Supplementary Information
The structure of the pyrrolysyl-tRNA synthetase:tRNA Pyl complex reveals the molecular basis of orthogonality
Kayo Nozawa 1* , Patrick O’Donoghue 2* , Sarath Gundllapalli 2* , Yuhei Araiso 1 ,
Ryuichiro Ishitani 4 , Takuya Umehara 2 , Dieter Söll 2,3,† , Osamu Nureki 1,4†
1 Department of Biological Information, Graduate School of Bioscience and Biotechnology, Tokyo
Institute of Technology, B34 4259 Nagatsuta-cho, Midori-ku, Yokohama-shi, Kanagawa 226-8501,
Japan; Departments of 2 Molecular Biophysics and Biochemistry, and 3 Chemistry, Yale University,
New Haven, Connecticut 06520-8114, USA; 4 Department of Basic Medical Sciences, Institute of
Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.
*These authors contributed equally to this work.
† To whom correspondence and requests for materials should be addressed:
E-mail: nureki@ims.u-tokyo.ac.jp, dieter.soll@yale.edu
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Supplementary discussion
In the following section we present a more detailed description of the unusual structure of tRNA Pyl . We then discuss the interactions between the protein and key structural elements of tRNA Pyl , i.e., the minimal core and acceptor stem that contribute to the specific and exclusive interaction of DhPylRS with tRNA Pyl .
Unusual structure of tRNA Pyl tRNA Pyl lacks many of the invariant structural elements conserved in the canonical tRNAs. The most prominent differences are as follows: (i) an elongated anticodon stem of 6 base pairs instead of 5, (ii) only one nucleotide, instead of two, at the junction connecting the acceptor and D stems
(iii) a short variable region of only 3 bases, (iv) a small D-loop of only 5 bases, (v) absence of the universally conserved G18G19D20 (D-loop) and T54Ψ55C56 (TΨC-loop) sequences and (vi) absence of the conserved modifications, such as m 22 G26, m 7 G46 and D47 1-3 (Supplementary Fig.
4b). tRNA Pyl folds into an L-shaped structure similar to that of canonical tRNAs (Supplementary
Fig. 4c,d). However, the tertiary interactions between the non-standard D-arm, the TΨC-loop and variable region are so different that tRNA Pyl has a quite compact core structure with re-organized tertiary base pairs (Supplementary Fig. 4c).
To facilitate a comparison between the structures of tRNA Pyl and canonical tRNA Phe , we divided the structure comprising the anticodon stem to the TΨC-loop into four regions, as shown in
Supplementary Fig. 5a: region I for the anticodon stem, region II for the augmented D helix, and regions III and IV for the D- and TΨC-loops, respectively. Hereafter, we will refer to the nucleotides at the same stacking level (starting from base pair 31:39 of the anticodon stem) as a base layer, as shown in Supplementary Fig. 5a; bases in the same base layer form a Watson Crick (WC) or non-WC base pair, while bases in the neighboring base layers make stacking interactions.
In region I, canonical tRNA Phe contains 6 base layers, consisting of a 5 base-paired anticodon stem (U31-A39 to G27-C43) and the non-WC base pair of G26-A44, while tRNA Pyl contains 7 base layers, consisting of an extended 6 base-paired anticodon stem (A31-U39 to C26b-G44) and the non-WC base pair of A26a-C45. Consequently, tRNA Pyl has a longer region-I than that of canonical tRNA Phe by one base layer (Supplementary Fig. 5a).
Region II of tRNA Pyl and tRNA Phe commonly contains 4 base layers, centered on the 4 WC base pairs of the D stem. In the canonical tRNA Phe , residues 45 and 46 of the variable region and residue 9 are inserted into the major groove of the D-helix, forming tertiary interactions with the
G10:C25, C13:G22, and U12:A23 base pairs, respectively (Supplementary Fig. 5a,b). In contrast, in tRNA Pyl , only two residues (i.e., A46 and G47) are inserted into the D-helix, making tertiary interactions with the U12:A23 and C13:G22 base pairs, respectively (Supplementary Fig. 5a,c).
The junction between the acceptor and D stems has only G9, which is flipped out and does not participate in the augmented D-helix (Supplementary Fig. 5c).
Region III of the canonical tRNA Phe contains complicated tertiary interactions among the D-,
TΨC- and variable regions, and the invariant U8 residue, which form four base layers consisting of the U8:A14 and G15:C48 base pairs, and the U59 and C60 bases in the TΨC-loop
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(Supplementary Fig. 5a,b). U47 in the long variable region is bulged out (Supplementary Fig. 5b).
In contrast, Region III of tRNA Pyl contains only the D- and TΨC-loops, because of the short variable region (Supplementary Fig. 5a,c). Position 14, which is an invariant adenosine forming a
WC base pair with the invariant U8 in canonical tRNAs (Supplementary Fig. 5a,b), is changed to a guanosine, which makes a WC base pair with C59 (TΨC-loop), due to the lack of U8 in tRNA Pyl
(Supplementary Fig. 5a,c). Position 15, which forms a WC base pair with nucleotide 48 of the variable region in canonical tRNAs (Supplementary Fig. 5a,b), flips out from the core structure because of the absence of nucleotide 48 in tRNA Pyl (Supplementary Fig. 5e). Moreover, nucleotide
60 of the TΨC-loop, which stacks with nucleotide 59 in canonical tRNAs (Supplementary Fig.
5a,b,f), stacks instead onto the G14-C59 base pair (Supplementary Fig. 5a,c,g). Altogether, these unusual interactions, due to the short D- and variable regions, form a very compact core structure with only two base layers in Region III of tRNA Pyl (Supplementary Fig. 5a).
Region IV of the canonical tRNA consists of the D- and TΨC-loops. The invariant G18 and
G19 residues of the D-loop are inserted into the stacking structure formed by the TΨC-loop, thus making non-WC and WC base pairs with the invariant Ψ55 and C56 residues, respectively
(Supplementary Fig. 5a,d). In contrast, in tRNA Pyl , G18 and Ψ55 are changed to A and G, respectively, which no longer make any hydrogen-bonding interactions (Supplementary Fig.
5a,e). The base moiety of G55 assumes the syn conformation by turning around in a reverse direction as compared to the canonical Ψ55 (Supplementary Fig. 5e). G19 and C56 are also changed to U19 and A56, respectively, in tRNA Pyl , which retains a WC interaction at the top of the acceptor-TΨC-helix (Supplementary Fig. 5a,e). The U19:A59 WC base pairing prevents U19 from being modified to dihydrouridine (D), which leads to a lack of the D that confers the nomenclature of the D-loop, in tRNA Pyl .
It should be mentioned here that the continuous stem structure of Regions I, II, and III in tRNA Pyl consists of 13 base layers, which is one layer shorter than that of the canonical tRNA
(Supplementary Fig. 5a). Furthermore, in the tRNA Pyl structure, the TΨC-loop is twisted by about
20 degrees around the acceptor-TΨC-helical axis due to the G14:C59 base pairing (Supplementary Fig. 4d), which places residues 59 and 60 at lower positions (Supplementary Fig. 5a,b).
Nevertheless, the flexible TΨC-helix, which is anchored to the minimal core by the short junction and the short variable region, recovers the twist (Supplementary Fig. 5a). This allows the 7:66 and
49:65 base pairs (at the junction of the TΨC and acceptor stems) to be located at the same spatial position in both tRNA Pyl and tRNA Phe (Supplementary Fig. 5f,g). Consequently, the flexibility of the TΨC-arm as well as the re-organization of the tRNA core allow the acceptor and anticodon arms of tRNA Pyl to adopt quite similar configurations to those of the canonical tRNA
(Supplementary Fig. 4d), which may enable the amber suppressor tRNA Pyl to function normally in the ribosomal translational machinery, like the other canonical tRNAs.
Recognition of the unusual core of tRNA Pyl by DhPylRS
The compact core of tRNA Pyl is recognized by the core binding surface (Fig. 1b,c). The long α2 helix in the tRNA binding domain 1 makes extensive contacts with the sugar backbones of the Dstem base pairs (G10:C25 to C13:G22) from the minor groove sides (Fig. 3c). The side chains of
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Asn47 and Glu50 provide base-specific hydrogen-bonding interactions with the 2-carbonyl group of U24 and the 2-amino group of G10, respectively (Fig. 3d). Furthermore, the short α1 helix in the tRNA binding domain 1 also interacts with the phosphate and sugar moieties of G10 to C13
(Fig. 3d). The C terminus (286–288) of the C terminal tail recognizes the bottom of the D-stem
(Fig. 3c): the main chain carbonyl group of Asn286 hydrogen-bonds with the ribose moiety of
G10, while Asn288, the very C-terminal residue of the enzyme, penetrates into the junction between the D and anticodon stems, to hydrogen-bond with the N1 and O2’ atoms of A26a.
However, the latter interaction is specific to the D. hafniense enzyme, since Asn288 is not conserved in the PylRSs from other organisms (Supplementary Fig. 1). The guanine base of G9, at the junction between the acceptor and D stems, is flipped out and stacks on the guanidinium group of the conserved Arg140 on the α6 helix from another subunit (Fig. 3c). This core binding surface recognizes the D stem and the flipped G9 of the tRNA Pyl -specific unusual configurations. The present observations are consistent with the previous footprint analysis suggesting that DhPylRS binds the phosphate backbones of the TΨC-loop, the D-stem, and the acceptor stem of tRNA Pyl 4 .
Acceptor helix recognition
The acceptor stem of tRNA Pyl is shape complementary to the U-shaped concave structure (Fig.
3b). The α1 helix approaches the minor groove of the acceptor stem to recognize the G4:C69 base pair (Fig. 3b): the side chains of Gln20 and Glu24 hydrogen-bond to the 2-amino group of G4 and to the 2’OH of C69, respectively. The side chain of Lys124 on the bulge domain hydrogen-bonds with the phosphate group of A66 (Fig. 3b). The side chains of His269, Ser278 and Tyr279 in the C terminal tail, forming the bottom of the U-shaped concave structure, hydrogen-bond with the phosphate groups of C71, C69 and C70, respectively (Fig. 3b). Thus, the U-shaped concave structure tightly embraces the acceptor helix to direct its 3’-terminus to the motif 2 loop (Arg160–
Asn170), which appears in a drastically altered conformation in the tRNA complex structure and becomes intercalated into the major groove of the acceptor stem (Fig. 3a, Fig. 4). The side chain and main-chain carbonyl groups of the Gln164 hydrogen-bond with the 4-amino groups of C71 and C72, respectively (Fig. 3a). The N1 and O6 atoms of the discriminator G73 hydrogen-bond with the main-chain carbonyl group of Glu162 and the main-chain amino group of Gln164, respectively (Fig. 3a). These base-specific recognitions of the G1:C72 base pair as well as the discriminator G73 are consistent with results from previous biochemical experiments with DhPylRS 4 .
Discrimination from canonical tRNAs
The anticodon arm (A27–U43) is not recognized by DhPylRS, resulting in very poor electron densities especially at the anticodon loop. This observation is also consistent with the previous mutational analyses, in which the anticodon mutations of tRNA Pyl (CUA to UUU, CUU or AAA) did not affect the aminoacylation activity by DhPylRS 4 . The present complex structure reveals that two features of DhPylRS contribute to the exclusive discrimination of tRNA Pyl from all of the other canonical tRNAs: 1) shape complementary recognition of the unusual minimal core by the core-binding surface and 2) recognition of the acceptor helix by the U-shaped concave structure.
Sequence specific recognition of tRNA Pyl identity elements (described above and in the main text)
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also contribute to the specificity of DhPylRS for tRNA Pyl .
Supplementary Figure Captions
Supplementary Figure 1. Alignment of known PylRS sequences. Note that PylRS has been found in seven distinct microbial species, 5 archaea and 2 bacteria. There are sequences from three strains of D. hafniense of which PylRS from strain PCP-1 is the basis of our crystal structure. The translation of the PylSn gene is included for the bacterial representatives. The PylSn for strain
PCP-1 has not been sequenced, so it is not included in the alignment. Asterisks denote DhPylRS residues in contact with tRNA Pyl . Colored bars show the domain organization of DhPylRS with the same coloring scheme as in Fig. 1.
Supplementary Figure 2. Time course of in vitro aminoacylation activity with Cyc of D. hafniense tRNA Pyl transcript by DhPylRS (●), D. hafniense tRNA Pyl transcript by DhPylRS and DhPylSn (◊),
D. hafniense tRNA Pyl transcript in competition with total E. coli tRNA by DhPylRS (  ), total E. coli tRNA by DhPylRS (  ), Methanosarcina barkeri tRNA Pyl transcript by truncated M. mazei PylRS
(185-454 aa) 1 (  ) and M. barkeri tRNA Pyl transcript by full length M. barkeri PylRS (  ) as visualized by thin layer chromatography.
Supplementary Figure 3. DhPylRS activity in vivo was tested by transforming the E. coli FTP5822 strain 5,6 with pylS in the pCBS vector and/or pylT in the pTECH vector. E. coli strains transformed with the genes indicated are streaked. 1) M. barkeri pylS and M. barkeri pylT; 2) D. hafniense pylS and D. hafniense pylT; 3) D. hafniense pylS, empty pTECH vector; 4) empty pCBS vector, D.
hafniense pylT; 5) empty pCBS vector, empty pTECH vector. (a) Minimal medium with Cyc and without tryptophan (after 55 hr growth), (b) minimal medium without Cyc or tryptophan (after
55 hr growth), (c) minimal medium with tryptophan (after 24 hr growth). (d) Growth curves in
M9 minimal liquid medium of E. coli strain FTP5822 transformed with M. barkeri pylS and pylT
(blue filled circle, ● ), D. hafniense pylS and pylT ( ○ ), D. hafniense pylS, pylSn, and pylT (◊), D.
hafniense pylS ( ▼ ), or D. hafniense pylT (

). All strains are grown in the presence of 10 mM Cyc. As controls, E. coli strain FTP5822 transformed with D. hafniense pylS and pylT and grown in minimal medium in the presence of tryptophan ( ■ ) and absence of Cyc and Trp (□) are shown. The curves shown are based on the average of duplicate measurements.
Supplementary Figure 4. tRNA Pyl structure. (a) The interactions between DhPylRS and tRNA Pyl are shown schematically on this interaction map. (b) The secondary and tertiary structures of tRNA Pyl . The acceptor, D-, anticodon, and TΨC-arms, and the variable region are colored red, yellow, green, purple, and sky blue, respectively. (c) Comparison of tRNA Pyl and canonical tRNA Phe . The strikingly distinct D-arms are highlighted in red. (d) Superimposition of tRNA Pyl onto tRNA Phe , based on the acceptor and anticodon stems.
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Supplementary Figure 5. Detailed comparison of the tRNA Phe and tRNA Pyl structures. Coloring is as defined in Supplementary Fig. 4. (a) The tertiary base arrangement diagrams of tRNA Phe and tRNA Pyl . Regions I-IV are differently colored, and the base layers are distinguished by contrasting color intensity. (b,c) Stereo views of the base layers in regions II and III of tRNA Phe and tRNA Pyl .
(d,e) Stereo views of regions III and IV of tRNA Phe and tRNA Pyl , highlighting the TΨC-loop:Dloop interactions. (f,g) Stereo views of the core regions including the TΨC- and D-loops and the variable regions in tRNA Phe and tRNA Pyl .
Supplementary Figure 6. Evolutionary conservation in the DhPylRS:tRNA Pyl interface. The complex is shown (a) with and (b) without one of the tRNAs. Residues in contact with the tRNAs are colored according to sequence conservation among the ten known PylRS sequences. Most of the residues that comprise the interface are strictly conserved (blue), decreasing conservation is indicated by pink (moderately conserved) and red (non-conserved) coloring. For reference, Pyl-
AMP has been placed in the active site pocket according to homology with the MmPylRS (2zim)
7 .
Supplementary Figure 7. DhPylRS active site with Pyl (ball and stick representation) placed according to homology with MmPylRS 7 . Trp139 replaces the much smaller Leu309 of MmPylRS.
Residues within 3.6 Å of the modeled Pyl are highlighted (stick representation) with Tyr217 from the Pyl recognition loop shown in the foreground, bent away from the active site of DhPylRS.
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