Functional and Crystal Structure Analysis of Active Site Adaptations of a Potent Anti-Angiogenic Human tRNA Synthetase

Abstract

Higher eukaryote tRNA synthetases have expanded functions that come from enlarged, more differentiated structures that were adapted to fit aminoacylation function. How those adaptations affect catalytic mechanisms is not known. Presented here is the structure of a catalytically active natural splice variant of human tryptophanyl-tRNA synthetase (TrpRS) that is a potent angiostatic factor. This and related structures suggest that a eukaryote-specific N-terminal extension of the core enzyme changed substrate recognition by forming an active site cap. At the junction of the extension and core catalytic unit, an arginine is recruited to replace a missing landmark lysine almost 200 residues away. Mutagenesis, rapid kinetic, and substrate binding studies support the functional significance of the cap and arginine recruitment. Thus, the enzyme function of human TrpRS has switched more to the N terminus of the sequence. This switch has the effect of creating selective pressure to retain the N-terminal extension for functional expansion.

Crystal structure of tetrameric form of human lysyl-tRNA synthetase: Implications for multisynthetase complex formation

“…In this instance, the sequences enable interaction of LysRS with p38 in a way that does not disturb the essential aminoacylation function. For human TrpRS, functional expansion (for potent angiostatic activity) required not only the acquisition of sequences in evolution but also a structural adaptation of the active-site region that enabled the enzyme to maintain its aminoacylation function, while also being able to regulate the activation of its cytokine function (31). In contrast to TrpRS, no active-site adaptation was needed for the p38 interaction, probably because the interaction itself can be regulated in a simple way (competition with the dimer-tetramer equilibrium of LysRS).…”

An alternative conformation of human TrpRS suggests a role of zinc in activating non-enzymatic function

“…HIGH and KMSKS ( 170 HVGH 173 and 349 KMSAS 353 in human TrpRS) are two conserved sequence motifs of class I tRNA synthetases involved in ATP recognition and catalysis; a third and less conserved motif in the catalytic domain ( 310 AIDQ 313 in human TrpRS) also participates in ATP binding. 22,23,25 The KMSAS region (Thr338-Thr362) is resolved in the WT T2-TrpRS crystal structure as flexible loops and turns; 4,22,25 however, it becomes completely disordered in subunit A of H130R T2-TrpRS, while in subunit B it folds into an α helix (Thr338-Ser351) (Fig. 3A, B).…”

“…4,22,26 However, in the H130R T2-TrpRS structure, it is folded as a loop (Ala376-Asp397) and flipped 180° onto the active site pocket of subunit A, while this loop is disordered from Gly379 to Gly391 in subunit B (Fig. 3B).…”

“…1 In vertebrates, TrpRS have an N-terminal extension of about 150 amino acids beyond the core catalytic architecture. 2,3 The extension is composed of a vertebrate-specific extension (VSE), also known as WHEP domain (named after the tRNA synthetases (i.e., tryptophanyl (W), histidyl (H), and glutamyl-prolyl (EP) tRNA synthetases) first identified to contain this domain), followed by an eukaryotic specific extension (ESE) 4 (Fig. 1A).…”

“…16 Interestingly, both bovine and human TrpRS were shown to bind zinc to enhance their aminoacylation activities. 17,18,19 However, no zinc was found in the crystal structures of TrpRS, 4,10,20,21,22,23 suggesting that the zinc-bound conformation may be different. Interestingly, Wakasugi has identified a single amino acid substitution in TrpRS (H130R) that eliminates zinc-induced stimulation and renders constitutively high enzymatic active, 24 suggesting H130R TrpRS may mimic a zinc-bound conformation.…”