Structures, Properties, and Dynamics of Intermediates in eEF2

Article pubs.acs.org/jcim

Structures, Properties, and Dynamics of Intermediates in eEF2Diphthamide Biosynthesis Jean-Marc Billod,†,‡ Patricia Saenz-Mendez,†,§ Anders Blomberg,∥ and Leif A. Eriksson*,† †

Department of Chemistry and Molecular Biology and ∥Department of Marine Sciences, University of Gothenburg, 405 30 Göteborg, Sweden ‡ Department of Chemical and Physical Biology, Center for Biological Research, CIB-CSIC, 28040 Madrid, Spain § Computational Chemistry and Biology Group, Facultad de Química, Universidad de la República, 11800 Montevideo, Uruguay S Supporting Information *

ABSTRACT: The eukaryotic translation Elongation Factor 2 (eEF2) is an essential enzyme in protein synthesis. eEF2 contains a unique modification of a histidine (His699 in yeast; HIS) into diphthamide (DTA), obtained via 3-amino-3-carboxypropyl (ACP) and diphthine (DTI) intermediates in the biosynthetic pathway. This essential and unique modification is also vulnerable, in that it can be efficiently targeted by NAD+-dependent ADP-ribosylase toxins, such as diphtheria toxin (DT). However, none of the intermediates in the biosynthesis path is equally vulnerable against the toxins. This study aims to address the different susceptibility of DTA and its precursors against bacterial toxins. We have herein undertaken a detailed in silico study of the structural features and dynamic motion of different His699 intermediates along the diphthamide synthesis pathway (HIS, ACP, DTI, DTA). The study points out that DTA forms a strong hydrogen bond with an asparagine which might explain the ADP-ribosylation mechanism caused by the diphtheria toxin (DT). Finally, in silico mutagenesis studies were performed on the DTA modified protein, in order to hamper the formation of such a hydrogen bond. The results indicate that the mutant structure might in fact be less susceptible to attack by DT and thereby behave similarly to DTI in this respect.

■

are required.5,10−12 The second step involves the formation of the methyl ester of diphthine, a previously neglected intermediate, recently reported.13 This intermediate results from a tetramethylation process catalyzed by diphthine synthase Dph5 using SAM as the methyl donor, which differs from the trimethylation reaction originally proposed.14,15 It is an unusual reaction in that the promiscuous methyltransferase (Dph5) catalyzes both N-trimethylation and O-methylation on the same substrate.16 The third step is the formation of DTI by a novel demethylation reaction, which is required to convert methylated diphthine into DTI, the actual substrate for the final formation of DTA. This additional demethylation reaction requires Dph7.13 Finally, the carboxyl group of diphthine is amidated in an ATP-dependent process catalyzed by the synthesis factor Dph6.4,7−9,17,18 Once fully modified, DTA can however be efficiently targeted by NAD+-dependent ADP ribosylase toxins including the diphtheria toxin (DT), Pseudomonas aeruginosa exotoxin A, and the Vibrio cholera cholix toxin. Thus, DTA, the unique and necessary post-translationally modified histidine residue of eEF2, is also the site of ADP-ribosylation activity of such lethal

INTRODUCTION Eukaryotic translation Elongation Factor 2 (eEF2) is a GTPase that operates during protein synthesis in facilitating the movement of the peptidyl tRNA-mRNA complex from the A site of the ribosome to the P site, a process known as translocation. It plays a major role all along the synthesis, assisting in elongating the developing polypeptide chain by one amino acid at a time.1 eEF2 carries a ubiquitous yet unique modification of one of its histidine residues (His699 in yeast, His715 in mammals), into a diphthamide unit (DTA). The absence of the DTA modification in eEF2 has been associated with altered translational fidelity in both yeast and mammals,2,3 strongly suggesting that the DTA modification to eEF2 plays an important role in general cell physiology with implications on cell proliferation and development.4 Cells generate diphthamide (2-[3-carboxyamido-3trimethylamino)propyl]histidine) by modifying the specific histidine residue in a four-step biosynthetic pathway that involves seven enzymes (Dph1-Dph7).5−9 The first step is the transfer of the 3-amino-3-carboxypropyl (ACP) group from Sadenosylmethionine (SAM) to the imidazole ring of His (Figure 1) generating the ACP-modified intermediate of eEF2. To perform this step four diphthamide synthesis factors (Dph1 to Dph4) and the S-adenosyl methionine (SAM) methyl donor © 2016 American Chemical Society

Received: April 21, 2016 Published: August 15, 2016 1776

DOI: 10.1021/acs.jcim.6b00223 J. Chem. Inf. Model. 2016, 56, 1776−1786

Article

Journal of Chemical Information and Modeling

Figure 1. Four-step pathway to obtain diphthamide. Adapted with permission from ref 16. Copyright 2014 John Wiley & Sons Ltd.

Figure 2. Proposed mechanism of diphthamide modification catalyzed by DT. Adapted with permission from ref 21. Copyright 2005 Elsevier Ltd.

toxins.19 The reaction mechanism involves the transfer of an ADP-ribose moiety of NAD+ to the N3 atom of the diphtamide imidazole ring in eEF2 (Figure 2).20−22 The ADP-ribosylation probably follows an SN1 mechanism,23,24 where the nicotinamide moiety is first released and an oxocarbenium ion intermediate is formed, which is then attacked by the diphthamide nucleophile.25,26 ADP-ribosylation of eEF2 inhibits the translocation reaction, irreversibly inactivating the translation function of eEF2 and leading to cell death.20,27,28 Diphtheria toxin expression results

in accumulation of cells that fail to separate following mitosis.29 The importance of the diphthamide quaternary nitrogen is wellknown, as the unmethylated precursor (ACP) cannot be ADPribosylated by the toxins. Also, the trimethylated precursor DTI, lacking the amide group, is a substrate for inhibitory ADPribosylation, albeit at a slower rate than DTA.21,30,31 The name diphthamide clearly emphasizes the pathological target function to the diphtheria toxin, although its physiological role is unclear, despite the fact that DTA is present in EF2 of all eukaryotes and archae.32 Also, the 1777



■

evolutionary pathway that has led DT to ribosylate a posttranslationally modified site on eEF2 remains speculative.33 Even though the role of the DTA modification for the function of eEF2 is as yet not clearly established, it has been described that DTA binds adenines 1492 and 1493 in 16S/18S rRNA. These two adenines are universally conserved in rRNA and essential for tRNA recognition at the A site of the ribosome.25,34−36 Moreover, apparently the diphthamidemodified residue on eEF2 stabilizes a stacked conformation of the two bases. There is a similarity between the orientation of the diphtheria toxin and the helix containing the conserved adenine bases. Hence, ribosome mimicry by DT ensures recognition of eEF2. eEF2 from Saccharomyces cerevisiae is a 93 kDa single polypeptide chain composed of 842 amino acids. The structure was identified based on cryo-electron microscopy in complex with the 80S ribosome at 17.5 Å resolution as the counterpart of prokaryotic elongation factor EF-G.37,38 Employing X-ray crystallography, the structures of eEF2 alone and in complex with sordarin were solved at 2.9 and 2.1 Å resolution, respectively.39 The protein contains 6 domains, namely the G-domain (residues 2−218 and 329−345), G′-domain (residues 219−328), domain II (residues 346−481), domain III (residues 482−558), domain IV (residues 559−726 and 801−842), and domain V (residues 727−800).39 Upon visualizing the atomic arrangements in eEF2, it appears that three histidine residues located near the C-terminus of the protein, His583, His694 and His699 shown in Figure 3,40 are rather closely spaced and might form a “π-interacting triad”.

Article

METHODOLOGY

Protein Data and Homology Modeling. The protein structure of eEF2 from S. cerevisiae retrieved from the Protein Data Bank44 (PDB id 1N0V39) shows His699 in its unmodified form, and it has a missing loop from Val40 to Gly67 as shown in Figure 3, thus failing to properly describe the 3D structure of the entire protein although the amino acid sequence is wellknown. To obtain a complete eEF2 structure, the full amino acid sequence from yeast (Figure S1, Supporting Information) was employed to build a homology model 45,46 using YASARA.47,48 We used the sequence of eEF2 to perform a BLAST search for templates,49 followed by homology modeling of the loop using the crystal structures of the selected templates. Since we used several templates, alternative alignments were created for each template using a stochastic approach,50 and the models were built from these alignments. After side chains of the missing parts were built, the various loop structures were optimized by exploring a large number of conformations. Loops and side chains were then subjected to a combined steepest descent and simulated annealing minimization (i.e., backbone atoms kept fixed). Finally, a fully unrestrained simulated annealing minimization was run for the entire model. Details regarding the settings used in YASARA are included in Table S1 (Supporting Information). Molecular Dynamics Simulations. The composition of the four systems studied herein is described in Table S2 (Supporting Information). In each case, the protein was first centered in a periodic box with the box size adapted to extend 1.0 nm from the protein edges. Water molecules were added, and the counterion concentration was modified to have a charge-neutral system using the genion algorithm of Gromacs.51,52 The parameters used for the MD simulation are displayed in Table S3 (Supporting Information). The system was subjected to equilibration at T = 300 K and P = 1 bar, and a trajectory of 50 ns was run at the same conditions. The time step was set to 2 fs, using the Lincs algorithm to constrain all bonds53 and the leapfrog algorithm for integration.54 Electrostatic forces were treated using particle-mesh Ewald (PME) summation,55,56 and the cutoff was 10 Å both for electrostatics and van der Waals interactions. The simulation was performed with a velocity rescaling thermostat (a Noose-Hover thermostat)57,58 with a 0.1 ps coupling constant and a Parinello-Rahman barostat59 (NPT ensemble) with a 2.0 ps coupling constant. The Amber ff99sb60 force field was used throughout all molecular dynamics simulations, containing updated backbone ϕ and ψ torsions and improved side chain torsion angles.61 Including a modified amino acid in the system requires the calculation of new parameters and constants to be included in the force field. Determination of missing parameters in the ff99sb force field was performed through optimization of the particular system to a minimum using ground state Hartree− Fock calculations with a 6-31G(d) basis set. It was then possible to use antechamber,62 to extract the data from the output file and convert them into the Amber ff99 force field parameters required in Gromacs. All ab initio calculations reported herein were carried out employing Gaussian 09.63

Figure 3. eEF2, PDB id 1N0V,39 with an arrow pointing out the missing loop. The three histidine residues (His583, His694, and His699) are displayed in a ball-and-stick representation to the far left.

Molecular dynamics simulations of elongation factor of prokaryotic organisms (EF-G) have been described before.41,42 Structural analysis of eEF2 (with or without post-translational modifications) has been described mostly regarding the interaction between toxins and eEF2.43 With the aim of understanding structural characteristics and possibly providing further insight regarding the differential susceptibility toward bacterial toxins of unmodified eEF2, and on the intermediates leading to the diphthamide unit (ACP, DTI and DTA), we have carried out a combination of homology modeling, MD simulations, and in silico mutagenesis calculations. In addition, the structural information obtained assists in providing clues into the mechanisms of action of the different toxins attacking eEF2.

■

RESULTS AND DISCUSSION Multiple Template Homology Modeling. In the current case the homology modeling is simple since besides one loop (residues 40 to 67) and the first amino acid of the sequence, the

1778


Article


Figure 4. RMSD of all atoms of residue 699 in yeast EF2. The red bar indicates the separation between equilibration and production phase (cf. Figure S2).

yeast EF2 protein 3D structure is already known.39 A number of possible templates were identified by iterations to extract a position specific scoring matrix (PSSM) whereafter the PDB was searched for a match (i.e., hits with an E-value below the homology modeling cutoff defined in Table S1). YASARA identified 601 hits that were used as templates to build 15 3D models of the missing loop. The top ranked model of the missing loop is based on chain A of the template 3B78.64 Aiming to improve the accuracy of the final model, further refinement was achieved combining the best parts of different models, i.e. the lower quality regions of the top scoring model were iteratively replaced with corresponding high-quality fragments from other models. Once the homology model was finished, the YASARA program assessed the quality of the derived model through the estimation of the Z-score for the system. In the current case, only the missing loop of eEF2 was modeled, meaning that the quality score only takes this segment into account (Table S4, Supporting Information). The final, complete 3D model of eEF2 was used as the starting point for all the following calculations. Molecular Dynamics Simulations and Structural Analysis. RMSD. We have calculated the root-mean-square deviation (RMSD) of the four eEF2 systems studied, i.e., carrying unmodified-His (HIS), ACP, DTI, and DTA (Figure S2, Supporting Information). To this end, the structure of each frame was rotated and translated in order to minimize the RMSD to the reference structure (first frame), and then the deviation was calculated. HIS and DTI showed a rapid equilibration stage during the first 4 ns of the simulation after which the protein seemed to be stable, whereas ACP and DTA required a longer time to reach satisfactory stabilization. Hence, the first 4 ns of the HIS and DTI trajectories and the first 15 and 10 ns of the ACP and DTA ones, respectively, were not taken into account in the subsequent analyses. Using the same

method, it is possible to calculate RMSD for a certain part of the system (e.g. the unmodified/modified histidine residue). Thus, again the RMSD was minimized for the whole structure, and then the deviation was calculated for a single residue only. The RMSDs of the atomic positions of the atoms of residues His699, ACP699, DTI699, and DTA699 are shown in Figure 4. The graphs show that the modified His-residues suffer fluctuations, thus describing several possible atomic arrangements during the MD simulation. In particular, the RMSDs of ACP699 and DTI699 suggest the existence of two stable states that the atoms oscillate between. One could envisage that this is also the case for DTA699; however, the major fluctuations occur during the equilibration stage and the atoms in this modification remain very stable during the production phase. His699 of the unmodified eEF2 shows only a slight variation relating to one single state during the entire simulation trajectory. Distances. In order to explore the variability in the local structural organization of the “histidine triad”, His583, His694, and modified His699, the distance between the C3 carbon of the side chain in the modified residue 699 and carbon CE1 of the imidazole ring of His583 and His694 was analyzed (Figure 5A). We can observe (Figure 5B, plots I and III) that the distance between the modified His699 variants ACP699 and DTI699 and His583 oscillates between 0.5 and 1.5 nm, characteristic of the existence of two stable states for each structure. Less variation is observed between the ACP699 side chain and His694 (II), while the distances from DTI699 and DTA699 to His694 (IV, VI), and DTA699 to His583 (V), remain stable at 0.8−1.0 nm throughout the simulations. Taking a closer look at each His699 modification, some specific behavior can be identified. For ACP we observed the formation of two hydrogen bonds, the first one being formed 1779


Article


Figure 5. (A) Selected distances for the ACP, DTA, and DTI derivatives shown as dashed lines. (B) Fluctuation of the two distances outlined in part A over time. I, II: ACP699-HIS583/HIS694; III, IV: DTI699-HIS583/HIS694; V, VI: DTA699-HIS583/HIS694.

between nitrogen NE2 of the imidazole ring of His583 and one of the ammonium hydrogen atoms of ACP699. The other hydrogen bond is between hydrogen HE2 from His694 and one of the carboxylate oxygen atoms of the ACP699 side chain (Figure 6A). Since the two carboxylate oxygen atoms are equivalent, as are the three ammonium hydrogens, we plot in Figure 6B the distance from C4 to HE2 (His694) and from N3 to NE2 (His583) over time. Two separated states can be clearly identified for ACP699. Plots III and IV in Figure 6B, as well as Figures 7 and 8C, represent the amount of states counted each 0.005 nm distance step (distance density) in the production runs. The peak on the left of Figures 6B:III and 6B:IV characterize the hydrogen bonded state. The larger and wider distribution on the right is the range of distances where the ACP699 side chain is free to move. For DTI, unlike ACP and DTA, no hydrogen bond was identified but instead a structural cavity close to His583 preventing DTI699 to move during a certain time. Analyzing the same two distances represented by dashed lines in Figure

5A over time, the distributions shown in Figure 7 were obtained. The peak on the left in Figure 7A represents the amount of states where DTI699 is trapped in the cavity and the one to the right where DTI699 is free to move. As seen from Figure 7B, there is no correlation between the distance of DTI699 and His694. As seen in Figures 4 and 5, the RMSD and distance plots between the DTA substituent and the two unmodified His units indicate a very stable system. In this case, a hydrogen bond could be identified between the amide nitrogen of Asn581 and the amide carboxylic oxygen of the modified side chain (Figure 8A). This distinct and stable hydrogen bond between DTA699 (O4) and Asn581 (HND2) efficiently prevented diphthamide of moving freely during the simulation. Variation of the distance was plotted over time between the two atoms involved in the formed hydrogen bond (Figure 8B). RMSF. Calculating the Root Mean Square Fluctuations (RMSF) is a useful tool for characterizing local changes along the protein chain over time. As expected, the rebuilt missing 1780


Article


Figure 6. (A) Distances N3-NE2 and C4-HE2 between ACP699 and HIS583 (NE2) and HIS694 (HE2), respectively. Hydrogen bonds are represented in dashed lines, and hydrogen atoms not involved in hydrogen-bonding are not shown. (B) I and II represent the distance fluctuation over time in the production phase, and III and IV show the distance density related to I and II, respectively.

Figure 7. A and B represent the distance density of Plots III and IV of Figure 5B, respectively.

loop in the yeast EF2 structure between residues 40 and 67 displays significant motion relative to the starting structure; shown in Figure S3 (Supporting Information). This analysis explains the lack of resolved structure of the loop in the reported X-ray crystal structure. As can be seen in Figure 3, the modeled loop is spatially far away from the region we are interested in, and it is therefore not expected that the aspects studied herein will be influenced by the particular conformation of the loop.

Taking all the above data into account, several interesting observations can be made. The eEF2 with three unmodified histidine residues (i.e., His583, His694, and His699) does not display any significant variation in the structure during the entire simulation, meaning that only one stable conformation could be detected and no hydrogen bond is formed between the histidine residues. For ACP, the analyzed distances and RMSD reveal the existence of two stable states, between which the system oscillates. The predominant state is when the 1781


Article


Figure 8. (A) Interaction between DTA699 and Asn581. Arrow indicates the hydrogen bond stabilizing the side chain of DTA699. (B) Distance fluctuation over time between HND2 of Asn581 and O4 of DTA699. (C) Distance density related to 8B.

Asn581 as well as His/DTA699, His583, and His694 appear highly conserved through the eukaryotes, as shown in Figures S4 and S5 (Supporting Information). All sequences employed for the alignment were obtained from Uniprot,65 and the alignment was performed employing the Clustal Omega online tool.66 It is evident that Asn581 (Saccharomyces numbering) is highly conserved and, thus, likely to play a similar structural role in anchoring DTA also in other organisms. We have also observed, based on protein−protein docking and MD simulations (work in progress), that the only conformation of the modified histidine able to efficiently interact with DT is that where DTA is in the anchored position and interacting with Asn581. That is, DTA is being held in the proper conformation for being susceptible for attack by DT, while DTI, ACP, and HIS are not. In order to verify this hypothesis, we performed a specific in silico point mutation in eEF2, namely Asn581Ala, thereby removing the possibility for formation of the hydrogen bond between this residue and DTA. After the mutation was performed, an MD simulation was carried out using the same conditions as before. RMSD was calculated (Figure S6, Supporting Information), showing that the system was equilibrated after ca. 3 ns. To explore the mobility of the DTA side chain relating to the interaction with residue 581, the distance between DTA699 and residue 581 (Asn or Ala in the wild-type or mutant eEF2, respectively) was analyzed (Figure 9). Regarding the mutant, from 10 to 35 ns, the DTA side chain is mostly locked in the pocket next to residue 581, as in the wild-type protein. However, after 35 ns, the tail is free to move,

modified tail of ACP699 is free to move, and the other state is when the modified side chain is involved in a hydrogen bond formation with His583 (9.2% of the time), during part of which a hydrogen bond to His694 is also formed (7.7% of total simulation time). His583 is more likely to create bonds with ACP699 than His694 due to its higher mobility. Given that His694 and ACP699 are located only five residues apart on the same backbone segment, the formation of a hydrogen bond between them requires an unfavorable torsion of the backbone in order to place both residues within the proper distance for interaction. On the other hand His583 is located on a different region and thus able to get closer to ACP without inducing structural distortions. The case of DTI is unique in the sense that the DTI699 modified side chain does not form any hydrogen bond with His583 or His694. However, the DTI699 tail remains trapped in a structural cavity nearby His583 (47.8% of the time) without forming any hydrogen bond. This phenomenon possibly provides a favorable orientation explaining the minor ADP-ribosylation by the diphtheria toxin observed for this form. Finally, the fully modified histidine side chain (DTA699) is not influenced by the two histidine residues of the triad but interacts through a strong hydrogen bond with Asn581. The hydrogen bond is strong enough to keep the residue in this conformation throughout the simulation. This could provide an explanation to the vulnerability toward the formation of ADPribosyl-diphthamide by e.g. the diphtheria toxin, in that the hydrogen bond with Asn581 locks DTA in a static conformation, making it susceptible for attack. Moreover, 1782


Article


Figure 9. Fluctuation of the distance between residues DTA699 and Asn/Ala581. (A) Wild-type eEF2. (B) Mutated eEF2. (C) Selected snapshots of the two states represented by arrows 1 and 2 in (B), left: 25 ns (point 1) and right: 45 ns (point 2).

and a conformational change is observed. The situation is in the case of the mutant hence more similar to DTI, with two clear states, one bound/locked and one free. Representations of the two conformations are shown in Figure 9C. In order to provide a more reliable conclusion, we extended the MD simulation of the mutant to 150 ns (Figure S7; Supporting Information). The extended simulation clearly shows that the DTA side chain is changing back and forth between two different conformations, one locked (susceptible to toxins) and one free, similar to DTI; which may indicate that the Asn581Ala DTA mutant just like DTI may be less prone to be ADP-ribosylated than wild-type DTA.

It is well-known that ADP-ribosylation by the diphtheria toxin is only possible when residue 699 is a diphthamide and, to a lesser degree, when it is a diphthine. According to our results, this requirement might be due to a favorable orientation enabled by the formation of a strong hydrogen bond to Asn581 in the case of diphthamide and by a cavity restriction observed during part of the time for diphthine. These conclusions are supported by our in silico mutation studies on DTA. The mutation Asn581Ala hampered the formation of a hydrogen bond with DTA, allowing the side chain to alternate between two different conformations, as in DTI. This conformational change might protect DTA from being exposed to the diphtheria toxin for sufficient time in the position required to enable attack. These results could hence serve as a starting point for exploration of the mechanism that occurs with NAD+dependent ADP ribosylase toxins, e.g., using multiscale methodologies such as quantum mechanics/molecular mechanics (QM/MM). QM/MM is a layer-based approach to deal with the entire system, describing the reactive part at QM-level (electronic description), while the environment is classically represented (MM). The QM/MM approach is currently well developed and implemented, being acknowledged as a state-ofthe-art technique,67−74 and highly suitable to address the inhibition mechanism of eEF2 by ADP-ribosylating toxins.

■

CONCLUSIONS A particular histidine in eukaryotic translation Elongation Factor 2 (corresponding to His699 in yeast and His715 in mammals) can be modified into diphthamide following a fourstep pathway including three stable, isolable, and well-known intermediates (Figure 1). In the first step His699 incorporates a 3-amino-3-carboxypropyl into the imidazole ring to give the derivative ACP. Subsequently, the ACP amino group becomes trimethylated in a process involving two independent steps yielding diphthine (DTI). DTI is ultimately amidated giving the final diphthamide (DTA) modification of active eEF2.16 1783


Article


■

(11) Zhu, X.; Dzikovski, B.; Su, X.; Torelli, A. T.; Zhang, Y.; Ealick, S. E.; Freed, J. H.; Lin, H. Mechanistic Understanding of Pyrococcus horikoshii Dph2, a [4Fe−4S] Enzyme Required for Diphthamidebiosynthesis. Mol. BioSyst. 2011, 7, 74−81. (12) Liu, S.; Wiggins, J. F.; Sreenath, T.; Kulkarni, A. B.; Ward, J. M.; Leppla, S. H. Dph3, a Small Protein Required for Diphthamide Biosynthesis, is Essential in Mouse Development. Mol. Cell. Biol. 2006, 26, 3835−3841. (13) Lin, Z.; Su, X.; Chen, W.; Ci, B.; Zhang, S.; Lin, H. Dph7 Catalyzes a Previously Unknown Demethylation Step in Diphthamide Biosynthesis. J. Am. Chem. Soc. 2014, 136, 6179−6182. (14) Mattheakis, L. C.; Shen, W. H.; Collier, R. J. DPH5, a Methyltransferase Gene Required for Diphthamide Biosynthesis in Saccharomyces cerevisiae. Mol. Cell. Biol. 1992, 12, 4026−4037. (15) Zhu, X.; Kim, J.; Su, X.; Lin, H. Reconstitution of Diphthine Synthase Activity in Vitro. Biochemistry 2010, 49, 9649−9657. (16) Schaffrath, R.; Abdel-Fattah, W.; Klassen, R.; Stark, M. J. The Diphthamide Modification Pathway From Saccharomyces cerevisiae Revisited. Mol. Microbiol. 2014, 94, 1213−1226. (17) Abdel-Fattah, W.; Scheidt, V.; Uthman, S.; Stark, M.; Schaffrath, R. Insights into Diphthamide, Key Diphtheria Toxin Effector. Toxins 2013, 5, 958−968. (18) Moehring, J. M.; Moehring, T. J. The Post-Translational Trimethylation of Diphthamide Studied in Vitro. J. Biol. Chem. 1988, 263, 3840−3844. (19) Foley, B. T.; Moehring, J. M.; Moehring, T. J. Mutations in the Elongation Factor 2 Gene Which Confer Resistance to Diphtheria Toxin and Pseudomonas Exotoxin A: Genetic and Biochemical Analyses. J. Biol. Chem. 1995, 270, 23218−23225. (20) Oppenheimer, N. J.; Bodley, J. W. Diphtheria Toxin. Site and Configuration of ADP-Ribosylation of Diphthamide in Elongation Factor 2. J. Biol. Chem. 1981, 256, 8579−8581. (21) Yates, S. P.; Jørgensen, R.; Andersen, G. R.; Merrill, A. R. Stealth and Mimicry by Deadly Bacterial Toxins. Trends Biochem. Sci. 2006, 31, 123−133. (22) Iglewski, B. H.; Liu, P. V.; Kabat, D. Mechanism of Action of Pseudomonas aeruginosa Wxotoxin Aiadenosine Diphosphate-Ribosylation of Mammalian Elongation Factor 2 in vitro and in vivo. Infect. Immunol. 1977, 15, 138−144. (23) Beattie, B. K.; Merrill, A. R. In Vitro Enzyme Activation and Folded Stability of Pseudomonas aeruginosa Exotoxin A and Its CTerminal Peptide. Biochemistry 1996, 35, 9042−9051. (24) Armstrong, S.; Merrill, A. R. Toward the Elucidation of the Catalytic Mechanism of the Mono-ADP-Ribosyltransferase Activity of Pseudomonas aeruginosa Exotoxin A. Biochemistry 2004, 43, 183−194. (25) Jørgensen, R.; Merrill, A. R.; Yates, S. P.; Marquez, V. E.; Schwan, A. L.; Boesen, T.; Andersen, G. R. Exotoxin A−eEF2 Complex Structure Indicates ADP Ribosylation by Ribosome Mimicry. Nature 2005, 436, 979−984. (26) Parikh, S. L.; Schramm, V. L. Transition State Structure for ADP-Ribosylation of Eukaryotic Elongation Factor 2 Catalyzed by Diphtheria Toxin. Biochemistry 2004, 43, 1204−1212. (27) Van Ness, B. G.; Howard, J. B.; Bodley, J. W. ADP-Ribosylation of Elongation Factor 2 by Diphtheria Toxin. NMR Spectra and Proposed Structures of Ribosyl-Diphthamide and its Hydrolysis Products. J. Biol. Chem. 1980, 255, 10710−10716. (28) Sitikov, A. S.; Davydova, E. K.; Bezlepkina, T. A.; Ovchinnikov, L. P.; Spirin, A. S. Eukaryotic Elongation Factor 2 Loses its NonSpecific Affinity for RNA and Leaves Polyribosomes as a Result of ADP-Ribosylation. FEBS Lett. 1984, 176, 406−410. (29) Mateyak, M. K.; Kinzy, T. G. ADP-Ribosylation of Translation Elongation Factor 2 by Diphtheria Toxin in Yeast Inhibits Translation and Cell Separation. J. Biol. Chem. 2013, 288, 24647−24655. (30) Moehring, T. J.; Danley, D. E.; Moehring, J. M. In Vitro Biosynthesis of Diphthamide, Studied with Mutant Chinese Hamster Ovary Cells Resistant to Diphtheria Toxin. Mol. Cell. Biol. 1984, 4, 642−650. (31) Chen, J.-Y. C.; Bodley, J. W. Biosynthesis of Diphthamide in Saccharomyces cerevisiae. Partial Purification and Characterization of a

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jcim.6b00223. Figures S1−S7 and Tables S1−S4 (PDF)

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +46 31 786 9117. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS J.M.B. gratefully acknowledges the French Ministry of Higher Education and Research and the Erasmus Programme for financial support. P.S.M. received funding from the People Programme (Marie Curie Actions) of the European Union’s Seventh Framework Programme (FP7/2007-2013) under REA grant agreement no. 608743. L.A.E. gratefully acknowledges funding from the Swedish Research Council (Grant agreement 2014-3914) and the Faculty of Science at the University of Gothenburg. We also acknowledge the generous allocation of computing time at the C3SE supercomputing center, via the Swedish National Infrastructure for Computing (SNIC).

■

REFERENCES

(1) Andersen, G. R.; Nissen, P.; Nyborg, J. Elongation Factors in Protein Biosynthesis. Trends Biochem. Sci. 2003, 28, 434−441. (2) Ortiz, P. A.; Ulloque, R.; Kihara, G. K.; Zheng, H.; Kinzy, T. G. Translation Elongation Factor 2 Anticodon Mimicry Domain Mutants Affect Fidelity and Diphtheria Toxin Resistance. J. Biol. Chem. 2006, 281, 32639−32648. (3) Liu, S.; Bachran, C.; Gupta, P.; Miller-Randolph, S.; Wang, H.; Crown, D.; Zhang, Y.; Wein, A. N.; Singh, R.; Fattah, R.; Leppla, S. H. Diphthamide Modification on Eukaryotic Elongation Factor 2 is Needed to Assure Fidelity of Mrna Translation and Mouse Development. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 13817−13822. (4) Schaffrath, R.; Stark, M. J. R. Decoding the Biosynthesis and Function of Diphthamide, an Enigmatic Modification of Translation Elongation Factor 2 (EF2). Microbial Cell 2014, 1, 203−205. (5) Liu, S.; Milne, G. T.; Kuremsky, J. G.; Fink, G. R.; Leppla, S. H. Identification of the Proteins Required for Biosynthesis of Diphthamide, the Target of Bacterial ADP-Ribosylating Toxins on Translation Elongation Factor 2. Mol. Cell. Biol. 2004, 24, 9487−9497. (6) Uthman, S.; Liu, S.; Giorgini, F.; Stark, M. J. R.; Costanzo, M.; Schaffrath, R., Diphtheria Disease and Genes Involved in Formation of Diphthamide, Key Effector of the Diphtheria Toxin. In Insight and Control of Infectious Disease in Global Scenario; Roy, P. K., Ed.; 2012.10.5772/31680 (7) Su, X.; Chen, W.; Lee, W.; Jiang, H.; Zhang, S.; Lin, H. YBR246W is Required for the Third Step of Diphthamide Biosynthesis. J. Am. Chem. Soc. 2012, 134, 773−776. (8) Su, X.; Lin, Z.; Chen, W.; Jiang, H.; Zhang, S.; Lin, H. Chemogenomic Approach Identified Yeast Ylr143w as Diphthamide Synthetase. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 19983−19987. (9) Uthman, S.; Bar, C.; Scheidt, V.; Liu, S.; ten Have, S.; Giorgini, F.; Stark, M. J.; Schaffrath, R. The Amidation Step of Diphthamide Biosynthesis in Yeast Requires DPH6, a Gene Identified Through Mining the Dph1-Dph5 Interaction Network. PLoS Genet. 2013, 9, e1003334. (10) Zhang, Y.; Zhu, X.; Torelli, A. T.; Lee, M.; Dzikovski, B.; Koralewski, R. M.; Wang, E.; Freed, J.; Krebs, C.; Ealick, S. E.; Lin, H. Diphthamide Biosynthesis Requires an Organic Radical Generated by an Iron−Sulphur Enzyme. Nature 2010, 465, 891−896. 1784


Article


(51) Berendsen, H. J. C.; van der Spoel, D.; van Drunen, R. GROMACS: A Message-Passing Parallel Molecular Dynamics Implementation. Comput. Phys. Commun. 1995, 91, 43−56. (52) Lindahl, E.; Hess, B.; van der Spoel, D. GROMACS 3.0: a Package for Molecular Simulation and Trajectory Analysis. J. Mol. Model. 2001, 7, 306−317. (53) Hess, B.; Bekker, H.; Berendsen, H. J. C.; Fraaije, J. G. E. M. Lincs: a Linear Constraint Solver for Molecular Simulations. J. Comput. Chem. 1997, 18, 1463−1472. (54) Van Gunsteren, W. F.; Berendsen, H. J. C. A Leap-frog Algorithm for Stochastic Dynamics. Mol. Simul. 1988, 1, 173−185. (55) Darden, T.; York, D.; Pedersen, L. Particle Mesh Ewald: An N· log(N) Method for Ewald Sums in Large Systems. J. Chem. Phys. 1993, 98, 10089. (56) Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.; Pedersen, L. G. A Smooth Particle Mesh Ewald Method. J. Chem. Phys. 1995, 103, 8577. (57) Nosé, S. A Unified Formulation of the Constant Temperature Molecular Dynamics Methods. J. Chem. Phys. 1984, 81, 511. (58) Hoover, W. G. Canonical Dynamics: Equilibrium Phase-Space Distributions. Phys. Rev. A: At., Mol., Opt. Phys. 1985, 31, 1695−1697. (59) Parrinello, M. Polymorphic Transitions in Single Crystals: a New Molecular Dynamics Method. J. Appl. Phys. 1981, 52, 7182. (60) Wang, J.; Cieplak, P.; Kollman, P. A. How Well Does a Restrained Electrostatic Potential (RESP) Model Perform in Calculating Conformational Energies of Organic and Biological Molecules? J. Comput. Chem. 2000, 21, 1049−1074. (61) Lindorff-Larsen, K.; Piana, S.; Palmo, K.; Maragakis, P.; Klepeis, J. L.; Dror, R. O.; Shaw, D. E. Improved Side-Chain Torsion Potentials for the Amber ff99sb Protein Force Field. Proteins: Struct., Funct., Genet. 2010, 78, 1950−1958. (62) Wang, J. M.; Wang, W.; Kollman, P. A. Antechamber: an Accessory Software Package for Molecular Mechanical Calculations. Abstr. Pap. Am. Chem. S. 2001, 222, U403−U403. (63) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, B.; Mennucci, B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, X.; Hratchian, H. P.; Izmaylov, A. F.; Bloino, J.; Zheng, G.; Sonnenberg, J. L.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.; Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.; Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.; Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.; Cossi, M.; Rega, N.; Millam, N. J.; Klene, M.; Knox, J. E.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas, Ö .; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09; Gaussian, Inc.: Wallingford, CT, 2009. (64) Jørgensen, R.; Wang, Y.; Visschedyk, D.; Merrill, A. R. The Nature and Character of the Transition State for the ADPRibosyltransferase Reaction. EMBO Rep. 2008, 9, 802−809. (65) The Uniprot Consortium. UniProt: a hub for protein information. Nucleic Acids Res. 2015, 43, D204−D212. (66) Sievers, F.; Wilm, A.; Dineen, D.; Gibson, T. J.; Karplus, K.; Li, W.; Lopez, R.; McWilliam, H.; Remmert, M.; Soding, J.; Thompson, J. D.; Higgins, D. G. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 2011, 7, 539−545. (67) Warshel, A. Computer Modeling of Chemical Reactions in Enzymes and Solutions; John Wiley and Sons: New York, 1997. (68) Warshel, A.; Levitt, M. Theoretical studies of enzymatic reactions: dielectric, electrostatic and steric stabilization of the carbonium ion in the reaction of lysozyme. J. Mol. Biol. 1976, 103, 227−249. (69) Warshel, A.; Sharma, P. K.; Kato, M.; Xiang, Y.; Liu, H.; Olsson, M. H. M. Electrostatic Basis for Enzyme Catalysis. Chem. Rev. 2006, 106, 3210−3235.

Specific S-Adenosyl-methionine:elongation Factor 2 Methyltransferase. J. Biol. Chem. 1988, 263, 11692−11696. (32) Phan, L. D.; Perentesis, J. P.; Bodley, J. W. Saccharomyces cerevisiae Elongation Factor 2. J. Biol. Chem. 1993, 268, 8665−8668. (33) Collier, R. J. Understanding the Mode of Action of Diphtheria Toxin: a Perspective on Progress During the 20th Century. Toxicon 2001, 39, 1793−1803. (34) Khade, P. K.; Shi, X.; Joseph, S. Steric Complementarity in the Decoding Center is Important for tRNA Selection by the Ribosome. J. Mol. Biol. 2013, 425, 3778−3789. (35) Ramrath, D. J. F.; Lancaster, L.; Sprink, T.; Mielke, T.; Loerke, J.; Noller, H. F.; Spahn, C. M. T. Visualization of two Transfer RNAs Trapped in Transit During Elongation Factor G-Mediated Translocation. Proc. Natl. Acad. Sci. U. S. A. 2013, 110, 20964−20969. (36) Taylor, D. J.; Nilsson, J.; Merrill, A. R.; Andersen, G. R.; Nissen, P.; Frank, J. Structures of Modified eEF2. 80s Ribosome Complexes Reveal the Role of GTP Hydrolysis in Translocation. EMBO J. 2007, 26, 2421−2131. (37) Gomez-Lorenzo, M. G.; Spahn, C. M. T.; Agrawal, R. K.; Grassucci, R. A.; Penczek, P.; Chakraburtty, K.; Ballesta, J. P. G.; Lavandera, J. L.; Garcia-Bustos, J. F.; Frank, J. Three-Dimensional Cryo-Electron Microscopy Localization of EF2 in the Saccharomyces cerevisiae 80s Ribosome at 17.5 A Resolution. EMBO J. 2000, 19, 2710−2718. (38) Spahn, C. M. T.; Gomez-Lorenzo, M. G.; Grassucci, R. A.; Jorgensen, R.; Andersen, G. R.; Beckmann, R.; Penczek, P. A.; Ballesta, J. P. G.; Frank, J. Domain Movements of Elongation Factor eEF2 and the Eukaryotic 80S Ribosome Facilitate tRNA Translocation. EMBO J. 2004, 23, 1008−1019. (39) Jorgensen, R.; Ortiz, P. A.; Carr-Schmid, A.; Nissen, P.; Kinzy, T. G.; Andersen, G. R. Two Crystal Structures Demonstrate Large Conformational Changes in the Eukaryotic Ribosomal Translocase. Nat. Struct. Biol. 2003, 10, 379−385. (40) Perentesis, J. P.; Phan, L. D.; Gleason, W. B.; LaPorte, D. C.; Livingston, D. M.; Bodley, J. W. Saccharomyces cerevisiae Elongation Factor 2. Genetic Cloning, Characterization of Expression, and GDomain Modeling. J. Biol. Chem. 1992, 267, 1190−1197. (41) Li, W.; Trabuco, L. G.; Schulten, K.; Frank, J. Molecular Dynamics of EF-G During Translocation. Proteins: Struct., Funct., Genet. 2011, 79, 1478−1486. (42) Wieden, H. J.; Mercier, E.; Gray, J.; Steed, B.; Yawney, D. A Combined Molecular Dynamics and Rapid Kinetics Approach to Identify Conserved Three-Dimensional Communication Networks in Elongation Factor Tu. Biophys. J. 2010, 99, 3735−3743. (43) Armstrong, S.; Yates, S. P.; Merrill, A. R. Insight into the Catalytic Mechanism of Pseudomonas aeruginosa Exotoxin A. Studies of Toxin Interaction with Eukaryotic Elongation Factor-2. J. Biol. Chem. 2002, 277, 46669−46675. (44) Protein Data Bank. http://www.rcsb.org/pdb/home/home.do (accessed July 25, 2016). (45) Kaczanowski, S.; Zielenkiewicz, P. Why Similar Protein Sequences Encode Similar Three-Dimensional Structures? Theor. Chem. Acc. 2010, 125, 643−650. (46) Krieger, E.; Nabuurs, S. B.; Vriend, G. Homology Modeling. In Struct. Bioinfo.; Bourne, P. E., Weissig, H., Eds.; Wiley-Liss, Inc.: 2003; pp 507−521. (47) Krieger, E. Yet Another Scientific Artificial Reality Application (YASARA); 2004. (48) Krieger, E.; Joo, K.; Lee, J.; Lee, J.; Raman, S.; Thompson, J.; Tyka, M.; Baker, D.; Karplus, K. Improving Physical Realism, Stereochemistry, and Side-Chain Accuracy in Homology Modeling: Four Approaches that Performed Well in CASP8. Proteins: Struct., Funct., Genet. 2009, 77, 114−122. (49) Altschul, S. F.; Madden, T. L.; Schäffer, A. A.; Zhang, J.; Zhang, Z.; Miller, W.; Lipman, D. J. Gapped BLAST and PSI-BLAST: a New Generation of Protein Database Search Programs. Nucleic Acids Res. 1997, 25, 3389−3402. (50) Mückstein, U.; Hofacker, I. L.; Stadler, P. F. Stochastic Pairwise Alignments. Bioinformatics 2002, 18, S153−S160. 1785


Article

Journal of Chemical Information and Modeling (70) Olsson, M. H. M.; Siegbahn, P. E. M.; Blomberg, M. R. A.; Warshel, A. Exploring pathways and barriers for coupled ET/PT in cytochrome c oxidase: A general framework for examining energetics and mechanistic alternatives. Biochim. Biophys. Acta, Bioenerg. 2007, 1767, 244−260. (71) Adamczyk, A. J.; Cao, J.; Kamerlin, S. C. L.; Warshel, A. Catalysis by dihydrofolate reductase and other enzymes arises from electrostatic preorganization, not conformational motions. Proc. Natl. Acad. Sci. U. S. A. 2011, 108, 14115−14120. (72) Frushicheva, M. P.; Cao, J.; Warshel, A. Challenges and Advances in Validating Enzyme Design Proposals: The Case of Kemp Eliminase Catalysis. Biochemistry 2011, 50, 3849−3858. (73) Marti, S.; Andres, J.; Moliner, V.; Silla, E.; Tuñon, I.; Bertran, J. Predicting an Improvement of Secondary Catalytic Activity of Promiscuos Isochorismate Pyruvate Lyase by Computational Design. J. Am. Chem. Soc. 2008, 130, 2894−2895. (74) Repič, M.; Vianello, R.; Purg, M.; Duarte, F.; Bauer, P.; Kamerlin, S. C. L.; Mavri, J. Empirical valence bond simulations of the hydride transfer step in the monoamine oxidase B catalyzed metabolism of dopamine. Proteins: Struct., Funct., Genet. 2014, 82, 3347−3355.

1786


Structures, Properties, and Dynamics of Intermediates in eEF2

Recommend Documents