PMC

(A) Phospho-mimetic LysRS^S207D has enhanced Ap₄A synthetic activity.
(B) Phospho-mimetic LysRS^S207D has enhanced ATP hydrolysis.
(C) Phospho-mimetic LysRS^S207D loses aminoacylation activity for tRNA^Lys. Four concentrations of tRNA were used for this assay. Error bars are standard deviations (SDs) from triplicates.
(D) Phosphorylation of Ser207 redirects the enzymatic flow of LysRS from aminoacylation to Ap₄A synthesis to activate transcription.
(E) Functional replacement assays in yeast show that LysRS^S207D is defective in essential translational functions in cells. The expression of endogenous yeast LysRS can be switched off by the addition of doxycycline to the yeast growth medium. Ten-fold serial dilutions of freshly grown yeast cells were spotted onto selective media SCM-HIS containing 2% raffinose with or without doxycycline and galactose.

(A) Schematic diagram of the structure of the human LysRS dimer.
(B–C) Deuterium uptake profiles for LysRS^S207D and LysRS^WT. Segments with an increased deuterium exchange rate are mapped in orange onto the 3-dimensional LysRS structure (C).
(D) Solution envelopes of LysRS^WT versus LysRS^S207D calculated from their small angle x-ray scattering curves. The 3-dimensional structure of LysRS^S207D is modeled by docking the N-domains into its extended solution envelope.
(E) Cartoon depicting the relationship between the two conformations of LysRS^WT versus LysRS^S207D.
(F–H) Structural model of the eGFP fragment complementation construct that fuses the N-terminal half of eGFP (eGFP_N) and C-terminal half of eGFP (eGFP_C) to the ends of human LysRS for monitoring the conformational change in HEK293T cells (F). Confocal images were taken 24 h after HEK293T cells were transfected with eGFP_N-LysRS-eGFP_C (G). GFP fluorescence was quantified by the ratio of green fluorescence to DAPI nucleic acid stain (H).
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(A) In vivo Ap₄A assay shows that the structural-opening mutant LysRS^G540Y produced high level of Ap₄A independent of mast cell activation. RBL cells were transfected with siLysRS and with plasmids expressing knockdown-resistant versions of LysRS^WT, LysRS^S207D or LysRS^G540Y. After 24 h, cells were stimulated with the IgE-DNP trigger. The means and standard errors of the means for three experiments are shown. P<0.05.
(B) Isothermal titration calorimetry assays for binding of the MITF bHLH-Zip domain to LysRS^WT, LysRS^S207D or the C-terminal aminoacylation domain of LysRS.
(C) Model for the phosphorylation-dependent translation and transcription switch of LysRS driven by structural opening. In quiescent mast cells, LysRS is associated with p38 in a closed form and is retained in the cytoplasmic MSC. Antigen activation phosphorylates Ser207 and triggers an open form of LysRS. By opening up the structure, phosphorylated LysRS is released from the MSC, translocates from cytoplasm to the nucleus, binds to MITF and generates Ap4A to activate MITF transcription functions. Thus, by selecting two distinct conformers, phosphorylation could switch the functional of LysRS between translation and transcription.
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(A–B) Two orthogonal views of the human LysRS:p38/AIMP2 complex structure. Sequence of p38/AIMP2 is color-coded as in (C).
(C) Sequence alignment of the N-terminus of p38/AIMP2 from Drosophila to human. Motifs are colored as Motif 1 (yellow), Gly linker (gray), turn (red) and Motif 2 (green). Spheres represent two similar sets of residues (yellow/green) implicated in the LysRS:p38/AIMP2 interaction.
(D–E) The interface of the LysRS-p38/AIMP2 complex. The surface representation of LysRS is shown. D, Motif 1 of p38/AIMP2 and its interaction with the bottom groove of LysRS. E, Motif 2 of p38/AIMP2 and its interaction with the symmetric groove on the LysRS dimer.
(F) The functional LysRS-p38/AIMP2 complex. Side view of free human LysRS structure (pdb: 3bju, gray) superimposed onto human LysRS:p38/AIMP2 complex, showing the close similarity of LysRS structures with and without binding to p38/AIMP2.
(G) Model of the human multi-tRNA synthetase complex. Dimerization of p38/AIMP2 is through the two helical regions and a C-terminal GST-domain outside of the LysRS binding site. The N-terminal linker (residues 32–48) is disordered as found in the crystal structure. p38/AIMP2 binds to at least 8 of the 11 members of the MSC and forms the core of the MSC. This model represents the canonical functional state of LysRS in the MSC for charging tRNA.
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(A) Close-up view of the domain-domain interface in the human LysRS dimer.
(B) Sequence alignment of the LysRS anticodon-binding domain (N-) and the aminoacylation domain (C-) interface. Phosphorylation site Ser207 and the opposite side G540 mutations analyzed in (C, D) are highlighted in boxes.
(C–D) Aminoacylation assays show that mutations at the dimer interface affect tRNA charging. Mutations with positive charge (S207R) are less effective at inhibiting aminoacylation activity, suggesting that the negative charge of the phosphate group on LysRS_S207 is also important for introducing repulsion between the domain interfaces.
(E) Electrophoretic mobility shift assay shows that LysRS^S207D binds tRNA with an affinity similar to that of LysRS^WT. Increasing concentrations of the protein (0, 0.1, 0.2, 0.4, 0.8, 1.6, 2.4, 3.2, 5.0, 6.25, and 7.5 μM, respectively) were used.
(F) Mechanism that Ser207-phosphorylation switches off the aminoacylation function of LysRS. Top, a docking model of human LysRS in complex with tRNA by superimposition of the human LysRS structure (pdb3bju) with the yeast AspRS-tRNA^Asp complex structure (pdb1asy) shows that the “closed” form of wild type LysRS places the 3′-CCA end of tRNA into the catalytic site. Bottom, the N-terminal tRNA anticodon-binding domain of human LysRS^p207 is modeled on the SAXS envelope of LysRS^S207D..
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(A) View of the LysRS:p38/AIMP2 interface showing that the Ser207 phosphorylation site is at the inter-domain interface that forms the p38/AIMP2 binding groove. Ser207 is 13 Å away from the bound p38/AIMP2.
(B) Co-immunoprecipitation analyses showing that mutations of LysRS predicted to affect the p38/AIMP2 interaction (V101D, V101W, V101R), a mutation causing structural opening (G540Y) and the phospho-mimetic mutation (S207D), all dissociate LysRS from the endogenous MSC in HEK293T cells.
(C) Pull-down assay showing LysRS^S207D loses the ability to bind a recombinant version of p38/AIMP2.
(D) Mechanism of Ser207 phosphorylation-directed release of LysRS from the MSC: The flipped-out anticodon-binding domain in LysRS^pS207 opens the binding groove for p38/AIMP2 and prevents LysRS-p38/AIMP2 interaction.
(E) Confocal immunofluorescence microscopy showing the nuclear localization of the endogenous LysRS (red) and MITF (green) in quiescent RBL mast cells and following an IgE-DNP trigger. The blue signal is nuclear DAPI. The picture was taken by a confocal microscopy in Z-mode. The amount of LysRS and MITF signal in the cross section is quantified at the bottom.
(F) Nuclear localization of LysRS following RBL mast cell activation. Western blot analysis of LysRS in cytoplasmic versus nuclear fractions in quiescent mast cells versus in cells activated by the IgE-DNP trigger for 5 min.
(G) Confocal live imaging quantification of LysRS nuclear translocation. Means ROI (region of illuminated) of nuclear eGFP-LysRS^wt versus eGFP-LysRS^S207D at quiescent state (Q) and 5, 10, 15 min after immune activation. The means and standard errors of the means for 20 representative cells are shown (P<0.05).
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