1. Valyl-tRNA Synthetase Josh Jarodsky http://www.rcsb.org/pdb/education_discussion/molecule_of_the_month/images/aaRS.gif

2. General Information Valyl-tRNA Synthetase = ValRS ValRS is an enzyme E.C. 6.1.1.9 PDB: 1IVS Seen in all Domains of Life: Archaea – Haloferax volcanii Bacteria - Escherichia Coli Eukaryote - Homo Sapiens, Saccharomyces cerevisiae Important for translation of mRNA for protein synthesis PDB: 1IVS Valine-tRNA Ligase, BRENDA Enzyme Database, http://www.brenda-enzymes.org/enzyme.php?ecno=6.1.1.9

3. Reaction of ValRS Binds L-Valine & tRNA(Val) to form Valyl-tRNA ATP cofactor http://blopig.com/blog/wp-content/uploads/2013/04/Translation.jpghttp://blopig.com/blog/wp-content/uploads/2013/04/Translation.jpgModified version of http://www.bio.davidson.edu/genomics/2005/Drysdale/MFYG.htmlhttp://www.bio.davidson.edu/genomics/2005/Drysdale/MFYG.html

4. Structure: PDB ID: 1IVS ValRS+AMS+tRNA(Val) Asymmetric Homodimer o 2 Subunits o 4 Domains per sub unit Large crescent shaped binding pocket 37 Alpha helices; 380 residues 42 Beta strands; 129 residues PDB: 1IVS Asymmetric Mirror Plane Secondary Structure:

5. Structure: ValRS+Val+AMS PDB: 1IVS ValRS L-Valine+AMS

6. Structure: ValRS+tRNA(Val) PDB: 1IVS tRNA(Val) ValRS

7. Structure: ValRS+tRNA(Val)+ATP+L-Valine PDB: 1IVS

8. Primary Structure and Amino Acid Sequence Alignment Comparison of 10 Organisms Large amounts of conservation: ATP binding site L-Valine binding site tRNA(Val) anticodon binding sites tRNA(Val) binding sites etc http://www.brenda-enzymes.org/sequences.php?f[stype_ec]=1&f[ec]=6.1.1.9

9. Interesting Conserved Amino Acids Lysine, Methionine, Serine, Lysine, Serine Characteristic of Catalytic site of aminoacyl-tRNA synthetases Hountondji, C. et al. (2002) Crucial Role of Conserved Lysine 277 in the Fidelity of tRNA Aminoaclation by Escherichia coli Valyl-tRNA Synthetase. BioChem, 41, 14856-14865.

10. Lys, Met, Ser, Lys, Ser PDB: 1IVS Lys528-Ser532 L-Valine + AMS (Yellow) tRNA (Orange) Ser530 Lys531 Lys528 Met533 Ser532

11. Binding Site: ValRS+tRNA(Val) PDB: 1IVS tRNA(Val)

12. Binding Site: ValRS+tRNA(Val) PDB: 1IVS Arg570 – tRNA Backbone interaction CCA Terminal Binding Anti-Codon Interactions Fukai, S. et al. (2000) Structural Basis for Double-Sieve Discrimination of L-Valine from L-Isoleucine and L-Threonine by the Comple of tRNA Val and Valyl-tRNA Synthetase. Cell, 103, 793-803.

13. CCA Terminal Interactions: C974(Grey) –side chain interactions with: Glu261,281 (Magenta) Leu278 (Red) C975(Grey) –”Contacts with:” Glu261 (Magenta) Phe264 (Blue) A976(Grey)- H-Bond with: Tyr337 (Green) A976 (Grey)-Sandwiched between: Leu269 (Red) Phe264 (Blue) Binding Site: CCA Terminal PDB: 1IVS Fukai, S. et al. (2000) Structural Basis for Double-Sieve Discrimination of L-Valine from L-Isoleucine and L-Threonine by the Comple of tRNA Val and Valyl-tRNA Synthetase. Cell, 103, 793-803. Glu281 Leu278 Leu269 Phe264 Glu261 Tyr337

14. Anticodon Interactions Asn584 o H-Bonds with hydroxyl groups on: C933 A934 o Side Chain interactions with: Phe588 (Blue) Leu650 (Red) Cys646 (Yellow) C935 o Hydrogen Bonding N3 to Lys581 Phe588 PDB: 1IVS Anti-Codon: CAC Minor Groove Interactions Fukai, S. et al. (2000) Structural Basis for Double-Sieve Discrimination of L-Valine from L-Isoleucine and L-Threonine by the Comple of tRNA Val and Valyl-tRNA Synthetase. Cell, 103, 793-803. Lys581 Asn584 Phe588 Cys646 Leu650

15. Interesting Conserved Amino Acids Lysine270 Involved in editing Edits by nucleophilic attack Second part of the “Double Sieve” Hountondji, C. et al. (2002) Crucial Role of Conserved Lysine 277 in the Fidelity of tRNA Aminoaclation by Escherichia coli Valyl-tRNA Synthetase. BioChem, 41, 14856-14865.

16. Lysine270 tRNA Interaction PDB: 1IVS Lys270

17. Lysine270 tRNA Interaction Lys270 (Yellow) Up and Down stream ValRS AA (Green) C975 (Red) Down stream tRNA Nucleic Acids (Orange) Interaction: o Amine group of Lys270 would preform a nucleophilic attack on improper amino acid PDB: 1IVS Lys270 C975 Hountondji, C. et al. (2002) Crucial Role of Conserved Lysine 277 in the Fidelity of tRNA Aminoaclation by Escherichia coli Valyl-tRNA Synthetase. BioChem, 41, 14856-14865

18. Double Sieve Fukai, S. et al. (2000) Structural Basis for Double-Sieve Discrimination of L-Valine from L-Isoleucine and L- Threonine by the Comple of tRNA Val and Valyl-tRNA Synthetase. Cell, 103, 793-803. Valine Threonine Isoleucine

19. ValRS Proofreading and Editing Pathways Gruic-Sovulj, I. et al. (2007) Hydrolysis of non-cognate aminoacyl-adenylates by a class II aminoacyl- tRNA synthetase lacking an editing domain. FEBS Letters, 581, 26, 5110-5114

20. Why should we care? Without ValRS: Valine would not be added to any protein sequence This would change proteins sequence leading to: Non-native conformations Malfunctioning protein Nonfunctional protein Malfunctioning ValRS: This would change proteins sequence leading to: Non-native conformations Malfunctioning protein Nonfunctional protein All of which could result in the death of the affected organism

21. Conclusion Valyl-tRNA Synthetase is an important enzyme o Seen in all domains of life o Large sections of conserved amino acids Structure o Asymmetric homodimer o Large crescent shape o Mainly alpha helices Binding o L-Valine and ATP via KMSKS o tRNA Backbone Minor groove CCA terminal Anti-codon Editing o Multiple pathways of editing pre and post transfer of tRNA o Lysine270 Why its Important? o Accurate translation of mRNA for correct Protein sequence o Proper conformations and functionality

22. Questions? http://www.rcsb.org/pdb/education_discussion/molecule_of_the_month/images/aaRS.gif

23. Work Cited 1http://www.rcsb.org/pdb/education_discussion/molecule_of_the_month/ima ges/aaRS.gifhttp://www.rcsb.org/pdb/education_discussion/molecule_of_the_month/ima ges/aaRS.gif 2PDB 1IVS 3http://blopig.com/blog/wp-content/uploads/2013/04/Translation.jpghttp://blopig.com/blog/wp-content/uploads/2013/04/Translation.jpg 4http://www.bio.davidson.edu/genomics/2005/Drysdale/MFYG.htmlhttp://www.bio.davidson.edu/genomics/2005/Drysdale/MFYG.html 5 http://www.brenda- enzymes.org/sequences.php?f[stype_ec]=1&f[ec]=6.1.1.9 http://www.brenda- enzymes.org/sequences.php?f[stype_ec]=1&f[ec]=6.1.1.9 6Hountondji, C.; Lazennec, C.; Beauvallet, C.; Dessen, P.; Pernollet, J.; Plateau, P.; Blanquet, S. (2002) Crucial Role of Conserved Lysine 277 in the Fidelity of tRNA Aminoaclation by Escherichia coli Valyl-tRNA Synthetase. BioChem, 41, 14856-14865. 7Fukai, S.; Nureki, O.; Sekine, S.; Shimada, A.; Tao, J.; Vassylyev, D.; Yokoyama, S. (2000) Structural Basis for Double-Sieve Discrimination of L-Valine from L- Isoleucine and L-Threonine by the Comple of tRNA Val and Valyl-tRNA Synthetase. Cell, 103, 793-803. 8Gruic-Sovulj, I., Rokov-Plavec, J., Weygand-Durasevic, I. (2007) Hydrolysis of non-cognate aminoacyl-adenylates by a class II aminoacyl-tRNA synthetase lacking an editing domain. FEBS Letters, 581, 26, 5110-5114