Structural Basis for the Recognition of para ‐Benzoyl‐ L ‐phenylalanine by Evolved Aminoacyl‐tRNA Synthetases

Abstract

A wide array of amino acids with novel chemical and biological properties have been genetically encoded in both prokaryotic and eukaryotic organisms, [1-3] which include the efficient photo-cross-linker para-benzoyl-l-phenylalanine (pBpa, Figure 1). Orthogonal tRNA/aminoacyl-tRNA syn-thetase (aaRS) pairs that selectively recognize pBpa have been evolved from both Methanococcus jannaschii (Mj) and Escher-ichia coli (Ec) tyrosyl-tRNA synthetases (TyrRS) in bacteria and yeast, respectively. [4, 5] To understand the structural basis for selective recognition of the large benzo-phenone side chain by these mutant enzymes, we now report the X-ray crystal structure of the mutant MjTyrRS-pBpa complex. A model of the corresponding mutant EcTyrRS-pBpa complex was also generated and shares many features with the M. jannaschii structure. In contrast to previous structural studies of evolved aminoacyl-tRNA synthe-tases, these mutant enzymes bind the relatively large side chain of pBpa in a deep hydrophobic cavity with relatively little change in the polypeptide backbone. To evolve a pBpa-specific Mj aminoacyl-tRNA synthetase (MjpBpaRS) in E. coli, a pool of MjTyrRS variants was generated with random mutations at five active-site amino acid residues (Y32, E107, D158, I159, and L162). Alternating rounds of positive and negative selection were then used to identify mutant aaRSs that aminoacylate an amber suppressor tRNA CUA with pBpa, but not with endogenous host amino acids. [6, 7] After five rounds of selection, six MjpBpaRSs were identified that showed sequence convergence. In most of these clones, Y32 was mutated to alanine or glycine; D158 was mutated to threonine; I159 was mutated to serine; and L162 was conserved; Y32G/D158T/I159S emerged as the consensus set of mutations. A similar method was used to evolve an EcTyrRS that selectively charges its cognate suppressor tRNA CUA with pBpa in yeast (EcpBpaRS). Positive and negative selections of a library that contains random mutations at five active-site residues (Y37, N126, D182, F183, and L186) afforded two clones selective for pBpa. In both clones, Y37 was mutated to glycine, N126 was conserved, D182 was mutated to glycine, F183 was either conserved or mutated into tyrosine, and L186 was mutated to alanine or methionine. Given the structural homology between archael and bacterial tyrosyl-tRNA synthetases, and the fact that the mutations to each are quite similar, it is likely that MjpBpaRSs and EcpBpaRSs bind the side chain of pBpa in a similar fashion. To address this question, the X-ray crystal structure of the MjpBpaRS (Y32G/D158T/I159S)-pBpa complex was determined, and the corresponding structure of the EcpBpaRS (Y37G/D182G/L186A)-pBpa complex was mod-eled. MjpBpaRS was crystallized in the presence of 1 mm pBpa by the hanging-drop vapor-diffusion method. The protein crystals belong to the space group P4 3 2 1 2 and contain one molecule per asymmetric unit (Table 1). There is one molecule of pBpa per protein bound at the active site as evidenced by the strong Fo-Fc electron density in the active site when pBpa is omitted from structure refinement (Fig-ure 2 a). MjpBpaRS is composed of five regions: the Ross-mann-fold catalytic domain, the short N-terminal region, the connective polypeptide 1 domain, the C-terminal domain, and the KMSKS loop. Its overall structure is very close to that of the wild-type MjTyrRS-tyrosine complex [8] with only a 0.47 Š root-mean-square (rms) deviation in Ca. The only significant changes are in the KMSKS motif loop from G203 to G210. This loop region is disordered in the wild-type structure, but traceable in MjpBpaRS. The active-site superposition of MjpBpaRS and wild-type MjTyrRS is shown in Figure 2 b. The two structures are almost identical to each other with the exception of the side chains of the three mutated residues. Although the overall structure of MjpBpaRS is very close to wild-type MjTyrRS, their substrate-binding interactions are quite distinct. The three active-site mutations alter hydrogen-bonding and packing interactions with the bound amino acid. In wild-type MjTyrRS, Y32 and D158 form two hydrogen bonds with the phenolic oxygen atom of the tyrosine substrate. These two hydrogen bonds are eliminated in MjpBpaRS by the Y32G and D158T mutations, consistent with the loss in selectivity for tyrosine. The Y32G mutation also deepens the substrate side-chain binding pocket to accommodate the para-benzoyl group of pBpa-this sub-stituent occupies the same site as the phenyl group of Y32 in wild-type MjTyrRS (Figure 2 b). In the mutant MjpBpaRS Figure 1. Structure of para-ben-zoyl-l-phenylala-nine.