move required 30 h on a Silicon Graphics R4000 while the truncated model required only 10 h. On a Cray Y-MP, the truncated model simulations required only 0.66 h. To create movies, AMBER output files where used to generate SYBYL history files. SYBYL Programming Language (SPL) scripts were written to pull up each of the conformations in the history file. The script created the correct coloring and ribbons for each of the conformations. The script then issues a single command to the operating system that calls scrsave (utility supplied by Silicon Graphics with IRIX). Scrsave allows command line arguments that specify the portion of the screen to capture and generates a ‘.RGB file of this region of the screen. The SPL script repeats this for each conformation in the history file. After all the *.RGB files are created, the Silicon Graphics program moviemaker is used to generate
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the SGI movie files. These were converted to MPEG files with the program mv2mpeg.
3. Results and discussion 3.1. Truncated model description In these simulations the focus is on the local motions of the loop. As bsLDH is a large protein, 317 residues with 4922 atoms, the first objective was to develop a truncated model that allows for more efficient use of computer time. One possibility was to use a model in which all the residues within a given radius of the loop were included. However, this presents a problem as the loop moves from the open to the closed conformation. Residue 89’s alpha carbon, the residue that moves the farthest in going frtm the open to the closed conformation, moves -14 A. The question was, which conformation to use to determine the atoms to include, the open, closed, or some intermediate conformation? The solution was to run repeated simulations in which atoms included were varied and then to compare loop motion results to loop motion results from full simulations in which all the protein atoms are allowed to move. In this
Fig. 2. A truncated model of bsLDH. The green (lighter) ribbon represents the backbone of the amino acid residues who’s atoms are allowed to move. The blue (darker) ribbon represents the backbone of amino acids residues who’s atoms are held in their crystallographic positions. The ligands from the holo dfLDH crystal structure are included as spacetilling models to identify the active site but are not included in the simulations. Movies 1 and 2 demonstrate the motions of the full protein simulation and the truncated model simulation respectively.
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Fig. 3. BsLDH conformations at various times during the truncated model simulation. These conformations occurred at: 0.0, Panel A, 1.0, Panel B, 2.0, Panel C, 3.0, Panel D and 4.0, Panel E, psec. The coordinates of these files, in pdb format, are included in the files struct2.pdb, stru&.pdb, stwt4.pdb, structS.pdb, and struct6.pdb of the ECCC2 archives.
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way, the final residues to include was based on information from both open and closed conformations (the development of the model is presented in considerable detail in another manuscript currently in preparation). The final model allows atoms in residues 74-118, 175-184, and 216-236 to move. The numbering system in this article starts with the first residue numbered 1 with no gaps in the primary structure numbering. This model results in 1156 atoms requiring 0.66 h on a Cray Y-MP for each simulation. Two separate criteria were used to access the validity of the truncated model for describing motions of the active site loop. The first was the difference in the position of the homologous loop in dfLDH. By aligning the dfLDH open and closed crystal structures, the difference in loop position as the loop changed from the open to closed position was calculated. These calculated differences were compared to the differences generated in the simulations to demonstrate that our model gave a range of motions consistent with the dfLDH crystallography data. Our second criterion was that the loop movement in the truncated model match the movement observed in a bsLDH simulation in which all the protein atoms are allowed to move. Both full protein and truncated model simulations suggest that the same conformation changes occur in the active site region. We believe that this consistency provides strong support for the method of developing and refining the truncated model. Fig. 2 displays the backbone of the regions allowed to move (green or lighter ribbon) and the regions that remain at their crystallographic position (blue or darker ribbon) as well as ligands to identify the active site. Movies 1 and 2 allow a comparison between a full protein simulation and a simulation using the truncated model. From this figure and these movies it is apparent that the regions of protein directly adjacent to the loop are included. The motion of the loop is similar in the two simulations as is motion of the helix to the right of the loop. Fig. 3 displays the conformation of the loop as the simulation progresses. 3.2. Intra-loop amino acid triad While examining the conformation of the loop, it was clear that motions within the loop were restricted. During the transition from the open to the closed conformation the loop appears to behave as a lid in that
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Fig. 4. Triad of intra-loop amino acid residues that contribute to the loop’s internal structure. The green ribbon represents the backbone of the loop while the side chains of Lys-87, Glu-90, and Asp-94 to the left, center, and right, are displayed in blue, red, and orange respectively. This figure represents loop structure at the beginning of a simulation. As the simulation proceeds Lys-87 moves to a position between Glu-90 and Asp-94. Movie 3 demonstrates this motion.
the loop residues, residues 85-96, seemed to move as a single unit maintaining its overall conformation. There are 3 charged residues located on the side of the loop facing away from the active site, Lys-87, Glu-90, and Asp-94. Movie 3 and Fig. 4 demonstrate that these three residues are close enough for charge/ charge interactions to occur. In fact, the side chain of Lys-87 moves over to a position between Glu-90 and Asp-94 over the course of the simulation. These three residues seem to provide a set of electrostatic interactions that restrict the loop’s internal mobility. This internal stability is apparent when comparing movie 3 with movies 5, 6, and 7 presented in the next section. 3.2.1. Arg-92 amino acid triad Arg-92 is an important residue in catalysis. As such
it seemed appropriate to examine the motions of this residue. During the model development phase of
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position between Glu-90 and Asp-94. However, when Lys-87 is changed to Gln, the side chain does not position itself in this location (Movie 5). A similar pattern is seen when Glu-90 is changed to Gln (Movie 6). However, when Asp-94 is changed to Asn, the side chain of Lys-87 does seem to go to a position close to Glu-90 (Movie 7). These results lend evidence in support of the idea of an amino acid triad, consisting of Lys-87, Glu-90, and Asp-94, that is held together by electrostatic interactions. Since these three residues span the middle of the loop, it is likely that they provide a structural framework that provides internal rigidity to the loop. Fig. 5. Amino acid triad which includes Arg-92, is important in the positioning of this residue, and is important for loop closure. The green ribbon represents the backbone of amino acid residues who’s atoms are allowed to move (the loop and residues 175-184) while the side chains of Arg-92, Glu-178, and Asp-181 are displayed in blue (on upper loop), orange (to the left on the lower loop), and green (to the right on the lower loop) respectively. Movie 4 demonstrates the motions of this triad as the loop goes from the open to the closed conformation. The movie presents the backbone as sticks to provide a more detailed view of the motions in the loop backbone.
this study, it became clear that Glu-178 and Asp-181 interacted with Arg-92. In fact, if the atoms of residues 175-184 where not allowed to move, the conformation of Arg-92 was considerably different than in the full simulation. As a result these residues were included. This led to the identification of a second triad of residues required to optimally position Arg92 for binding of substrate (Fig. 5 and Movie 4). Arg92 is conserved over the MDH’s and LDH’s that make up the family of 2-hydroxy-acid dehydrogenase enzymes [14]. This, along with the role that this residue plays in catalysis, suggests that this residue is extremely important for correct function of the enzyme. Glu-178 and Asp-181 determine the position of Arg-92 more precisely than if either of these was absent. 3.3. Intra-loop amino acid substitutions Residues Lys-87, Glu-90, and Asp-94 make up the triad that restricts the flexibility of the loop. These three residues were individually substituted with Gln, Gln, and Asn, respectively, to eliminate charges while maintaining hydrophilicity. In the native simulation, Movie 3, Lys-87’s side chain moves to a
3.4. Arg-92 amino acid substitutions Earlier, a triad of residues that may be important in determining the position of Arg-92 was identified (Fig. 5 and Movie 4). To further investigate this, amino acid substitution of the 3 residues involved in this triad were simulated. Movie 8 displays the results obtained when Arg-92 was changed to Gln. The results obtained are drastically different than in the native simulation. The most obvious difference is that the loop does not close. This impacts the environment of the active site. When the loop is in the closed conformation, the active site is sealed off from the solvent. This provides the appropriate environment for catalysis [15]. If the loop does not close, the active site is fully solvent exposed, creating a less favorable catalytic environment. Movie 9 displays the simulation when Asp-181 is changed to Asn. In this case the side chain of Arg-92 no longer positions itself as in the full simulations. In fact, the side chain appears to interact with Glu-178. However, this positions the charged portion of Arg92’s side chain further from the active center. Results for the substitution of Glu-178 with Gln gave similar results (results not presented). However, its’ effect is significantly less as the charged portion of Arg-92 is still in approximately the same position as in the native simulation. The main difference between the native and Gln-178 simulation is that the orientation of the side chain of Arg-92 is altered. In the native simulation the middle of Arg-92’s side chain bends towards Glu-178. In the substituted simulation the Arg-92 points straight at Asp-181.
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4. Conclusions A truncated model of bsLDH has been presented and used to examine the motions of the bsLDH active site peptide loop. To develop the model and obtain initial results in the computationally simplest system possible we have not included either solvent or ligands in the simulations reported here. However, we have made several important observations. Simulations in which both solvent and ligands are included are underway to further investigate the conclusion presented here. Electrostatic interactions between amino acids side chain in each of two amino acid triads were identified. Those in the first triad limit the internal flexibility of the loop while those in the second triad position the catalytically important residue Arg-92. The first triad is composed of Lys-87, Glu-90, and Asp-94 while the second triad is made up of Arg-92, Glu-178, and Asp181. Mutations of the residues involved in these triads further verify these results and suggest that Arg-92 may also have a role in loop closure. The results of these simulations suggest a mechanism to link loop motions to catalysis. The key to this is Arg-92 that acts in two capacities. First it has a role in loop closure. When the Arg-92 to Asp-181/Glu-178 interaction is absent the loop does not close. As a result the active site environment is solvent exposed. Additionally, when this triad is absent, Arg-92 will not be optimally positioned for substrate catalysis. The other triad is important in limiting loop flexibility which may also be crucial to Arg-92’s position.
Acknowledgements This work was supported by grants from the Ohio Affiliate of the American Heart Association, the Ohio Supercomputing Center, and the Biomedical Sciences Program of Wright State University. This article describes the motions of the bsLDH active site loop obtained from molecular dynamics simulations. To demonstrate these motions, movies are extensively used. The movies are provided in 2 formats, SGI movie files and MPEG movies. The SGI movies are much larger than the MPEG files. However, the quality is much better. The MPEG’s are
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included for those who do not have access to a Silicon Graphics workstation. The SGI movies are provided as 3 tarred and compressed files, movies1.tar.Z which contains Movies 1 and 2 (26 MB), movies2.tar.Z which contains Movies 3 and 4 (15 MB), and movies3.tar.Z which contains Movies 5 thru 9 (13 MB). These files can be uncompressed with the command: uncompress movies* .tar.Z on a Silicon Graphics workstation and then the tar archive can be extracted with the command: tar xvf movies*.tar A directory named movies will be created with the movies in that directory. To view these movies use the command: movieplayer [filename] We have numbered our model with the first residue #l, and each residue thereafter numbered in sequence with no gaps. This is in contrast to some papers which use a numbering system relative to dfLDH. This provides consistency in numbering across all the structures included here.
References [l] J. Birktoft, G. Rhodes and L. Banaszak, Biochemistry, 28 (1989) 6065. [2] M. Hall and L. Banaszak, J. Mol. Biol., 231 (1993) 213. [3] A. Waldman, K. Hart, A. Clarke, D. Wigley, D. Barswow, T. Atkinson and J. Holbrook, Biochem. Biophys. Res. Comm., 150 (1988) 752. [4] M. Philippopoulos, Y. Xiang and C. Lim, Protein Engineering, 8 (1995) 565. [S] U. Grau, W. Trommer and M. Rossmann, J. Mol. Biol., 151 (1981) 289. [6] A. Clarke, D. Wigley, W. Chia, D. Barstow, T. Atkinson and J. Holbrook, Nature, 324 (1986) 699. [7] C. Letner, P. Tung and G. Alter, FASEB J., 8 (1991) A1285. [8] K. Pionteck, P. Chakrabarti, H. Schar, M. Rossmann, and H. Zuber, Proteins, 7 (1990) 74. [9] D. Kitchen, F. Hirata, J. Westbrook, R. Levy, D. Kotke and M. Yarmush, J. Comp. C&em., ll(l990) 1169. [lo] E. Abola, F. Bernstein, S. Bryant, T. Koetzle and J. Weng, (1987) “Protein Data Bank”, in F.H. Allen, G. Bergerhoff, and R. Sievers (Eds.), “Crystallographic Databases - Information Content, Software Systems, Scientific Applications”, Data
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Commission of the International Union of Crystallography, Bonn/Cambridge/Chester, (1987), pp. 107-132. [ll] Tripes Associates, Inc., SYBYL Molecular Modeling Software, Version 6.0., (1992). [12] D. Pearlman, D. Case, J. Caldwell, G. Seibel, U. Singh, P. Weiner and P. Kollman, AMBER 4.0, University of California, San Francisco (1991).
[13] S. Weiner, P. Kollman, D. Nyugen and D. Case, J. Camp. Chem., 7 (19860 230. [14] E. Honka, S. Fabry, T. Niermann, P. Palm and R. Hensel, Eur. J. Biochem., 188 (1990) 623. [15] C. Abad-Zapatero, J. Griffith, J. Sussman and M. Rossmann, J. Mol. Biol., 198 (1987) 445.
Journal of Molecular Structure (Tbecchem) 368 (1996) 205-212
Molecular dynamics of local protein motions in lactate dehydrogenase’ Charles Letner, Gerald Alter* Wright State University, School of Medicine, Department of Biochemistry and Molecular Biology Dayton, OH 45435, USA
Received 1 November 1995; accepted 29 March 1996
Abstract The conformation and dynamics of an active-site loop in Bacillus stearothermophilus lactate dehydrogenaae has been examined. A series of molecular dynarniea calculations were performed in which only this loop and neighboring amino acid residues were allowed to move. Though the remainder of the protein was held rigid, it was still included in energy calculations.
In this initial study, solvent water was not included. Several interesting features were observed. Two triads of electrostatically interacting amino acid side chains were identified.Their functional significancewas assessed in simulationsby changing the
charge properties of each triad’s amino acid side chains. Resultsindicate one triad reduces the loop’s internal flexibility while the other mediates both loop closing and the precise positioning of the catalytically important arginine 92 side chain. These results link loop motions with the catalytic function of lactate dehydrogenase. Keywords:
Active site; Lactate dehydrogenase; Loop; Molecular dynamics; Protein dynamics
1. Introduction
The various isozymes of lactate dehydrogenases (LDH) all share a common feature, a flexible peptide loop that is adjacent to their active site. During a catalytic cycle this loop goes through a large conformational change as it moves from an open to a closed conformation. In the closed conformation the loop contributes catalytically important amino acid side chains to the active site. In the open conformation these side chains move away from the active site. Fig. 1 demonstrates the magnitude of this change obtained from crystallography data. Comparing the * Ccrresponding author. ’ Presented at the 2nd Electronic Computational Chemistry Conference, November 1995. This issue along witb any supplimentary material can be accessed from the THBOCHBM HomePage at URL:http://www.elsevier.co.rrJ/lccste/thecchem
open, apo, conformation (green or lighter ribbon) to the closed, holo, conformation (blue or darker ribbon) a considerable change in conformation is apparent upon ligand binding. The crystal data for malate dehydrogenase (MDH), a structurally homologous protein, give results consistent with this observation [1,2]. Kinetic data from Bacillus stearothermophihs LDH (bsLDH) indicate that the rate limiting step for catalysis is loop closure [3,4]. The link between catalysis and loop residues resides with Arg-92 of bsLDH. This residue, a loop residue, has been proposed by [5] to decrease the pKa of His-179 as the loop moves from the open to the closed conformation bringing Arg-92 closer to His179. This histidine acts as an acid catalyst in the utilization of pyruvate and a base catalyst in the utilization of lactate. A more recent mechanism proposes that Arg-92 acts to polarize the carbonyl of the
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Fig. 1. Superposition of the apo and holo crystal structures of dfLDH. The green (lighter) ribbon corresponds to the dogfish LDH (dfLDH) backbone conformation in the absence of ligands ([l], PDB code 6LDH). The blue (darker) ribbon represents the dfLDH backbone conformation when the enzyme is crystallized in the presence of oxamate (a substrate analog) and NADH ([l], PDB code 1LDM). The oxamate and NADH from the ternary complex of oxamate, NADH, and dfLDH are represented as space-filling models.
substrate [6]. That the loop can influence catalysis is further supported by kinetic experiments in the presence of Ficoll 70. In these experiments, MDH and LDH kinetics are altered in crowded solutions resulting from high concentrations of Ficoll 70 [7]. Holbrook [4] used a mutant bsLDH and stop flow fluorescence to examine loop motions in crowded solutions. These experiments indicated that loop motions are altered by solution crowding (Holbrook personal communication). From these results we have proposed that the loop is the structural feature responsible for alterations of catalysis seen in crowded solutions. To further establish a mechanism for the influence of loop motions on catalysis, we have undertaken a theoretical study of loop motion in bsLDH. This enzyme was chosen since a body of kinetic information including stop flow experiments examining loop motions have been reported [3]. Further, the consequences of specific mutations in bsLDH have been reported and a bsLDH clone is available. This offers the possibility for verification of computational results presented in this paper by mutation experiments. A complete molecular dynamics simulation study of the loop’s role in bsLDH catalysis requires simulations performed in the presence as well as the
absence of active site ligands while the protein is adequately hydrated. As a first step towards achieving this goal, we have determined how the molecular model of the protein may be simplified so that computing resources can be conserved. We have also searched for probable electrostatic interactions important for the loop’s conformation and dynamics. We report results of these initial studies, performed using in vacua simulations, in this communication. Results from these molecular dynamics simulations suggest probable charge/charge interactions that may have importance in loop conformation and in the positioning of Arg-92. We take advantage of the unique features of this conference to present movies generated from these simulations.
2. Methods The crystal structure for bsLDH ([8], PDB code 1LDB) was obtained from the Brookhaven Protein Database 19,101.The loop is not defined in this structure and was modeled using the loop search routine of SYBYL [ll]. The details are described in another manuscript currently in preparation. Molecular dynamics simulations were calculated with the MINMD module of AMBER 4.0 [12] with the allatom force field [13]. To pre-condition the modeled structure, energy minimization was conducted for 2 000 steps of steepest descent and 8 000 steps of conjugate gradient minimization. A distance dependent dielectric function was used with the dielectric constant set to 1.0. The non-bonded cutoff was set to 8 A and was updated every 25 steps. The resulting structure was used as the input for molecular dynamics. Molecular dynamics simulations were run at a temperature of 50 K. As the loop closes early in the simulation (within 5 ps) and remains closed for the rest of the simulation, it was not possible to increase the temperature to a more physiologically relevant temperature. This is probably owing to the unphysiological conditions of the simulation, particularly the absence if water. Under these conditions, it is likely that the polar/charged loop is more stable in the polar/ charged active site than in a vacuum, which would surround it when it is in an open conformation. In initial 100 ps simulations the loop is seen to close in 5 ps and remain in that general conformation for the
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remaining 95 ps. The simulations presented here were run for 10000 steps with a 0.002 ps time step for a total simulation time of 20 ps. SHAKE was applied to all carbon-hydrogen bonds so that time step of 0.002 ps could be used. As in Fe minimization, the non-bonded cutoff was set to 8 A, the non-bonded list was updated every 25 steps, and a distance dependent dielectric function with the dielectric constant set to 1.0 was used. For the truncated model, the BELLY command of MINMD was used to allow only the atoms of residues 74-118,175184, and 216-236 to move. In this way the atoms of the stationary residues still contribute to the force felt by each atom allowed to move. However, the force on each stationary atom is not calculated. Model verification was done by comparisons of the truncated model to the dfLDH crystal data and full 20 ps simulations allowing all atoms to move. The results of both comparisons demonstrate that the truncated model gave conformations consistent with the open and closed dfLDH and motions consistent with the full bsLDH simulation. Mutants were modeled using SYBYL by altering the native residue of the energy minimized structure to the desired mutant. The side chain torsional angles of the mutant residue were adjusted to match the torsional angles of the native residue. The structures obtained were used in molecular dynamics simulations without further alteration or minimization. Simulations in which all residues are allowed to move required 30 h on a Silicon Graphics R4000 while the truncated model required only 10 h. On a Cray Y-MP, the truncated model simulations required only 0.66 h. To create movies, AMBER output files where used to generate SYBYL history files. SYBYL Programming Language (SPL) scripts were written to pull up each of the conformations in the history file. The script created the correct coloring and ribbons for each of the conformations. The script then issues a single command to the operating system that calls scrsave (utility supplied by Silicon Graphics with IRIX). Scrsave allows command line arguments that specify the portion of the screen to capture and generates a ‘.RGB file of this region of the screen. The SPL script repeats this for each conformation in the history file. After all the *.RGB files are created, the Silicon Graphics program moviemaker is used to generate
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the SGI movie files. These were converted to MPEG files with the program mv2mpeg.
3. Results and discussion 3.1. Truncated model description In these simulations the focus is on the local motions of the loop. As bsLDH is a large protein, 317 residues with 4922 atoms, the first objective was to develop a truncated model that allows for more efficient use of computer time. One possibility was to use a model in which all the residues within a given radius of the loop were included. However, this presents a problem as the loop moves from the open to the closed conformation. Residue 89’s alpha carbon, the residue that moves the farthest in going frtm the open to the closed conformation, moves -14 A. The question was, which conformation to use to determine the atoms to include, the open, closed, or some intermediate conformation? The solution was to run repeated simulations in which atoms included were varied and then to compare loop motion results to loop motion results from full simulations in which all the protein atoms are allowed to move. In this
Fig. 2. A truncated model of bsLDH. The green (lighter) ribbon represents the backbone of the amino acid residues who’s atoms are allowed to move. The blue (darker) ribbon represents the backbone of amino acids residues who’s atoms are held in their crystallographic positions. The ligands from the holo dfLDH crystal structure are included as spacetilling models to identify the active site but are not included in the simulations. Movies 1 and 2 demonstrate the motions of the full protein simulation and the truncated model simulation respectively.
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Fig. 3. BsLDH conformations at various times during the truncated model simulation. These conformations occurred at: 0.0, Panel A, 1.0, Panel B, 2.0, Panel C, 3.0, Panel D and 4.0, Panel E, psec. The coordinates of these files, in pdb format, are included in the files struct2.pdb, stru&.pdb, stwt4.pdb, structS.pdb, and struct6.pdb of the ECCC2 archives.
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way, the final residues to include was based on information from both open and closed conformations (the development of the model is presented in considerable detail in another manuscript currently in preparation). The final model allows atoms in residues 74-118, 175-184, and 216-236 to move. The numbering system in this article starts with the first residue numbered 1 with no gaps in the primary structure numbering. This model results in 1156 atoms requiring 0.66 h on a Cray Y-MP for each simulation. Two separate criteria were used to access the validity of the truncated model for describing motions of the active site loop. The first was the difference in the position of the homologous loop in dfLDH. By aligning the dfLDH open and closed crystal structures, the difference in loop position as the loop changed from the open to closed position was calculated. These calculated differences were compared to the differences generated in the simulations to demonstrate that our model gave a range of motions consistent with the dfLDH crystallography data. Our second criterion was that the loop movement in the truncated model match the movement observed in a bsLDH simulation in which all the protein atoms are allowed to move. Both full protein and truncated model simulations suggest that the same conformation changes occur in the active site region. We believe that this consistency provides strong support for the method of developing and refining the truncated model. Fig. 2 displays the backbone of the regions allowed to move (green or lighter ribbon) and the regions that remain at their crystallographic position (blue or darker ribbon) as well as ligands to identify the active site. Movies 1 and 2 allow a comparison between a full protein simulation and a simulation using the truncated model. From this figure and these movies it is apparent that the regions of protein directly adjacent to the loop are included. The motion of the loop is similar in the two simulations as is motion of the helix to the right of the loop. Fig. 3 displays the conformation of the loop as the simulation progresses. 3.2. Intra-loop amino acid triad While examining the conformation of the loop, it was clear that motions within the loop were restricted. During the transition from the open to the closed conformation the loop appears to behave as a lid in that
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Fig. 4. Triad of intra-loop amino acid residues that contribute to the loop’s internal structure. The green ribbon represents the backbone of the loop while the side chains of Lys-87, Glu-90, and Asp-94 to the left, center, and right, are displayed in blue, red, and orange respectively. This figure represents loop structure at the beginning of a simulation. As the simulation proceeds Lys-87 moves to a position between Glu-90 and Asp-94. Movie 3 demonstrates this motion.
the loop residues, residues 85-96, seemed to move as a single unit maintaining its overall conformation. There are 3 charged residues located on the side of the loop facing away from the active site, Lys-87, Glu-90, and Asp-94. Movie 3 and Fig. 4 demonstrate that these three residues are close enough for charge/ charge interactions to occur. In fact, the side chain of Lys-87 moves over to a position between Glu-90 and Asp-94 over the course of the simulation. These three residues seem to provide a set of electrostatic interactions that restrict the loop’s internal mobility. This internal stability is apparent when comparing movie 3 with movies 5, 6, and 7 presented in the next section. 3.2.1. Arg-92 amino acid triad Arg-92 is an important residue in catalysis. As such
it seemed appropriate to examine the motions of this residue. During the model development phase of
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position between Glu-90 and Asp-94. However, when Lys-87 is changed to Gln, the side chain does not position itself in this location (Movie 5). A similar pattern is seen when Glu-90 is changed to Gln (Movie 6). However, when Asp-94 is changed to Asn, the side chain of Lys-87 does seem to go to a position close to Glu-90 (Movie 7). These results lend evidence in support of the idea of an amino acid triad, consisting of Lys-87, Glu-90, and Asp-94, that is held together by electrostatic interactions. Since these three residues span the middle of the loop, it is likely that they provide a structural framework that provides internal rigidity to the loop. Fig. 5. Amino acid triad which includes Arg-92, is important in the positioning of this residue, and is important for loop closure. The green ribbon represents the backbone of amino acid residues who’s atoms are allowed to move (the loop and residues 175-184) while the side chains of Arg-92, Glu-178, and Asp-181 are displayed in blue (on upper loop), orange (to the left on the lower loop), and green (to the right on the lower loop) respectively. Movie 4 demonstrates the motions of this triad as the loop goes from the open to the closed conformation. The movie presents the backbone as sticks to provide a more detailed view of the motions in the loop backbone.
this study, it became clear that Glu-178 and Asp-181 interacted with Arg-92. In fact, if the atoms of residues 175-184 where not allowed to move, the conformation of Arg-92 was considerably different than in the full simulation. As a result these residues were included. This led to the identification of a second triad of residues required to optimally position Arg92 for binding of substrate (Fig. 5 and Movie 4). Arg92 is conserved over the MDH’s and LDH’s that make up the family of 2-hydroxy-acid dehydrogenase enzymes [14]. This, along with the role that this residue plays in catalysis, suggests that this residue is extremely important for correct function of the enzyme. Glu-178 and Asp-181 determine the position of Arg-92 more precisely than if either of these was absent. 3.3. Intra-loop amino acid substitutions Residues Lys-87, Glu-90, and Asp-94 make up the triad that restricts the flexibility of the loop. These three residues were individually substituted with Gln, Gln, and Asn, respectively, to eliminate charges while maintaining hydrophilicity. In the native simulation, Movie 3, Lys-87’s side chain moves to a
3.4. Arg-92 amino acid substitutions Earlier, a triad of residues that may be important in determining the position of Arg-92 was identified (Fig. 5 and Movie 4). To further investigate this, amino acid substitution of the 3 residues involved in this triad were simulated. Movie 8 displays the results obtained when Arg-92 was changed to Gln. The results obtained are drastically different than in the native simulation. The most obvious difference is that the loop does not close. This impacts the environment of the active site. When the loop is in the closed conformation, the active site is sealed off from the solvent. This provides the appropriate environment for catalysis [15]. If the loop does not close, the active site is fully solvent exposed, creating a less favorable catalytic environment. Movie 9 displays the simulation when Asp-181 is changed to Asn. In this case the side chain of Arg-92 no longer positions itself as in the full simulations. In fact, the side chain appears to interact with Glu-178. However, this positions the charged portion of Arg92’s side chain further from the active center. Results for the substitution of Glu-178 with Gln gave similar results (results not presented). However, its’ effect is significantly less as the charged portion of Arg-92 is still in approximately the same position as in the native simulation. The main difference between the native and Gln-178 simulation is that the orientation of the side chain of Arg-92 is altered. In the native simulation the middle of Arg-92’s side chain bends towards Glu-178. In the substituted simulation the Arg-92 points straight at Asp-181.
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4. Conclusions A truncated model of bsLDH has been presented and used to examine the motions of the bsLDH active site peptide loop. To develop the model and obtain initial results in the computationally simplest system possible we have not included either solvent or ligands in the simulations reported here. However, we have made several important observations. Simulations in which both solvent and ligands are included are underway to further investigate the conclusion presented here. Electrostatic interactions between amino acids side chain in each of two amino acid triads were identified. Those in the first triad limit the internal flexibility of the loop while those in the second triad position the catalytically important residue Arg-92. The first triad is composed of Lys-87, Glu-90, and Asp-94 while the second triad is made up of Arg-92, Glu-178, and Asp181. Mutations of the residues involved in these triads further verify these results and suggest that Arg-92 may also have a role in loop closure. The results of these simulations suggest a mechanism to link loop motions to catalysis. The key to this is Arg-92 that acts in two capacities. First it has a role in loop closure. When the Arg-92 to Asp-181/Glu-178 interaction is absent the loop does not close. As a result the active site environment is solvent exposed. Additionally, when this triad is absent, Arg-92 will not be optimally positioned for substrate catalysis. The other triad is important in limiting loop flexibility which may also be crucial to Arg-92’s position.
Acknowledgements This work was supported by grants from the Ohio Affiliate of the American Heart Association, the Ohio Supercomputing Center, and the Biomedical Sciences Program of Wright State University. This article describes the motions of the bsLDH active site loop obtained from molecular dynamics simulations. To demonstrate these motions, movies are extensively used. The movies are provided in 2 formats, SGI movie files and MPEG movies. The SGI movies are much larger than the MPEG files. However, the quality is much better. The MPEG’s are
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included for those who do not have access to a Silicon Graphics workstation. The SGI movies are provided as 3 tarred and compressed files, movies1.tar.Z which contains Movies 1 and 2 (26 MB), movies2.tar.Z which contains Movies 3 and 4 (15 MB), and movies3.tar.Z which contains Movies 5 thru 9 (13 MB). These files can be uncompressed with the command: uncompress movies* .tar.Z on a Silicon Graphics workstation and then the tar archive can be extracted with the command: tar xvf movies*.tar A directory named movies will be created with the movies in that directory. To view these movies use the command: movieplayer [filename] We have numbered our model with the first residue #l, and each residue thereafter numbered in sequence with no gaps. This is in contrast to some papers which use a numbering system relative to dfLDH. This provides consistency in numbering across all the structures included here.
References [l] J. Birktoft, G. Rhodes and L. Banaszak, Biochemistry, 28 (1989) 6065. [2] M. Hall and L. Banaszak, J. Mol. Biol., 231 (1993) 213. [3] A. Waldman, K. Hart, A. Clarke, D. Wigley, D. Barswow, T. Atkinson and J. Holbrook, Biochem. Biophys. Res. Comm., 150 (1988) 752. [4] M. Philippopoulos, Y. Xiang and C. Lim, Protein Engineering, 8 (1995) 565. [S] U. Grau, W. Trommer and M. Rossmann, J. Mol. Biol., 151 (1981) 289. [6] A. Clarke, D. Wigley, W. Chia, D. Barstow, T. Atkinson and J. Holbrook, Nature, 324 (1986) 699. [7] C. Letner, P. Tung and G. Alter, FASEB J., 8 (1991) A1285. [8] K. Pionteck, P. Chakrabarti, H. Schar, M. Rossmann, and H. Zuber, Proteins, 7 (1990) 74. [9] D. Kitchen, F. Hirata, J. Westbrook, R. Levy, D. Kotke and M. Yarmush, J. Comp. C&em., ll(l990) 1169. [lo] E. Abola, F. Bernstein, S. Bryant, T. Koetzle and J. Weng, (1987) “Protein Data Bank”, in F.H. Allen, G. Bergerhoff, and R. Sievers (Eds.), “Crystallographic Databases - Information Content, Software Systems, Scientific Applications”, Data
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Commission of the International Union of Crystallography, Bonn/Cambridge/Chester, (1987), pp. 107-132. [ll] Tripes Associates, Inc., SYBYL Molecular Modeling Software, Version 6.0., (1992). [12] D. Pearlman, D. Case, J. Caldwell, G. Seibel, U. Singh, P. Weiner and P. Kollman, AMBER 4.0, University of California, San Francisco (1991).
[13] S. Weiner, P. Kollman, D. Nyugen and D. Case, J. Camp. Chem., 7 (19860 230. [14] E. Honka, S. Fabry, T. Niermann, P. Palm and R. Hensel, Eur. J. Biochem., 188 (1990) 623. [15] C. Abad-Zapatero, J. Griffith, J. Sussman and M. Rossmann, J. Mol. Biol., 198 (1987) 445.