Conformational flexibility of interdomain linker in bovine tyrosyl-tRNA synthetase studied by molecular dynamics simulation

Here we report a study of molecular dynamics of a YCD2 fragment of mammalian tyrosyl-tRNA synthethase (Asp322-Ser528), which includes the COOH-terminal cytokine-like domain, intermodular flexible linker, and H5-a-helix of catalytic core of synthetase. Our calculations show that while compact C-terminal domain was less flexible and relatively stable, the interdomain linker shows a high degree of conformational changes. After short relaxation time it forms a short helix-like structure, which may be involved in the regulation of domain interaction and modulation of protein activities.

Introduction.Bovine (Bos taurus) tyrosyl-tRNA syn thetase (TyrRS, EC 6.1.1.1)is one of the most studied mammalian aminoacyl-tRNA synthetases [1 ].So far, the full-length synthetase has not been crystallized and its experimental 3D structure has not been reported.Difficulties of obtaining crystals can be caused by presence of a labile peptide linker between N-terminal catalytic core and C-terminal domain.Mobility and flexibility of this linker may be required for adaptable orientation of domains necessary for tRNA aminoacylation reaction.The lack of expe rimentally determined structure justifies the use of computational methods for the study of TyrRS spatial composition and temporal behavior.Hence, the main aim of this work is to study the structure of inter domain linker and its conformational flexibility using molecular dynamics (MD) simulation techniques.
Bovine TyrRS exists as a homodimer formed by two 59.2 kDa subunits of 528 amino acid residues each.N-terminal catalytic core and C-terminal do main of a subunit are joined by a peptide linker (Fig. 1).N-terminal module forms a minimal 39 kDa TyrRS which reveals full catalytic activity in vitro [ 1,2].C-terminal domain is dispensable for catalytic activity of mammalian TyrRS [3].Presence of this domain is not a ubiquitous feature among TyrRSs.Up to date, it is found in human, mouse, rat, zebra fish, and fruit fly.There is no equivalent of C-domain in TyrRS of invertebrates (nematode Caenorhabditis elegans), plants, lower eukaryotes, and Archaebacteriae.This suggests that it has been attached to the TyrRS of chordates and insects common ancestor later in evolution.TyrRSs from Eubacieriac have similar composition, but their C-terminal domains have little or no homology to their counterpart in higher eukaryotes or insects, and probably emerged as a result of evolutionary convergence [4 ].A multiple alignment of C-domains guided by predicted secon dary structure revealed two independent sub-domains [3]: a /ї-pleated part (OB-fold, residues Val363- Lys470) and an a-helical A-subdomain (residues Gly471-Ser528, Fig. 1) [3,5].The TyrRS C-domain reveals 52.7 % identity to a mammalian cytokine (endothelial and monocyte activating polypeptide, EMAP II) [6,7], which activates both monocytes and endothelial cells -an effect first discovered in chemi cals induced cancerogenesis [8,9].EMAP II is a product of p43 (proEMAP II) cleavage.p43 is a non-synthetase protein involved in high-molecular weight multi-tRNA synthetases complex formation in higher eukaryotes [10].p43 interacts with ArgRS directly and modulates its aminoacylation activity [11].It has been experimentally shown that stressinduced cleavage of TyrRS by an unknown, possibly thiol protease results in acquisition of cytokine-like activity [12][13][14].In vitro experiments show that TyrRS C-domain induces a twofold increase of mono cyte chemotaxis and enhancement of human tissue factor expression [12][13][14].The described effects of the C-domain are similar to those exhibited by EMAP II cytokine.
Several organisms possess C-terminal domains homologous to that of TyrRS.There are experimental data showing involvement of Arclp (G4pl) from Saccharomyces cerevisiae (55.3 % identity) [15], human p43 (pro-EMAP II) (62.7 %) [3], and ARCE from Euplotes octocarinatus (52 %) [16] in non specific tRNA binding.These proteins direct tRNA to the active sites of corresponding ARSases [15,17].It is possible, that during the evolution C-terminal domain was transferred to several diverse proteins involved in translation (such as TyrRS, MetRS, p43, and Arclp) to enable their proper functioning in higher eukaryotes protein synthesis apparatus [3 ].
Bovine C-domain contributes about 50 % of TyrRS affinity to ribosomal RNAs [18].This binding has a certain specificity: among others poly(G) had the most inhibitory effect in the reaction of tRNA Tyr aminoacylation [18].In chemical modification expe riments TyrRS protected anticodon of tRNA Tyr from N-nitroso-N-ethylurea modification.Hence, the anti codon region is involved in tRNA Tyr recognition by mammalian TyrRS [19].
Despite all the experimental information gathered up to date, there is no clear understanding of C-domain and interdomain linker role and mode of action.Significance of its relation to both cytokines and tRNA-binding domains is unclear and needs explanation.The absence of experimentally derived structure and importance of studying the role of eukaryotic cytokines justify computational approach to TyrRS study.In the light of recent theories and observations concerning functions of flexible inter domain linkers [20] we are especially interested in gathering detailed information about temporal be havior of TyrRS linker to make an attempt of understanding its functioning in the whole protein.
Materials and Methods.The amino acid sequence of bovine TyrRS was reported earlier [21,22] and can be downloaded from Entrez (http://www.ncbi.nlm.nih.gov/entrez/),accession number Q29465.To build the model of the three-dimensional structure of TyrRS we used homology modeling techniques [23 ].Earlier, we performed homology modeling of 3D structures of N-and C-terminal domains of bovine TyrRS separately [5,24] using two PDB structures (1N3L, 95 % homology and 1NTG_A, 92 %) as templates.The putative conformation of bovine TyrRS interdomain linker was modeled using the C-Abl tyrosine protein kinase structure (10PL_A).Its frag ment connecting SH2 and SH3 domains has an unexpected sequence homology (57 % homology, 32 % identity) to bovine TyrRS interdomain linker (Fig. 2).The exact modeling procedure of the fulllength bovine TyrRS will be published elsewhere.
For molecular dynamics calculations we employed GROMACS 3.1.4with GROMOS96 force field [25,26].We used a virtual octahedral box.Minimal distance from its walls to the protein molecule was 1.0 nm.The box was filled with 16458 SPC-models of water molecules.A total protein charge of +6 was compensated by 38 sodium and 44 chloride ions replacing equal number of water molecules to simulate ionic strength of 0.15 M.
Algorithms of steepest descent and conjugated gradient were used to do original minimization of system energy.Minimal energy of 200 kJ/mol was attained after 3421 steps.We equilibrated water molecules during 100 ps, while keeping proteins coordinates bound to the box with the aid of ad- YCD2 fragment was experimentally obtained earlier as a protein having cytokine-like activities and RNA binding ability [28 ].In further discussions we use the numbering system starting from the first amino acid of YCD2, which corresponds to Asp322 of the full length TyrRS.We ran two 1875 and 2000 ps MD simulations, respectively.For further analysis one of them (1875 ps) was selected.Both MD simulation experiments display the same pattern of linker behavior.
Fig. 2 shows an alignment of the initial structure of YCD2 and its structure after 1875 ps of MD simulation.It may be pointed out that both Nterminal fragment and linker region (Pro21-Glu40) are the most flexible and reveal higher deviations from the initial structure.
Fig. 3 represents time course of C a -atoms RMSD.It increased up to 0.4 nm during first 400 ps quickly.Slower increase during the next 1000 ps brings RMSD to 0.5 nm.After that the system shows low amplitude fluctuations between 0.5 and 0.6 nm.This cor responds to the original re-arrangement and sta bilization of the structure, while the last stage reflects small system fluctuations.We suggest that the largest changes are contributed by more flexible linker area.To prove this assumption, we factored out and analyzed RMSD changes contributed by the linker only.The results can be seen in Fig. 4. It is clear that the RMSDs of the area between Asp і (322) and Glu40 (362) (C-terminal part of N-domain and the linker) show higher values (up to 0.6 nm) comparing to those of the rest of YCD2 (0.25 nm).This supports our hypothesis about high impact of the linker on overall protein mobility.hydrophilic and hydrophobic components.These are as decreased significantly during first 1.5 ns of the experiment, and then increased by a small amount.This behavior suggests compactization and stabili zation of the protein structure in the course of dynamics.
A number of hydrogen bonds formed by protein atoms displays the following trend: it drops after first 50 ps (protein relaxation), and increases significantly by the end of MD trajectory.For the whole YCD2 protein their number drops to 140, then rises to 155 and later fluctuates around this value (Fig. 6, a).The area Asp21-Val68 (343-389) displays a higher percentwise change comparing to the rest of YCD2from 18 to 25 (Fig. 6, b).The number of hydrogen bonds is a good indication of secondary structure formation.To get more insight into changes in secondary structure of our protein, we plotted a percentile of time a residue stays in helical con formation (Fig. 7).It is clear, that residues 1-20 Analysis of root-mean square fluctuations (RMSF) of C a -atoms, presented in Fig. 8, shows that N-terminal part of YCD2 and the linker area are most labile (0.3 nm), while C-domain residues remain relatively stable.We think that the linker undergoes compactization as a result of hydrogen bond for-RMSF, nm 0,5 -If mation.This leads to reduction of solvent accessible area.
The peculiarities of linker behavior -change in flexibility by formation of short helical structuremay suggest that it may be involved in regulation of TyrRS activity.Such behavior was observed in many proteins, where short interdomain connectors play an important role in function modulation [20].It is noteworthy that X-ray crystallographic studies of TyrRS from Thermus thermophilus revealed a for mation of an additional turn of helix in the flexible linker between N-terminal and C-terminal parts of the enzyme after its binding with cognate tRNA Tyr [29].
Acknowledgements.This research was supported by grant N 5.07/200 from the Ministry of Science and Education of Ukraine.

Fig. 1 .
Fig. 1.Domain organi zation of a single subunit of Bos taurus cytoplasmic TyrRS

Fig. 4 .
Fig. 4. Time course of RMSD (nm) of the linker area (upper curve), and the remaining part of YCD2 (lower curve)