2.9 Å crystal structure of ligand‐free tryptophanyl‐tRNA synthetase: Domain movements fragment the adenine nucleotide binding site

Abstract

The crystal structure of ligand-free tryptophanyl-tRNA synthetase~TrpRS! was solved at 2.9 Å using a combination of molecular replacement and maximum-entropy map0phase improvement. The dimeric structure~R ϭ 23.7, R free ϭ 26.2! is asymmetric, unlike that of the TrpRS tryptophanyl-59AMP complex~TAM; Doublié S, Bricogne G, Gilmore CJ, Carter CW Jr, 1995, Structure 3:17-31!. In agreement with small-angle solution X-ray scattering experiments, unliganded TrpRS has a conformation in which both monomers open, leaving only the tryptophan-binding regions of their active sites intact. The amino terminal aA-helix, TIGN, and KMSKS signature sequences, and the distal helical domain rotate as a single rigid body away from the dinucleotide-binding fold domain, opening the AMP binding site, seen in the TAM complex, into two halves. Comparison of side-chain packing in ligand-free TrpRS and the TAM complex, using identification of nonpolar nuclei~Ilyin VA, 1994, Protein Eng 7:1189-1195 shows that significant repacking occurs between three relatively stable core regions, one of which acts as a bearing between the other two. These domain rearrangements provide a new structural paradigm that is consistent in detail with the "induced-fit" mechanism proposed for TyrRS by Fersht et al.~Fersht AR, Knill-Jones JW, Beduelle H, Winter G, 1988, Biochemistry 27:1581-1587 Coupling of ATP binding determinants associated with the two catalytic signature sequences to the helical domain containing the presumptive anticodon-binding site provides a mechanism to coordinate active-site chemistry with relocation of the major tRNA binding determinants.

Full Implementation of the Genetic Code by Tryptophanyl-tRNA Synthetase Requires Intermodular Coupling

“…The two domains correspond roughly to the Rossmann dinucleotide binding fold containing the active site and a C-terminal helical domain containing the anticodon-binding determinants. However, the two catalytic signatures, TIGN and KMSKS, belong to the Rossmann fold yet move with the anticodon-binding domain (18). The Rossmann fold is itself a mosaic of three modular components: the first and second ␣-␤-␣ crossover connections (Fig.…”

Crystal Structure of Tryptophanyl-tRNA Synthetase Complexed with Adenosine-5′ Tetraphosphate: Evidence for Distributed Use of Catalytic Binding Energy in Amino Acid Activation by Class I Aminoacyl-tRNA Synthetases

“…Recombinant native and selenomethionylated TrpRS of Bacillus stearothermophilus were overexpressed in Escherichia coli and purified as previously described 12 . Binary native complex crystals were grown by microdialysis (5μL of 4mg/mL protein vs 2000 mL well).…”

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

“…The structural reaction profile for tryptophan activation by TrpRS has been elucidated by structural, biochemical, and biophysical studies. Four structural subclasses have been identified with catalysis of Trp-5Ј-AMP formation (10)(11)(12)(13)(14)(15). Relationships between structures and their catalytic relevance have been extensively documented (13,15), in particular, by how they rationalize the kinetic behavior of active-site mutations in the closely related tyrosyl-tRNA synthetase (16,17).…”