Paradox of mistranslation of serine for alanine caused by AlaRS recognition dilemma

Abstract

Mistranslation arising from confusion of serine for alanine by alanyl-tRNA synthetases (AlaRSs) has profound functional consequences. Throughout evolution, two editing checkpoints prevent disease-causing mistranslation from confusing glycine or serine for alanine at the active site of AlaRS. In both bacteria and mice, Ser poses a bigger challenge than Gly. One checkpoint is the AlaRS editing centre, and the other is from widely distributed AlaXps-free-standing, genome-encoded editing proteins that clear Ser-tRNA(Ala). The paradox of misincorporating both a smaller (glycine) and a larger (serine) amino acid suggests a deep conflict for nature-designed AlaRS. Here we show the chemical basis for this conflict. Nine crystal structures, together with kinetic and mutational analysis, provided snapshots of adenylate formation for each amino acid. An inherent dilemma is posed by constraints of a structural design that pins down the alpha-amino group of the bound amino acid by using an acidic residue. This design, dating back more than 3 billion years, creates a serendipitous interaction with the serine OH that is difficult to avoid. Apparently because no better architecture for the recognition of alanine could be found, the serine misactivation problem was solved through free-standing AlaXps, which appeared contemporaneously with early AlaRSs. The results reveal unconventional problems and solutions arising from the historical design of the protein synthesis machinery.

Figure 1. Plasticity of active site in AlaRS

a, Overall structure of the N-terminal catalytic fragment AlaRS_441-LZ of E. coli AlaRS, shown with the water-accessible cavity (grey). b, Global movement of catalytic domain upon ligand binding. c, Dynamic adjustments of active site pocket of AlaRS. Left, active site cavity of apo AlaRS_441-LZ with l-alanine and ATP placed by modeling (blue). Model of apo AlaRS_441-LZ is derived from structure of apo G237A AlaRS_441-LZ by mutating Ala237 back to Gly. Middle, active site cavity of AMPPCP complex with bound AMPPCP and l-alanine placed by modeling (gray). Right, active site cavity of Ala-AMP/PCP complex with bound Ala-AMP and βγ-PCP. The movements of two active site loops are indicated by arrows. d, Stereo view of ‘open to close’ active site conformational change upon Ala-SA binding. The difference map of the omitted Ala-SA ligand is contoured at 9.0 σ (green). e, Amino acid pocket with three different ligands bound in EcAlaRS_441-LZ. Three co-crystal structures of EcAlaRS_441-LZ with Ala-SA, Ser-SA, and Gly-SA were superimposed. The ligand, active site residues and cavity of each complex structure are colored as indicated. The small cavity containing 3 water molecules is seen below the amino acid moiety of all three bound ligands.

Figure 2. Alanine active site with intrinsic design defect that mis-binds serine

a-c, Interactions with l-alanine, l-serine and glycine as shown in their ligand-bound structures. Two extra interactions are formed between AlaRS and the γ-OH of serine: Asp235(COO⁻) -- Ser(OH) and Gly237(N) -- Ser (OH). A contact between Gly237(C_α) -- Ser(OH) is also shown (3.3 Å). Asp235 also forms a common hydrogen bond with α-amino group of all three amino acids. d, Diagram of the interactions between the amino acid moiety of Ser-SA and the synthetase. The aromatic ring of Trp170 stacks with the backbone of amino acid substrates.

Figure 3. Mutant enzyme with a smaller pocket retains serine binding affinity

a-c, Kinetic parameters for amino acid activation of G237A and WT AlaRS₄₄₁. k_cat/K_M ratios of noncognate serine and glycine were calculated against cognate alanine for each protein, separately. Error bars are s.d., n ≥ 3. d-f, G237A AlaRS_441-LZ structures in complex with three different ligands are compared with the corresponding WT AlaRS_441-LZ complex structures (white). For clarity, only the active site cavities of WT AlaRS_441-LZ structures are shown here.

Figure 4. Critical role of Asp235 shows dilemma for AlaRSs

a, Kinetic parameters for amino acid activation of D235A and D235N. b, Various designs of acidic residues that recognize α-amino group of amino acids in Class II AARSs, which structures are aligned by their amino acid substrates (orange, with α-amino groups in blue). The central β-sheets are shown (AlaRS in green, others in white). The acidic residues are shown with carboxyl groups in red. c, Ser-SA ligands in the active site of E. coli (E.c.) AlaRS and Pyrococcus horikoshii (P.h.) SerRS. Both acidic residues form bifurcated interactions with the α-amino group and γ-OH of serine.