has only one nucleotide, U20, between G19 and A21, while trna Glu CUC has two

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1 SPPLEMENTRY INFORMTION doi:1.138/nature9411 Supplementary Discussion The structural characteristics of trn Gln G in comparison to trn Glu 16 in trn Gln G is directed towards the G19 56 pair, or the outer corner of the L shape, while 16 in trn Glu is oriented towards the acceptor arm. trn Gln G has only one nucleotide,, between G19 and 1, while trn Glu has two nucleotides, -a, in the corresponding position (Fig. a and Supplementary Fig. 4). The base moiety of in trn Gln G points away from the trn body, while that in trn Glu is directed inward, and instead the base moiety of the following nucleotide, a, in trn Glu is directed outward. These characteristic differences in the D-loop structure should be evolutionally conserved between trn Gln and trn Glu, because all of the trn Gln species have one pyrimidine nucleotide and all of the trn Glu species have two or three nucleotides (mostly pyrimidines) between the invariant G19 and 1 (Supplementary Fig. 4) 4,8. The interaction between trn Gln G and the tail body of GatB in the transamidosome In comparison to the GluRS trn Gln binary complex, the base and sugar moieties of 16, the sugar moiety of G19, and the entire nucleotide of are largely 1

2 RESERH SPPLEMENTRY INFORMTION shifted toward the tail domain of GatB. Especially, the base moiety of protrudes into the pocket in the tail domain of GatB, where the N3 imino group of hydrogen bonds with the γ-carboxyl group of sp44 in a -specific manner. The trn Gln interactions of the helical domain of GatB (Supplementary Fig. 5), which are similar to those of the corresponding domain of GatE 5, are not specific to trn Gln. Therefore, these non-specific interactions may function to increase the overall affinity of the GatB tail body for trn Gln. The structural characteristics of the GluRS trn Gln G binary complex The significant differences between the T. maritima nondiscriminating (ND) GluRS trn Gln G binary complex and the T. thermophilus discriminating (D) GluRS trn Glu binary complex exist around the anticodon and D loops of the trns. The differences around the D loop are discussed in the main text. In the anticodon loop, the structures of the nucleotides adjacent to the anticodon (positions 33 and 37) are quite different from each other (Supplementary Figure 8). Both 33 and G37 in the T. maritima ND-GluRS trn Gln G complex are directed outwards from the anticodon loop, in stark contrast to the canonical trn structure. On the other hand, the structures of 33 and 37 in the T. thermophilus D-GluRS trn Glu complex are

3 SPPLEMENTRY INFORMTION RESERH canonical. superposition of the structures of the Rossmann-fold catalytic domains of the two binary complexes revealed that the positions of the anticodon-binding domains 1 and of the T. maritima binary complex are closer to the Rossmann-fold domain than those of the T. thermophilus binary complex. In the T. maritima binary complex, the flipping out of 33 and G37 allows the anticodon to be located closer to the Rossmann-fold domain, and recognized by the anticodon-binding domains 1 and. s for the anticodon recognition itself, many common features were identified in both binary complexes (Supplementary Figure 8). The carbonyl group and the ring nitrogen at positions and 3 of 34 hydrogen bond with the side-chain guanidinium group of the arginine residue (rg449 and rg435 in the T. maritima ND-GluRS trn Gln G complex and the T. thermophilus D-GluRS trn Glu complex, respectively) and the main-chain NH group of the leucine residue (Leu461 and Leu447), respectively. The carbonyl and imino groups at positions and 3 of 35 hydrogen bond with the main-chain NH and O groups of the threonine residue (Thr458 and Thr444), respectively. However, the nucleotides at the third position of the anticodon are recognized in distinct manners. The carbonyl group at position 6 of G36 hydrogen bonds with the side-chain amino group of Lys369 in the T. maritima ND-GluRS trn Gln G complex. Since the nondiscriminating GluRS must recognize 3

4 RESERH SPPLEMENTRY INFORMTION not only trn Gln but also trn Glu, the side chain of Lys369 in T. maritima ND-GluRS needs to recognize G36 and 36. The tertiary structure of the ND-GluRS trn Glu complex will clarify the recognition mechanism of 36. In contrast, the carbonyl group and the ring nitrogen at positions and 3 of 36 hydrogen bond cytosine-specifically with the guanidinium group of rg358 in the T. thermophilus D-GluRS trn Glu complex, which agrees with the fact that D-GluRS recognizes only the trn Glu with 36. Pivoting movements of the catalytic bodies of GluRS and GatB in the bacterial glutamine transamidosome model of the amidation states of the bacterial glutamine transamidosome was constructed, as described in the Supplementary Methods. The supplementary information about the catalytic-body movements in the bacterial glutamine transamidosome is provided here. In the glutamylation state, the distance between the active sites of GluRS and GatB is about 46 Å, as shown in Supplementary Fig. 6f. The angle between the productive and non-productive GluRS catalytic bodies is about 8, while that between the productive and non-productive GatB catalytic bodies is about 3 (Supplementary Fig. 6f). In the amidation state, the distance between the active sites 4

5 SPPLEMENTRY INFORMTION RESERH of GluRS and GatB is about 48 Å. Supplementary Figs. 6a and 6b show three vertical views of the glutamylation and amidation states. When the structure of the latch loop in GatB is straight, the productive-form GluRS and the productive-form GatB could both exist, resulting in the closed state (state V in Supplementary Fig. 6c). However, in the closed state, the 3'-end of Glu-tRN Gln is unable to move from GluRS to GatB, because there is no space for the conformational change of the trn between the two enzymes. When the Glu-tRN Gln with its 3'-end sequence extended into the GatB active site is superimposed onto this closed transamidosome, severe steric hindrance occurs between the acceptor arm and the connective-peptide domain of GluRS (step V in Supplementary Fig. 6c, and Supplementary Fig. 6d). Even if GluRS is shifted to the non-productive form (step VI in Supplementary Fig. 6c) from the closed form, utilizing the hinge between anticodon-binding domains 1 and, the space between the two enzymes is still not wide enough for the 3'-end of Glu-tRN Gln to move from the catalytic site in GluRS to that in GatB. Therefore, neither the combination of the non-productive GatB with the productive GluRS (described in the main text) nor that of the non-productive GluRS with the productive GatB (described here) provides sufficient space for the conformational change of the 3'-end sequence and the acceptor stem of 5

6 RESERH SPPLEMENTRY INFORMTION Glu-tRN Gln for the transfer of the substrate from the first active site to the second. In other words, the functional glutamine transamidosome must assume its intermediate state (Fig. 3c), where both GluRS and GatB are open in the non-productive form, for the two-step synthesis of Gln-tRN Gln. Both GluRS and GatB bind to the minor groove of the acceptor stem when they catalyze the reactions; GluRS extensively contacts the first fifth base pairs, and GatB contacts the first base pair, from the terminus. Therefore, it was previously thought that the two enzymes in their productive forms would compete for the terminal base pair on the minor groove of the acceptor arm (Supplementary Fig. 6e), and consequently, the transamidosome could not be formed. In contrast, in the actual transamidosome, the GluRS and GatB enzymes are both anchored simultaneously to the anticodon loop and the outer corner of the L shape, respectively, and have the flexibility to adopt the productive and non-productive forms, as described in the main text. This alternative-conformation mechanism of the transamidosome elegantly evades the postulated steric hindrance. The model construction of the glutamylation state of the archaeal glutamine transamidosome, the GluRS trn Gln GatDE complex 6

7 SPPLEMENTRY INFORMTION RESERH In order to gain more insight into the archaeal glutamine transamidosome, we constructed a model of the glutamylation state, as follows. First, the structures of the GatDE trn Gln complex (PDB ID: D6F) 5 and the GluRS trn Glu complex (PDB ID: 1N78) 13 were superimposed with respect to the whole trn structure. fter the superposition, the GatDE-bound trn was removed. In this initial model, severe overlap was observed between the connective-peptide domain of GluRS and the snrs-like insertion domain of GatE (Supplementary Figs. 7a and 7b). However, if the catalytic body of GatDE is rotated by about 3 degrees, with the boundary between the cradle and helical domains of GatE as a pivot point (Supplementary Fig. 7c), then the above-mentioned overlap disappeared (Supplementary Fig. 7d). This movement is analogous to the aforementioned movement of the catalytic body of GatB between the productive and non-productive forms in the bacterial glutamine transamidosome. In the resultant model of the glutamylation state of the archaeal glutamine transamidosome, the connective-peptide domain of GluRS is able to bind to the snrs-like insertion domain of GatE with a modestly-wide area. This fact is consistent with the results in the recently published report by Rampias et al. 1, who biochemically described the tight interactions among GluRS, trn Gln, and GatDE. 7

RESERH SPPLEMENTRY INFORMTION a trn Gln (µm) GluRS (µm) GatB (µm) 5 1 5 1 * * * * * * * * * * * *

5% native-pge 1% SDS-PGE Supplementary Figure 1 a, Gel mobility shift assay of trn Gln G against

The bands marked with asterisks were derived from the minor component of trn Gln (probably the

b, Two electrophoresis steps to identify the bacterial glutamine transamidosome.

8 RESERH SPPLEMENTRY INFORMTION a trn Gln (µm) GluRS (µm) GatB (µm) * * * * * * * * * * * * free trn b trn Gln (µm) GluRS (µm) GatB (µm) GatB GluRS Gat 6.5% native-pge 1% SDS-PGE Supplementary Figure 1 a, Gel mobility shift assay of trn Gln G against GluRS or GatB. The final concentrations of the samples are indicated at the top. The bands marked with asterisks were derived from the minor component of trn Gln (probably the artificially-formed multimer). b, Two electrophoresis steps to identify the bacterial glutamine transamidosome. The final concentrations of the samples for the first electrophoresis, 6.5% native PGE, are indicated at the top. The band that was excised and subjected to the second electrophoresis, 1% SDS-PGE, is indicated by the red dotted arrow. 8

state (model) (b), in which the loops from Gln134 to Ser14 in GatB (the latch loops) are in the bent

9 SPPLEMENTRY INFORMTION RESERH a b bent-loop conformation straight-loop conformation Supplementary Figure a and b, lose-up views of the interaction between GluRS and GatB in the glutamylation state (a) and the amidation state (model) (b), in which the loops from Gln134 to Ser14 in GatB (the latch loops) are in the bent conformation (a) and the straight conformation (model) (b), respectively, as indicated by the red arrows. 9

RESERH SPPLEMENTRY INFORMTION trngln 74 trngln 74 76 Glu-S 75 76 Glu-S 75 GluRS

sequence of trn Gln is directed toward the catalytic site in GluRS (stereo view).

The Fo - Fc annealed omit maps (3σ level) for 74 76 and Glu-S are also indicated.

10 RESERH SPPLEMENTRY INFORMTION trngln 74 trngln Glu-S Glu-S 75 GluRS GluRS Supplementary Figure 3 In the elucidated transamidosome structure, the 3'-end sequence of trn Gln is directed toward the catalytic site in GluRS (stereo view). 74, 75, 76, and GluRS-bound Glu-S are represented by stick models. The Fo - Fc annealed omit maps (3σ level) for and Glu-S are also indicated. The entire model is presented on the right, with the square indicating the expanded region. 1

11 SPPLEMENTRY INFORMTION RESERH G G 1 1 T. maritima trn Gln 1 G T. maritima trn Gln G G G G 18 G G G G G G G G G G G G G G G G G G G G G G G G 46 G G G 46 G G G G G46 a a G a b 3 3 G 4 4 3G 4 G G 16 1 G G G G G G 5 48 G G G G G 46 G 3G 4 76 G 34 G G G G 16 1 G 7 6 G 1 G G G G G 5 48 G G G T 46 3G 4 G G T. maritima trn Glu T. maritima trn Glu T. thermophilus trn Glu Supplementary Figure 4 The nucleotide sequences of the unmodified trns are represented as clover-leaf models. The sequences were obtained from trndb 9 ( 5. The sequences of trn Gln G and trn Gln G from T. maritima are indicated in the upper panel, and those of trn Glu and trn Glu from T. maritima, and trn Glu from T. thermophilus are in the lower panel. The nucleotides at the first base pair, position 16, and positions /a/b are colored red, green, and magenta, respectively. 11

RESERH SPPLEMENTRY INFORMTION GatB Helical domain Tyr319 GatB Helical domain Tyr319 Lys394 Lys391 6 Lys394 Lys391 6 G18 G5 G18 G5 56 G19 56 G19 Tail domain Tail domain

trn Gln G (yellow) and the tail body of GatB (salmon) in the glutamine transamidosome are shown.

12 RESERH SPPLEMENTRY INFORMTION GatB Helical domain Tyr319 GatB Helical domain Tyr319 Lys394 Lys391 6 Lys394 Lys391 6 G18 G5 G18 G5 56 G19 56 G19 Tail domain Tail domain Supplementary Figure 5 trn Gln recognition by the helical domain of GatB (stereo view). trn Gln G (yellow) and the tail body of GatB (salmon) in the glutamine transamidosome are shown. The G5 6 and G19 56 pairs and G18 are depicted by stick models, and the base-interacting side chains in the helical domain of GatB are also shown: Tyr319, Lys391, and Lys394 interact with G5, G19, and G5, respectively. The entire model is represented in the same manner as in Supplementary Fig

13 SPPLEMENTRY INFORMTION RESERH a b productive/non-productive glutamylation state non-productive/productive amidation state (model) Supplementary Figure 6 Structural alternation in the glutamine transamidosome. a and b, Three vertical views of the glutamylation state (a) and the amidation state (b) of the glutamine transamidosome, represented by ribbon models. 13

RESERH SPPLEMENTRY INFORMTION c Gln Glu Ia Ib II III IVa IVb d V V VI V V VI productive productive closed state with 3

-end-folded trn productive productive closed state with 3 -end-extended trn Supplementary Figure 6 (continued) c, The other

The putative synthetic pathway of Gln-tRN Gln is represented in the same manner as in Fig. 3c.

14 RESERH SPPLEMENTRY INFORMTION c Gln Glu Ia Ib II III IVa IVb d V V VI V V VI productive productive closed state with 3 -end-extended trn productive productive closed state with 3 -end-folded trn non-productive productive closed state with 3 -end-folded trn productive productive closed state with 3 -end-extended trn Supplementary Figure 6 (continued) c, The other possible states of the glutamine transamidosome. The putative synthetic pathway of Gln-tRN Gln is represented in the same manner as in Fig. 3c. The steps indicated at the top are the same as those in Fig. 3c. d, The 3 -end-extended trn Gln, placed in the transamidosome of the productive-form of GluRS and the productive-form of GatB. The severe steric hindrances are indicated by the red arrow. 14

SPPLEMENTRY INFORMTION RESERH e f ~3 GatB 46 Å ~8 GluRS productive non-productive glutamylation state non-productive productive amidation state (model)

The severe clashes between GluRS and GatB were identified, and the clashing atoms are indicated by red spheres.

15 SPPLEMENTRY INFORMTION RESERH e f ~3 GatB 46 Å ~8 GluRS productive non-productive glutamylation state non-productive productive amidation state (model) Supplementary Figure 6 (continued) e, The glutamylation and amidation forms, superimposed with respect to the acceptor stem of the trn Gln. The severe clashes between GluRS and GatB were identified, and the clashing atoms are indicated by red spheres. f, The distance between the catalytic sites in the glutamylation state and the angles of the domain movements are presented in the ribbon models of the glutamylation (left) and amidation (right) states. 15

RESERH SPPLEMENTRY INFORMTION a c ~3 d overlap no overlap b 4 GluRS trn GatDE complex in glutamylation state (model) hinge

1N78) Supplementary Figure 7 a and b, The structures of the GluRS trn binary complex (colored cyan) (PDB ID: 1N78) and the

stems. Two different views are represented. Severe overlap between GatD and GluRS is indicated by red dotted circles.

The pivot point is the boundary between the cradle and helical domains in GatE.

16 RESERH SPPLEMENTRY INFORMTION a c ~3 d overlap no overlap b 4 GluRS trn GatDE complex in glutamylation state (model) hinge GatDE trn complex in amidation state (PDB ID: D6F) overlap Helical domain GluRS trn complex in glutamylation state (PDB ID: 1N78) Supplementary Figure 7 a and b, The structures of the GluRS trn binary complex (colored cyan) (PDB ID: 1N78) and the GatDE trn binary complex (colored yellow) (PDB ID: D6F) were superimposed with respect to the phosphorus atoms of the D- and T- stems. Two different views are represented. Severe overlap between GatD and GluRS is indicated by red dotted circles. c, The possible rotational movement applied to the catalytic body of GatDE. The pivot point is the boundary between the cradle and helical domains in GatE. The resultant structure of GatDE is colored green. d, The model of the glutamylation state of the archaeal glutamine transamidosome. This model lacks overlapping between GluRS and GatDE, as indicated in the cyan dotted circle. 16

SPPLEMENTRY INFORMTION RESERH G37 trn Gln trn Glu Lys369 Thr458 G36 35 34 37 rg358 Thr444 36 35 33 GluRS rg449 Leu461 GluRS rg435 Leu447 34 33 T.

maritima ND-GluRS trn Gln G binary complex (cyan and purple) and the T.

17 SPPLEMENTRY INFORMTION RESERH G37 trn Gln trn Glu Lys369 Thr458 G rg358 Thr GluRS rg449 Leu461 GluRS rg435 Leu T. maritima ND-GluRS trn Gln G binary complex T. thermophilus D-GluRS trn Glu binary complex Supplementary Figure 8 Ribbon models of the T. maritima ND-GluRS trn Gln G binary complex (cyan and purple) and the T. thermophilus D-GluRS trn Glu binary complex (light blue and gray) are shown. The nucleotides at positions in the trn and the anticodon-interacting residues in GluRS are depicted by stick models. 17

RESERH SPPLEMENTRY INFORMTION Gat trn Gln GluRS Gat GatB Glu-S Ile94 Phe trn Gln GatB Pro15 Glu-S GluRS Gat Gat Supplementary Figure 9 Ribbon models of two T.

In the left panel, one unit of the functional glutamine transamidosome is indicated by the magenta dotted ellipse.

18 RESERH SPPLEMENTRY INFORMTION Gat trn Gln GluRS Gat GatB Glu-S Ile94 Phe trn Gln GatB Pro15 Glu-S GluRS Gat Gat Supplementary Figure 9 Ribbon models of two T. maritima glutamine transamidosomes, which were crystallized next to each other, are shown. In the left panel, one unit of the functional glutamine transamidosome is indicated by the magenta dotted ellipse. The Gat and GluRS derived from one fusion protein, and the associated Gat and GatB are colored. In the right panel, an expanded view around the linker between Gat and GluRS is shown. The disordered linker region between Ile94 of Gat and Pro15 of GluRS, which consists of 4 residues, is indicated by the black dotted line. 18

19 SPPLEMENTRY INFORMTION RESERH Supplementary Table 1: Data collection and refinement statistics Data collection GluRS trn Gln G binary complex glutamine transamidosome (GluRS trn Gln G GatB ternary complex) Space group P1 P1 ell dimensions a, b, c (Å) 63., 159.9, , 15.7, α, β, γ ( ) 9., 9.1, 9. 9., 9., 9. Resolution (Å) 5..9 (3..9) * ( ) Rsym.19 (.47).118 (.396) I/σI 11.7 (.) 15.8 (3.4) ompleteness (%) 88. (75.3) 93. (7.) Redundancy 3.5 (.8) 6.7 (5.5) Refinement Resolution (Å) No. reflections Twinning operator/fraction (h, -k, -l)/.5 Rwork/ Rfree No. complex molecules No. atoms./.8.195/ Protein 1544 (GluRS: ) 157 (Gat: 367, GatB: 3891, Gat: 757, GluRS: 3937) Nucleic cid Ligand 634 (trn Gln : ) 18 (Glu-S: 3 4) 1581 (trn Gln : 1581) 3 (Glu-S: 3) Ion 1 (Zn ion: 1) B-factors Protein [GatB atalytic body: Nucleic cid , GatB tail body: 1.7, GluRS: 15.] 97. Ligand Ion 81.7 R.m.s deviations Bond lengths (Å).3.8 Bond angles (º) *Highest resolution shell is shown in parentheses. 19

20 RESERH SPPLEMENTRY INFORMTION Supplementary Movie 1 The movie shows the transition of the glutamine transamidosome from the glutamylation state to the amidation state. t the beginning of the movie, GluRS in the productive form catalyzes the formation of Glu-tRN Gln with Glu-MP. The GluRS then undergoes a transition from the productive form to the non-productive form. sing the newly formed space between GluRS and GatB, the glutamylated 3' end of trn Gln dissociates from the active site in GluRS, and points toward GatB. GatB then undergoes the transition from the non-productive form to the productive form, and the glutamylated 3' end of trn Gln enters the active site in GatB. In the GatB active site, the glutamyl moiety is phosphorylated with TP (indicated by a stick model) and magnesium ions (indicated by the large white balls), and the amidation reaction consecutively occurs with ammonia (indicated by a blue ball) to produce glutaminyl-trn Gln. The movie repeats with the overall and expanded views (QuickTime; 9.7 MB). dditional reference in Supplementary Information 5. Juhling, F. et al. trndb 9: compilation of trn sequences and trn genes. Nucleic cids Res 37, D159-6 (9).

X-ray structures of fructosyl peptide oxidases revealing residues responsible for gating oxygen access in the oxidative half reaction

X-ray structures of fructosyl peptide oxidases revealing residues responsible for gating oxygen access in the oxidative half reaction Tomohisa Shimasaki 1, Hiromi Yoshida 2, Shigehiro Kamitori 2 & Koji