Independent saturation of three TrpRS subsites generates a partially assembled state similar to those observed in molecular simulations

Abstract

Two new crystal structures of Bacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS) afford evidence that a closed interdomain hinge angle requires a covalent bond between AMP and an occupant of either pyrophosphate or tryptophan subsite. They also are within experimental error of a cluster of structures observed in a nonequilibrium molecular dynamics simulation showing partial active-site assembly. Further, the highest energy structure in a minimum action pathway computed by using elastic network models for Open and Pretransition state (PreTS) conformations for the fully liganded TrpRS monomer is intermediate between that simulated structure and a partially disassembled structure from a nonequilibrium molecular dynamics trajectory for the unliganded PreTS. These mutual consistencies provide unexpected validation of inferences drawn from molecular simulations.conformational transition state ͉ domain movement ͉ induced fit ͉ minimum action pathway ͉ nonequilibrium molecular dynamics

Enhanced Amino Acid Selection in Fully Evolved Tryptophanyl-tRNA Synthetase, Relative to Its Urzyme, Requires Domain Motion Sensed by the D1 Switch, a Remote Dynamic Packing Motif

“…Relative domain motion during the TrpRS catalytic cycle. The Rossmann-fold and ABD move as rigid bodies relative to one another (12,14,17). Hinge bending and torsional motion of the anticodon-binding domain shown here reveal that the ABD assumes three distinct orientations relative to the Rossmann-fold domains that have been superimposed.…”

“…We used the recently described algorithm POVME (21) to assess the tryptophan binding pocket volumes (287 Å 3 , 102 Å 3 , and 92 Å 3 ) of these three states. These substantial changes suggest a possible mechanism to account for the association of specific substrate selection with domain movement, as well as the unique role of Ile-4: control over the tryptophan binding pocket volume by domain movement (10,17). 4 These structural sequences illustrate semi-quantitatively (e.g.…”

“…The Observed Effects Are Associated with Changes in the Volume of the Tryptophan Binding Pocket-TrpRS catalysis proceeds by a sequence of structural states represented by three distinct crystal structures (Protein Data Bank codes 1MAW, 1MAU, and 1I6L (7,17)). We used the recently described algorithm POVME (21) to assess the tryptophan binding pocket volumes (287 Å 3 , 102 Å 3 , and 92 Å 3 ) of these three states.…”

“…Transition between the first two structures corresponds to induced-fit, between the second and third to catalysis, and the restorative step to product release stimulated by acyl-transfer to cognate tRNA Trp . Superposition of the three representative crystal structures (Protein Data Bank codes 1MB2, 1MAU, and 1I6L) reveals that the conformational cycle (7,12,16,17) is well approximated by rigid-body movements of the ABD relative to the active site (Fig. 1).…”

“…These orientations are denoted LF (ligand-free, wheat), PreTS (pre-transition state; sky blue), and Products (magenta). Catalytic residues move with the ABD (11,17). FIGURE 2.…”