SUPPLEMENTARY INFORMATION

Transcription

1 Supplementary Information Supplementary Figure 1. The serine paradox. Nature-designed AlaRSs pin-down the -NH3 + group of alanine with an acidic Asp that is critical for binding and activation of alanine. This interaction is conserved through evolution, but creates a serendipitous interaction between the Asp carboxyl and the non-cognate Ser-OH. This interaction is a formidable obstacle for differentiating the larger Ser from Ala, and can lead to production of misacylated Ser-tRNA Ala and mistranslation. The serine paradox may have necessitated the appearance of freestanding AlaXps contemporaneous with early AlaRSs. 1

2 Supplementary Figure 2. Substrate specificity of three freestanding AlaXps. Left, equal amount of M.mazei AlaXp-Ib and yeast AlaXp-II (25 nm) were used in deacylation assay of Gly-tRNA Ala and Ser-tRNA Ala (5 μm), under 50 mm Hepes 7.5, 30 mm KCl, 200 mm NaCl, 10 mm MgCl2, 2 mm DTT at room temperature. Right, mouse AlaRS (20 nm) and mouse AlaXp-II (100 nm) were used in deacylation assay of Gly-tRNA Ala and Ser-tRNA Ala (5 μm) under the same condition. 2

3 doi: /nature08612 Supplementary Figure 3. Leucine-zipper mutations on WT EcAlaRS441-LZ. The central sevenstranded -sheet is numbered. The bound Ala-SA ligand is shown as blue sticks. Three residues located on the opposite side of the active site were mutated to leucines for the purpose of crystallization of the enzyme. 3

4 Supplementary Figure 4. Kinetic parameters of WT EcAlaRS441 and WT EcAlaRS441-LZ 4

5 Supplementary Figure 5. Omit electron density map of the ligands. The ligands are shown by the omit difference Fo-Fc map calculated with Refmac5 contoured at indicated levels. 5

6 Supplementary Figure 6. Kinetic parameters of D235 mutants. The experiments were done at ph 8.0, 23 C. None of these substitutions improved the Ala versus Ser discrimination. For the D235A mutant 6

7 enzyme, the kcat for alanine activation was decreased by 17-fold, while the KM was raised on the order of 10,000-fold. (This result is consistent with the earlier report that a D235A substitution is lethal 35. The KM for serine was increased more than our detectable limit, consistent with the loss of interaction between Asp235 and the Ser-OH. For the D235N mutant enzyme, kcat for alanine activation was decreased by 16-fold, along with a 3,560-fold increase of the KM for alanine. This result indicates the critical need of the negative charge for amino acid binding and activation. Interestingly, the D235N substitution had little effect on the KM for serine (although with a great decrease in kcat). Plausibly, the NH2 of Asn235 could form a different kind of H-bond with the Ser-OH by donating instead of accepting a proton from the Ser-OH. Consequently, the discrimination power (Ala versus Ser) is greatly decreased (to 5.2-fold compared to 310-fold for the native enzyme). 7

8 Supplementary Figure 7. D235 mutants lose specificity for the overall synthesis of the Ala/SertRNA Ala. Different amount of full-length E. coli AlaRS (with C666A/H700K editing defect mutation) wild type, D235N and D235A were used in charging assay of E. coli trna Ala(UGC), under 20 mm HEPES, ph 7.5, 20 mm KCl, 10 mm MgCl 2, 2mM DTT, 4 mm ATP, 0.05U/ml pyrophosphatase at 23 C. L-Alanine (100 M) (a) or L-serine (100 M) (b) were used in the assay. Because of the very low kcat/km of D235 mutants for activating amino acid, high concentration of enzymes had to be used and true turnovers could not be measured. 8

9 doi: /nature08612 Supplementary Figure 8. Various -amino recognition mechanisms of class II AARSs show dilemma for AlaRSs. Distinct designs of acidic residues have evolved to recognize -amino groups of amino acids in class II AARSs. All class II AARS structures are aligned by their amino acid substrates, with motif 1 in cyan, motif 2 in yellow, motif 3 in pink. The bound ligands together with the -amino grouprecognizing acidic residues are shown as sticks. In class I enzymes, in MetRS, IleRS, ValRS and GlnRS, a conserved aspartate at the C-terminus of the second -strand in the Rossmann-fold forms a hydrogen bond with the -NH of the amino acid substrate (with glutamate replacing this aspartate in GluRSs)36. However, class I CysRSs use a threonine (Thr68 in E. coli CysRS) instead of any acidic residue 37. Class IIc TrpRSs (bacterial) and TyrRSs (eukaryotic) use a neutral glutamine. In these cases, the importance of the carboxylate residue for binding amino acid substrate could be partially compensated by other interactions involving the larger 9

10 side chain of Trp (Tyr), or the special -SH of Cys. Class II SerRS (IIa) evolved two mechanisms to bind to the -NH, using a glutamate (most SerRSs (and also class II PheRS and LysRS -II )) or a zinc (methanogenic archae) 38,39. All class II ThrRSs also recognize the -NH through zinc 40. GlyRSs have an acidic pit (using four glutamates) to recognize the -NH group 41. AspRSs have an aspartate binding to the -NH indirectly through water 42, and the corresponding glutamate in AsnRS is used to recognize +43 the side chain of asparagine instead of its -NH 3. Both AspRS and AsnRS also have another glutamate, residing on a loop (as similarly seen in HisRS and ProRS), which directly binds to -NH. However, Asp235 of AlaRSs is located at a position distinct from any of the acidic residues mentioned above. The specialized SepRS and PylRS do not have any acidic residue to bind to the -NH group, probably because of the characteristic side chains of their substrates O-phosphoserine and pyrrolysine, respectively. The idiosyncratic nature of distinct -NH -recognizing groups in different AARSs was possibly developed to accommodate different amino acid side chains while still orienting the amino acid such that the -carboxyl group is free to attack the -phosphate of ATP. The analysis not only shows the need for acidic residues for amino acid recognition, but also that nature tried to adjust the position of these acidic residues so as to be compatible with different amino acids. As it turns out, D235 is probably at the best position for the needed acidic residue in AlaRS. It shows the true dilemma faced by this enzyme. 10

11 Supplementary Notes 35. Shi, J.P., Musier-Forsyth, K. & Schimmel, P. Region of a conserved sequence motif in a class II trna synthetase needed for transfer of an activated amino acid to an RNA substrate. Biochemistry 33, (1994). 36. First, E.A. Catalysis of the trna Aminoacylation Reaction. in The Aminoacyl-tRNA Synthetases (eds. Ibba, M., Francklyn, C. & Cusack, S.) (Eurekah, Georgetown, 2005). 37. Newberry, K.J., Hou, Y.M. & Perona, J.J. Structural origins of amino acid selection without editing by cysteinyl-trna synthetase. EMBO J 21, (2002). 38. Itoh, Y. et al. Crystallographic and mutational studies of seryl-trna synthetase from the archaeon Pyrococcus horikoshii. RNA Biol 5, (2008). 39. Bilokapic, S. et al. Structure of the unusual seryl-trna synthetase reveals a distinct zincdependent mode of substrate recognition. EMBO J 25, (2006). 40. Sankaranarayanan, R. et al. Zinc ion mediated amino acid discrimination by threonyl-trna synthetase. Nat. Struct. Biol. 7, (2000). 41. Arnez, J.G., Dock-Bregeon, A.C. & Moras, D. Glycyl-tRNA synthetase uses a negatively charged pit for specific recognition and activation of glycine. J. Mol. Biol. 286, (1999). 42. Moulinier, L. et al. The structure of an AspRS-tRNA(Asp) complex reveals a trna-dependent control mechanism. EMBO J 20, (2001). 43. Iwasaki, W. et al. Structural basis of the water-assisted asparagine recognition by asparaginyltrna synthetase. J. Mol. Biol. 360, (2006). 11

12 Supplementary Movie 1. Serine paradox is caused by AlaRS recognition dilemma. This movie shows formation of complexes of 3 different aminoacyl-amp analogs with WT and G237A mutant AlaRSs. Clearly seen in how universally conserved acidic residue (Asp235) causes the mis-binding of serine in the pocket for alanine. The renderings used 6 structures presented in this work (pdb3hxu, 3hxv, 3hxw, 3hxz, 3hy0, 3hy1). Supplementary Movie 2. Alanyl-adenylate formation mechanism. This movie shows the stepwise view of alanine/atp binding and activation. The alanine pocket is plastic, and Asp235 is important for alanine binding and its translocation to trna. The renderings used 3 structures presented in this work (pdb3hxx, 3hxy,3hy1) 12