Valyl-tRNA Synthetase

Josh Jarodskyhttp://www.rcsb.org/pdb/education_discussion/molecule_of_the_month/images/aaRS.gif

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: 1IVSValine-tRNA Ligase, BRENDA Enzyme Database, http://www.brenda-enzymes.org/enzyme.php?ecno=6.1.1.9

Reaction of ValRS

• Binds L-Valine & tRNA(Val) to form Valyl-tRNA

• ATP cofactor

http://blopig.com/blog/wp-content/uploads/2013/04/Translation.jpg Modified version of http://www.bio.davidson.edu/genomics/2005/Drysdale/MFYG.html

Structure:• PDB ID: 1IVS• ValRS+AMS+tRNA(Val)• Asymmetric Homodimer

o 2 Subunitso 4 Domains per sub unit

• Large crescent shaped binding pocket

• 37 Alpha helices; 380 residues

• 42 Beta strands; 129 residues

PDB: 1IVSAsymmetric Mirror Plane

Secondary Structure:

PDB: 1IVS

Structure: ValRS+Val+AMS

ValRS

L-Valine+AMS

PDB: 1IVS

Structure:ValRS+tRNA(Val)

tRNA(Val)

ValRS

Structure:ValRS+tRNA(Val)+ATP+L-

Valine

PDB: 1IVS

Primary Structure

and Amino Acid

Sequence

Alignment

Accession Number Organism A6L3G7 Bacteroides vulgatus Q9PK91 Chlamydia muridarum Q82ZW6 Enterococcus faecalis Q74JZ8 Lactobacillus johnsonii Q4A917 Mycoplasma hyopneumoniae Q3A253 Pelobacter carbinolicus A3PHJ6 Rhodobacter sphaeroides Q7X2N3 Sphingomonas elodea P0DG64 Streptococcus pyogenes serotype M3 P46216 Trichomonas vaginalis

• 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

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.

Lys, Met, Ser, Lys, Ser

PDB: 1IVS

• Lys528-Ser532

• L-Valine + AMS (Yellow)• tRNA (Orange)

Ser530

Lys531Lys528

Met533

Ser532

PDB: 1IVS

Binding Site:ValRS+tRNA(Val)

tRNA(Val)

Binding Site:ValRS+tRNA(Val)

PDB: 1IVS

Arg570 – tRNA Backbone interaction

CCATerminalBinding

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 tRNAVal and Valyl-tRNA Synthetase. Cell, 103, 793-803.

• 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: 1IVSFukai, S. et al. (2000) Structural Basis for Double-Sieve Discrimination of L-Valine from L-Isoleucine and L-Threonine by the Comple of tRNAVal and Valyl-tRNA Synthetase. Cell, 103, 793-803.

Glu281

Leu278

Leu269

Phe264 Glu261

Tyr337

Anticodon Interactions

• Asn584 o H-Bonds with hydroxyl groups on:

• C933• A934

• A934o Side Chain interactions with:

• Phe588 (Blue)• Leu650 (Red)• Cys646 (Yellow)

• C935o Hydrogen Bonding

• N3 to Lys581• Phe588PDB: 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 tRNAVal and Valyl-tRNA Synthetase. Cell, 103, 793-803.

Lys581

Asn584

Phe588

Cys646

Leu650

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.

PDB: 1IVS

Lysine270 tRNA Interaction

Lys270

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

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 tRNAVal and Valyl-tRNA Synthetase. Cell, 103, 793-803.

Valine

Threonine

Isoleucine

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

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

Conclusion• Valyl-tRNA Synthetase is an important enzyme

o Seen in all domains of lifeo Large sections of conserved amino acids

• Structureo Asymmetric homodimero Large crescent shapeo Mainly alpha helices

• Bindingo L-Valine and ATP via KMSKSo tRNA

• Backbone• Minor groove• CCA terminal• Anti-codon

• Editingo Multiple pathways of editing pre and post transfer of tRNAo Lysine270

• Why its Important?o Accurate translation of mRNA for correct Protein sequenceo Proper conformations and functionality

Questions?

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

Work Cited1 http://

www.rcsb.org/pdb/education_discussion/molecule_of_the_month/images/aaRS.gif2 PDB 1IVS3 http://blopig.com/blog/wp-content/uploads/2013/04/Translation.jpg4 http://www.bio.davidson.edu/genomics/2005/Drysdale/MFYG.html 5http://www.brenda-enzymes.org/sequences.php?f[stype_ec]=1&f[ec]=6.1.1.96 Hountondji, 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.

7 Fukai, 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 tRNAVal and Valyl-tRNA Synthetase. Cell, 103, 793-803.

8 Gruic-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