CRYSTAL STRUCTURE OF TRYPTOPHANYL-TRNA SYNTHETASE COMPLEXED TO INDOLMYCIN, A SPECIES-SPECIFIC INHIBITOR Yuhui Yin and Charles W. Carter, Jr., Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7260 USA

Indolmycin is a tryptophan analog containing indole joined to an oxazolone ring. It selectively inhibits prokaryotic Tryptophanyl-tRNA synthetases (TrpRS) with an affinity (K_i = 9x10^-6 M) at least 10³ fold greater than that for eukaryotic TrpRS and is thus a potential candidate for antibacterial drug design. We have solved the crystal structure of an indolmycin:ATP:TrpRS complex to provide a structural basis for increasing its binding affinity while preserving the species specificity. Tetragonal crystals of this complex were prepared using a high excess (10 mM) of ATP. The structure, solved at 3.0 Å resolution by molecular replacement, has several unexpected features. TrpRS contains two domains: a Rossmann fold containing the active site, and a helical domain. The indole moiety binds to the active site in a manner similar to that observed previously in the Trp-5'AMP complex, involving a hydrogen bond to Asp 132. A new hydrogen bond is formed between Asp 132 and the nearby His 43. The pH-dependence of this hydrogen bond may help explain a 13-fold increase in Vmax/Km observed at high pH. The oxazolone ring makes hydrogen bonds both to His 43 and to Gln 147 on the opposite side of the active site. The ATP molecule adopts an unusual orientation, with the adenine ring facing the active site opening and the triphosphate in the space occupied by the adenine moiety of the Trp-5'AMP adenylate intermediate (Doublié, et al., Structure, 3:17-31, 1995). It makes contacts with both of the "signature" sequences characteristic of class I aminoacyl-tRNA synthetases. The triphosphate shifts the KMSKS loop from its configuration in the adenylate complex, such that Lys 192 makes contact with the beta phosphate. The KMSKS loop rearrangement is correlated with an 8( rotation of the helical domain, relative to the Rossmann fold domain. This conformation resembles that of a ligand-free structure obtained by dissociation of the product, Trp-2',3'-ATP, from tetragonal crystals. The tertiary structure of the indolmycin:ATP:TrpRS complex therefore differs from that of the Trp-5'AMP intermediate complex. Movement of the helical domain relative to its position in that structure suggests that formation of the adenylate triggers a conformational change which might be important for tRNA recognition.

(This work supported by NIH GM48519-02)