A concerted tryptophanyl-adenylate-dependent conformational change in Bacillus subtilis tryptophanyl-tRNA synthetase revealed by the fluorescence of Trp92
Hogue, C.W.; Doublié, S.; Xue, H.; Wong, J.T.; Carter, C.W.; Szabo, A.G.
Journal of Molecular Biology 260(3): 446-466
ISSN/ISBN: 0022-2836 PMID: 8757806 DOI: 10.1006/jmbi.1996.0413
A semi-conserved tryptophan residue of Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) was previously asserted to be an essential residue and directly involved in tRNATrp binding and recognition. The crystal structure of the Bacillus stearothermophilus TrpRS tryptophanyl-5'-adenylate complex (Trp-AMP) shows that the corresponding Trp91 is buried and in the dimer interface, contrary to the expectations of the earlier assertation. Here we examine the role of this semi-conserved tryptophan residue using fluorescence spectroscopy. B. subtilis TrpRS has a single tryptophan residue, Trp92. 4-Fluorotryptophan (4FW) is used as a non-fluorescent substrate analog, allowing characterization of Trp92 fluorescence in the 4-fluorotryptophanyl-5'-adenylate (4FW-AMP) TrpRS complex. Complexation causes the Trp92 fluorescence to become quenched by 70%. Titrations, forming this complex under irreversible conditions, show that this quenching is essentially complete after half of the sites are filled. This indicates that a substrate-dependent mechanism exists for the inter-subunit communication of conformational changes. Trp92 fluorescence is not efficiently quenched by small solutes in either the apo- or complexed form. From this we conclude that this tryptophan residue is not solvent exposed and that binding of the Trp92 to tRNATrp is unlikely. Time-resolved fluorescence indicates conformational heterogeneity of B. subtilis Trp92 with the fluorescence decay being best described by three discrete exponential decay times. The decay-associated spectra (DAS) of the apo- and complexed-TrpRS show large variations of the concentration of individual fluorescence decay components. Based on recent correlations of these data with changes in the local secondary structure of the backbone containing the fluorescent tryptophan residue, we conclude that changes observed in Trp92 time-resolved fluorescence originate primarily from large perturbations of its local secondary structure. The quenching of Trp92 in the 4FW-AMP complex is best explained by the crystal structure conformation, in which the tryptophan residue is found in an alpha-helix. The amino acid residue cysteine is observed clearly within the quenching radius (3.6 angstroms) of the conserved tryptophan residue. These tryptophan and cysteine residues are neighbors, one helical turn apart. If this local alpha-helix was disrupted in the apo-TrpRS, this disruption would concomitantly relieve the putative cysteine quenching by separating the two residues. Hence we propose a substrate-dependent local helix-coil transition to explain both the observed time-resolved and steady-state fluorescence of Trp92. A mechanism can be further inferred for the inter-subunit communication involving the substrate ligand Asp132 and a small alpha-helix bridging the substrate tryptophan residue and the conserved tryptophan residue of the opposite subunit. This putative mechanism is also consistent with the observed pH dependence of TrpRS crystal growth and substrate binding. We observe that the mechanism of TrpRS has a dynamic component, and contend that conformational dynamics of aminoacyl-tRNA synthetases must be considered as part of the molecular basis for the recognition of cognate tRNA.