X-shaped structure of bacterial heterotetrameric tRNA synthetase suggests cryptic prokaryote functions and a rationale for synthetase classifications

Abstract AaRSs (aminoacyl-tRNA synthetases) group into two ten-member classes throughout evolution, with unique active site architectures defining each class. Most are monomers or homodimers but, for no apparent reason, many bacterial GlyRSs are heterotetramers consisting of two catalytic α-subunits and two tRNA-binding β-subunits. The heterotetrameric GlyRS from Escherichia coli (EcGlyRS) was historically tested whether its α- and β-polypeptides, which are encoded by a single mRNA with a gap of three in-frame codons, are replaceable by a single chain. Here, an unprecedented X-shaped structure of EcGlyRS shows wide separation of the abutting chain termini seen in the coding sequences, suggesting strong pressure to avoid a single polypeptide format. The structure of the five-domain β-subunit is unique across all aaRSs in current databases, and structural analyses suggest these domains play different functions on α-subunit binding, ATP coordination and tRNA recognition. Moreover, the X-shaped architecture of EcGlyRS largely fits with a model for how two classes of tRNA synthetases arose, according to whether enzymes from opposite classes can simultaneously co-dock onto separate faces of the same tRNA acceptor stem. While heterotetrameric GlyRS remains the last structurally uncharacterized member of aaRSs, our study contributes to a better understanding of this ancient and essential enzyme family.


Supplementary Figure S1. Measuring the oligomeric state of EcGlyRS-FL and EcGlyRS575
in solution using size-exclusion chromatography. The apparent molecular weights of EcGlyRS-FL and EcGlyRS575 in solution were measured using a HiLoad 16/60 Superdex 200 pg column (Cytiva) which was calibrated with standard proteins from Gel Filtration LMW Calibration Kit (Cytiva). EcGlyRS-FL was eluted as a sharp symmetric peak at 62.5 mL, which is corresponding to the molecular weight of MW SEC = 253.9 kDa. This number is close to the molecular weight deduced from the protein sequences of the predicted heterotetrameric form of EcGlyRS-FL (MW deduced = 225.2 kDa), indicating that EcGlyRS-FL is a heterotetramer in solution. Similarly, EcGlyRS575 was eluted at 64.6 mL with a MW SEC = 212.9 kDa, which is close to MW deduced = 199.2 kDa and supports that EcGlyRS575 forms a heterotetramer in solution. Synechococcus elongatus GlyRS (SeGlyRS, UniProtKB ID: Q31KD2) and Streptococcus pneumoniae GlyRS (SpGlyRS, UniProtKB ID: B8ZL21) were aligned using Clustal Omega program(1). The residue numbering and secondary structures corresponding to EcGlyRS are displayed above the sequences. The conservation scores were calculated by the program Jalview (2) and exhibited in various shades of purples. The signature motifs 1, 2, and 3 conserved in class II aaRSs are marked with salmon, yellow and blue arrows, respectively. Figure S5. The structural flexibility of the B2 domain. The structure superposition of the two protomers (colored the same as in Figure 1B) reveals that both protomers are almost identical except that their B2 domains undergo large conformation movement between two protomers.

Supplementary
Supplementary Figure. S6. Domain-swapping interactions between the residual ABD sequences of two EcGlyRS575 proteins from adjacent asymmetric units contribute to crystal packing. The yellow cube represents an asymmetric unit that contains one EcGlyRS575 molecule consisting of two protomers (colored in magenta for protomer1 and cyan for protomer 2). The two protomers of the adjacent EcGlyRS575 molecules are colored in gray and gold, respectively. The zoom-in views show the formation of domain-swapping interactions between adjacent EcGlyRS575 molecules. Figure S7. The phosphate groups of AMP-PNP rotate toward the β-subunit.

Structural comparison of the aminoacylation pockets between EcGlyRS and CjGlyRS-ATP-glycine
and CjGlyRS-ATP complexes (PDB: 3grl and 3ufg, colored yellow), and the major conformation differences of the phosphate groups are indicated by red arrows.  (2) and exhibited in various shades of purples. Figure S12. Binding of full length and truncated EcGlyRS to tRNA Gly is analyzed by using size-exclusion chromatography. The binding assays were performed by using a Superdex 200 increase 10/30 column (Cytiva), which was calibrated with standard proteins from Gel Filtration LMW Calibration Kit (Cytiva). Full and dash lines represent the absorption at a wavelength of 280 and 260 nm, respectively. The molar ratio for the EcGlyRS protein and tRNA Gly is 4:1. As shown in (A), after 30 min co-incubation at room temperature, EcGlyRS-FL and tRNA Gly were able to form a peak (gray) which shifts to the left compared to the peak of EcGlyRS-FL alone (blue). And the UV260 of the gray peak increased significantly compared to the blue peak, indicating the complex formation of EcGlyRS-FL and tRNA Gly . After using the same co-incubation process as EcGlyRS-FL, in contrast, the EcGlyRSΔB2 (B) and EcGlyRS575 (C) protein could not form a complex with the same amount of tRNA Gly , as their gray peaks in gel-filtration showed almost the same location and intensity with the blue peak. These results showed the importance of the B2 domain and ABD of

Supplementary Table 1. Statistics of X-ray diffraction data collection and structure refinement.
a Values in parentheses are for the highest resolution shell. b R merge = Σ h Σ l | I(h) l -<I(h)> | / Σ h Σ l I(h) l , where I(h) l is the lth observation of the reflection h and <I(h)> is the weighted average intensity for all observations l of reflection h. c R work =∑ h | | F obs (h) | -| F cal (h) | | /∑ h | F obs (h) |, where F obs (h) and F cal (h) are the observed and calculated structure factors for reflection h respectively. d R free was calculated as R work using 5.0% of the reflections which were selected randomly and omitted from refinement.

Data collection
Resolution (