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.

Fig. 1.

Ligand dependence of TrpRS conformations. (A) Pre-TS structures formed with Mg²⁺·ATP and tryptophanamide or with >10 mM Mg²⁺·ATP. (B) Product conformations formed by in situ synthesis of trp-5′-AMP or by cocrystallization with stable adenylate analogs. (C) Open structures, shown here with substoichiometric Mg²⁺·ATP. Wide and narrow arrows denote hinge bending and twisting, respectively, converting C to A to B. (D) WMP structures solved in this work.

Fig. 2.

Adenosine positions in the TrpRS WMP complexes. Monomers were superimposed by using 42 residues derived from the most constant portions of the Rossmann fold (10). (A) Comparison between the representative SeMet WMP structure, the simulated structure (magenta and wheat sticks, respectively) and the Open [1MAW(A)] and PreTS (1MAU) structures (blue and green lines, respectively). (B) Monomers a–f of the SeMet-substituted complex. The HIGH catalytic signature is green; the KMSKS signature is magenta. Tryptophan and P_i moieties superimpose closely; AMP assumes many different positions close to that observed in the singly liganded ATP complex (13). The active site aspartate, D146, interacts with the ribose 2′-OH group in the assembled PreTS active site, but is too far from the ribose in the WMP complexes to stabilize a unique structure.

Fig. 3.

Conformational space defined by internal conformational angles of TrpRS crystal structures and simulations. Open, PreTS, and Products complex crystal structures are indicated by blue, green, and red diamonds (7). Because the 3 Open state crystal structures are isomorphous, there are <18 distinct blue squares. Partially closed native and SeMet WMP structures are shown as black circles and amber squares. MD structures (large, filled red squares) and the origins of their trajectories (fine gray dashed arrows; OM25 and P48) are described in the text. Open squares denote structures along a minimum action pathway connecting the Open and PreTS structures. The red filled star represents the highest-energy structure along the simulated path. Dashed (unliganded) and solid (bound to Mg²⁺·ATP and tryptophan) semiempirical harmonic conformational energy wells were inferred from experimental conformational free-energy changes with and without ATP (15) and from simulations. Circles denote transition states along the forward and reverse induced-fit transition. Vertical dashed lines relate free-energy minima (gray; observed crystal structures) and maxima (simulations; red, blue arrows) to structures below.

Fig. 4.

The OM partial assembly trajectory. (A) Time courses of the 2 internal coordinates. The star denotes the initial configuration before energy minimization, which carries the system immediately to a configuration that recurs later in the trajectory (OM25). (B) Three structural clusters based on distances derived from the first 2 principal components (which map to the hinge and twist angles), represented by snapshots at 2,500 (OM25, red), 3,500 (OM35, blue), and 4,500 (OM45, green) ps. Radii of dashed ellipses are σ values from Table 2.