Breaking sieve for steric exclusion of a noncognate amino acid from active site of a tRNA synthetase

Abstract

The genetic code is fixed in aminoacylation reactions catalyzed by aminoacyl-tRNA synthetases. Amino acid discrimination occurs at two sites: one for amino acid activation and aminoacylation and one for editing misactivated amino acids. Although the active site sieves out bulkier amino acids, misactivation occurs with substrates whose side chains are smaller than the cognate one. Paradoxically, although alanyl-tRNA synthetase activates glycine as well as alanine, the sterically larger (than alanine) serine is also misactivated. Here, we report crystal structures of an active fragment of Aquifex aeolicus alanyl-tRNA synthetase complexed, separately, with Mg 2؉ -ATP, alanine, glycine, and serine. Ala and Gly are bound in similar orientations in a side-chain-accommodating pocket, where ␣-amino and carboxyl groups are stabilized by salt bridges, and the carboxyl by an H-bond from the side chain NH2 of Asn-194. In contrast, whereas the same two salt bridges stabilize bound Ser, H-bonding of the highly conserved (among class II tRNA synthetases) Asn-194 side chain NH 2 to the Ser OH, instead of to the carboxyl, forces pocket expansion. Significantly, in the Mg 2؉ -ATP complex, Asn-194 coordinates a Mg 2؉ -␣-phosphate bridge. Thus, the sieve for Ser exclusion is broken because of selective pressure to retain Asn-194 for Mg 2؉ -ATP and Ala binding.alanyl-tRNA synthetase ͉ amino acid discrimination ͉ editing ͉ genetic code accuracy ͉ ATP binding A minoacyl-tRNA synthetases (AARSs) establish the genetic code by attaching specific amino acids to the 3Ј ends of tRNAs bearing the anticodons cognate for those amino acids (1-3). These modular enzymes fall into two classes (I and II), based on the architectures of their catalytic domains (4). For both classes, aminoacylation proceeds in two steps catalyzed by one active site. A bound amino acid such as Ala is activated with ATP to form the enzyme-bound aminoacyl adenylate intermediate (Ala-AMP), releasing inorganic pyrophosphate (step 1). The aminoacyl moiety of the adenylate is then transferred to the 2Ј-or 3Ј-OH of the ribose of A76, the universally conserved 3Ј nucleotide of tRNA, with the release of AMP (step 2). Misactivation of amino acids occurs occasionally when a wrong amino acid fits into the active site, usually because it is smaller than the cognate one. Here, we describe structural features in a specific AARS (complexed with three different amino acids) that explain a longstanding paradox about activation of a larger amino acid.AlaRS ϩ Ala ϩ ATP ¡ AlaRS(Ala-AMP) ϩ PPi

Structural and Functional Studies of the Biotin Protein Ligase from Aquifex aeolicus Reveal a Critical Role for a Conserved Residue in Target Specificity

“…39 Analysis of the structures of Staphylococcus aureus threonyl-tRNA synthetase bound to ATP and Zn 2+ and to an adenylate analogue (threonyl-AMS) and the structures of the MgATP and alanine complexes of A. aeolicus alanyltRNA synthetase supports this common binding mode in class II tRNA synthetases. 40,41 The structure of the AaBPL:biotin:ATP complex has revealed the importance of Arg40 from the glycine-rich motif in the stabilisation of the two ligands. The side chain of this conserved arginine plays an important role in the neutralisation of the negative charges of the triphosphate chain of ATP as well as positioning the biotin carboxylate in close proximity to the α-phosphate.…”

Crystallographic Studies on Multiple Conformational States of Active-site Loops in Pyrrolysyl-tRNA Synthetase

“…3a and b), as observed in all of the class II aaRS structures. 16,19,23,[25][26][27]31,38,41 The structure of the present apo form of PylRS(c270) was compared with that of the AMPPNP-bound form, to reveal the ATP-induced conformational changes. Although the overall structures were essentially the same (rmsd of the C α carbon atoms of 0.59 Å over 254 residues), the apo The residues that move significantly upon AMPPNP binding, Asn280, Asp281, Thr282, Glu283 (the ordering loop) and Asp334, Gly335, and Lys336 (the motif-2 loop), are colored vermilion and green, respectively.…”

Molecular basis of alanine discrimination in editing site

“…The inclusion of noncognate serine and glycine in the AlaRS activation site was reported in ref. 10 and was recently confirmed structurally (11). The structures of AlaRS complexed with amino acids suggested that serine hydroxyl is accommodated by an induced fit of Asn-194 that allows expansion of the activation pocket and that the smaller glycine is also included, based simply on a canonical size-sieving model.…”