Structural Basis for the Recognition ofpara-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][2][3] which include the efficient photo-cross-linker para-benzoyl-l-phenylalanine (pBpa, Figure 1). Orthogonal tRNA/aminoacyl-tRNA synthetase (aaRS) pairs that selectively recognize pBpa have been evolved from both Methanococcus jannaschii (Mj) and Escherichia coli (Ec) tyrosyl-tRNA synthetases (TyrRS) in bacteria and yeast, respectively. [4,5] To understand the structural basis for selective recognition of the large benzophenone 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 synthetases, 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 modeled.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 d...

A Biosynthetic Route to Photoclick Chemistry on Proteins

“…On the other hand, while not making direct contact with p -Tpa, the Val164Gly mutation acquired during the second round of mutation converts the GVD loop, which is located close to the critical p -Tpa contact helix α8 where two important mutations Asp158Gly and Leu162Val reside,16 into a more flexible GGD loop, presumably to allow the synthetase to tolerate all these mutations without loss of the aminoacylation activity. Overall, the substrate specificity of M. jannaschii tyrosyl-tRNA synthetase was altered by active site mutations to favor p -Tpa over tyrosine without major backbone structural reorganization, which was also observed in other Mj TyrRS engineering work 14,18…”

An Enhanced System for Unnatural Amino Acid Mutagenesis in E. coli

“…In doing so, we have identified a number of aaRS/tRNA CUA pairs (e.g., oNiF and HQA) with suboptimal catalytic efficiencies and fidelities that can lead to diminished yields and decreased fidelities, which vary with the site of incorporation. Background incorporation by these aaRS/tRNA CUA pairs in rich media can likely be reduced by further evolution of these aaRS (e.g., increased stringency of the negative selection improved active-site libraries guided by X-ray structures of mutant aaRS, 33,[46][47][48] or selection in rich media) or by increasing amino acid concentration. 21 Alternatively, decreased fidelity can be minimized by the use of minimal media 49 ; however, this generally results in decreased yields of mutant proteins.…”

A Critical Examination of Escherichia coli Esterase Activity

“…Furthermore, an obvious paradigm has now emerged regarding how the amber suppression method (24) continually succeeds (3) in surmounting nature's original solution for achieving exquisite fidelity in translation. The PDB data base now has at least nine M. jannaschii Tyr aaRS structures bound with various nonnatural amino acids, and together they provide the method with an obvious structural rationale for success (31)(32)(33)(34)(35). For example, the minimum requirement needed to reduce wild-type enzyme affinity for tyrosine involves mutating Tyr-32 and Asp-158 to amino acids less capable of hydrogen bonding to the tyrosine hydroxyl group.…”