Structural Overview
Structural insights into ValRS come from a fluorine-19 NMR study on E.coli ValRS[3] and a crystal structure of T. thermophilus ValRS complexed with tRNA(val) and a valine-AMP analog solved at 2.9Å[4]. The major structural elements of ValRS, like other class-Ia aminoacyl-tRNA synthetases, are a helical insertion into the N-terminal half of a Rossmann fold domain and an α-helix bundle domain near the C-terminus. Additionally, ValRS has a large editing domain important in the discrimination between valine and structurally similar amino acids.
The ValRS active site first catalyzes the adenylation of a valine then the transfer of Val-AMP to the acceptor stem of tRNA(val). A canonical Rossman fold forms the core of the active site and creates a binding pocket for valine and ATP[5]. Two highly conserved consensus sequences, , are especially important for catalyzing the adenylation reaction. Upon valine and ATP binding, these sequences adopt a closed conformation which stabilize the pyrophosphate moiety to drive the transfer reaction.
A positively charged (SC-fold) domain contacts the D-loop of tRNA(val), creating a space between the anticodon recognition and editing domains where the tRNA is "pinched" and held onto. This positive patch lines up with the negative backbone of the D-stem, with the Arg570 side chain contacting the oxygens of C11 and C25, the aromatic nitrogen of Trp571 forming weak bonds with the oxygens of U12, and the side chain of Arg566 hydrogen bonding with the backbone of C13.
tRNA(val) Binding
The anticodon of tRNA(val) is recognized and bound by a highly conserved α-helix bundle domain near the C-terminus. Upon binding to ValRS, the anitcodon loop of tRNA(val) becomes extensively unwound and deformed, leading to disordered anticodon. In this state, the first base of the anticodon (C34) is wound out towards the solvent and does not form any interactions with ValRS (which is likely important in wobble during translation). Instead, A35 and C36 become the primary identity elements of the tRNA(val) anticodon. A35 and C36 are spatially stabilized through stacking interactions which may be important in tRNA discrimination as both the A34-C35 and A36-C37 sequences are quite rare in natural tRNA species[6]. is recognized by van der Waals interactions with the side chains of Phe588 and Leu650 and hydrogen bonding between the 6-NH2 group and the side chains of Glu651/Arg587 and the backbone of Cys646. C36 is recognized by hydrogen bonding with the side chains of Lys581 and Asn584.
A coiled-coil domain at the C-terminus is also important in binding the of tRNA(val) and stabilizing the protein/tRNA complex. The primary residue contacted is A20 which forms hydrogen bonds between its base and the side chain of Asn847 as well as between is 2' -OH and the side chain of Arg818. This interaction is also stabilized by van der Waals forces with Leu815. Arg818 and Arg843 are highly conserved and form ionic bonds with the backbone phosphates of A20 and A21. Mutating Arg818 and Arg843 greatly increases the Km, suggesting these ionic interactions are especially important in stabilizing the complex.
Amino Acid Discrimination
To differentiate valine from isosteric threonine, ValRS has a double-sieve mechanism where both valine and threonine are identified by their shape by the first sieve, then threonine is rejected based on its hydrophilicity by the second sieve[7]. The binds valine and threonine as a part of the Rossman fold which holds the active site for amino acid activation. This sieve forms hydrogen bonds with the phosphate backbone of the activated amino acid (Pro42, Asn44, Asp81) and the amino acid fits snugly in a hydrophobic pocket formed by Pro41, Pro42, Trp456, Ile491 and Trp495. This pocket is a very tight fit, and the addition of any additional group (e.g. the additional methyl group of a Leu/Ile residue) will cause steric hindrance and inhibit binding.
The second sieve is present in the editing domain where the incorrect attachment of threonine to tRNA(val) is corrected. This domain is distant (>30Å) from the first sieve and the mechanism of this movement is not well understood, but is thought to be accomplished by a bending of the acceptor stem of tRNA(val). In this , interactions between the threonyl hydroxyl group and the protein trigger hydrolysis of the threonine moiety. The methyl groups of threonine is sandwiched between Asp279 and Arg216 and the hydroxyl group is thought to be recognized by forming hydrogen bonds with Asp276 and Asp279. This mechanism is generally less well understood than that of the first sieve, but it is expected that this interaction can only occur when threonine inhabits the editing domain pocket and triggers the eventual hydrolysis of the threonine to regenerate an uncharged tRNA(val).
