Methyl transfer by substrate signaling from a knotted protein fold


Proteins with knotted configurations, in comparison with unknotted proteins, are restricted in conformational space. Little is known regarding whether knotted proteins have sufficient dynamics to communicate between spatially separated substrate-binding sites. TrmD is a bacterial methyltransferase that uses a knotted protein fold to catalyze methyl transfer from S-adenosyl methionine (AdoMet) to G37-tRNA. The product, m1G37-tRNA, is essential for life and maintains protein-synthesis reading frames. Using an integrated approach of structural, kinetic, and computational analysis, we show that the structurally constrained TrmD knot is required for its catalytic activity. Unexpectedly, the TrmD knot undergoes complex internal movements that respond to AdoMet binding and signaling. Most of the signaling propagates the free energy of AdoMet binding, thereby stabilizing tRNA binding and allowing assembly of the active site. This work demonstrates new principles of knots as organized structures that capture the free energies of substrate binding and facilitate catalysis.

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Figure 1: Ternary crystal structure and dynamics of the TrmD–tRNA–SFN complex.
Figure 2: Molecular simulations of the bent versus open shape of AdoMet.
Figure 3: Signaling strengths from individual protein-ligand contacts mapped to the ternary structure of HiTrmD.
Figure 4: Mutations leading to fold changes in kinetic parameters of TrmD methyl transfer.
Figure 5: Effects of the Y115A mutation.
Figure 6: The active sites and the ligand binding stoichiometries of the wild-type and Y115A structures of TrmD.
Figure 7: Diagram of substrate signaling in TrmD.

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This work was supported by US National Institutes of Health grants GM108972 and GM114343 (to Y.-M.H.); European Molecular Biology Organization (EMBO) installation grant 2057 and National Science Center grant Sonata BIS 2012/07/E/NZ1/01900 (to J.I.S.); and Targeted Proteins Research Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan, JSPS KAKENHI grant no. 20247008 (to T.I. and S.Y.). We thank I. Masuda, R. Takase, R. Matsubara, S. Maharjan, and K. Donaldson for preparation of figures.

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T.C., R.S., and G.L. performed kinetic analysis; A.P.P. and J.I.S. performed computational molecular simulation analysis; T.I. and S.Y. performed structural analysis; E.A.T. performed sequence-conservation analysis; and J.I.S. and Y.-M.H. prepared the manuscript. All authors discussed and commented on the manuscript.

Corresponding author

Correspondence to Ya-Ming Hou.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Conformations of AdoMet in TrmD and Trm5.

(a) The bent conformation of AdoMet when bound to TrmD (PDB 1UAK)19 and (b) the extended open conformation of AdoMet when bound to Trm5 (PDB 2ZZN)20. (c) Structure of TrmD-SFN-tRNA (PDB 4YVI) and (d) structure of Trm5-AdoMet-tRNA (PDB 2ZZN). D1, D2, and D3 are three domains of Trm5. (e) Docking and simulation analysis of AdoMet in the structure of Trm5 with tRNA. The AdoMet in the existing crystal structure is shown in orange (PDB 2ZZN), whereas the docked and simulated AdoMet in the bent shape is shown in green and the simulated open shape is shown in light blue. The distance between the methyl group of AdoMet and the N1 atom of G37 is indicated on the dotted line of each structure in the respective color. Drawn by PyMol. Comparison of TrmD and Trm5 structures show that (1) TrmD is an obligate homodimer, whereas Trm5 is an active monomer, (2) TrmD binds AdoMet in the trefoil-knot, whereas Trm5 binds AdoMet in the open space of a Rossmann-fold, (3) TrmD active-site is located in the deep cleft of the dimer interface, whereas Trm5 active-site is in an easily accessible region between D2 and D3, and (4) TrmD binds only the anticodon-stem loop domain of tRNA, whereas Trm5 binds the entire L-shape of tRNA, particularly holding on the tertiary core region of the L. Moreover, the best superposition of the active site in TrmD and in Trm5 (48 residues) gives an r.m.s.d. of 6.7 Å, indicating that the two active sites are distinct from each other. These are fundamental differences that contribute to the different placement of the active site in TrmD and in Trm5.

Supplementary Figure 2 R.m.s.d. of Cα atoms of both chains in wild-type and mutated structures of TrmD.

(a) Wild-type and mutated ternary complex structures. (b) TrmD structures: apoenzyme, TrmD with two AdoMets, and TrmD with two AdoMets and one tRNA. Trm5 structure: Trm5 with AdoMet and tRNA. (c) Three different trajectories of TrmD with two AdoMets and one tRNA. (d) Three different trajectories of TrmD mutant Y115A with two AdoMets and one tRNA. (e) Hydrogen bonds between all amino acids in wild-type and mutated ternary complex structures. (f) Hydrogen bonds between all amino acids in TrmD structures: apoenzyme, TrmD with two AdoMets, and TrmD with two AdoMets and one tRNA. (g) Hydrogen bonds between all amino acids in three different trajectories of TrmD with two AdoMets and one tRNA. (h) Hydrogen bonds between all amino acids in three different trajectories of TrmD mutant Y115A with two AdoMets and one tRNA. All simulations reached equilibrium within 20 ns and some remained stable up to 400 ns (the longest time performed for simulation).

Supplementary Figure 3 Analysis of r.m.s.f. across TrmD or Trm5.

(a) Root Mean Square Fluctuation (r.m.s.f.) of wild-type TrmD structures (the apo-enzyme and the ternary complex with tRNA). Grey area indicates the knot region (residues 83 to 141), whereas orange dashed lines indicate the most important hydrogen (H) bonds between the protein and AdoMet. The knot region has the lowest difference in its fluctuations among different mutant proteins and also is the most rigid part of the protein regardless of the presence or absence of ligands. (b) Comparison of r.m.s.f. between ternary structures of TrmD (knotted protein) and Trm5 (unknotted protein) based on Cα atoms. Grey area represents the active site in TrmD (residues 83 to 141) and light green area represents the active site in Trm5 (residues 200 to 250). The averages from selected areas are presented. (c) R.m.s.f. of a second TrmD in the apo-enzyme and the ternary complex form. (d) Comparison of the second TrmD ternary complex with the Trm5 ternary complex.

Supplementary Figure 4 Mixing controls for determination of kobs.

(a) Three types of mixing were performed: (1) premixing of AdoMet and tRNA, followed by mixing with TrmD (shown in red), (2) premixing of TrmD and tRNA, followed by mixing with AdoMet (shown in blue), and (3) premixing of TrmD and AdoMet, followed by mixing with tRNA (shown in green). Plots of kobs as a function of TrmD concentration. Three independent measurements were performed for each experiment. (b) Fitting the average values of each experiment in (a) to determine Kd (AdoMet) and kchem of methyl transfer. These data showed that different mixing orders generated similar values of Kd (AdoMet) and kchem. Errors bars are s.d. (n = 3 independent experiments).

Supplementary Figure 5 The hydrogen-bond frequency between TrmD residues and AdoMet, determined by simulation analysis of the G55A mutant versus the wild-type enzyme.

(a) Analysis of TrmD residues in the wild-type structure (upper panel) and in the G55A mutant structure (lower panel), showing the decrease in frequency at Y86, G113, and L138. These residues are in the AdoMet-binding pocket. “Holo” refers to the enzyme-AdoMet binary complex. Data for 3 enzyme binary complexes with AdoMet and 3 enzyme ternary complexes with tRNA are shown. (b) Analysis of the protein G55 residue for interaction with G27 in the tRNA (G55-G27), the protein E116 residue for interaction with G37 in the tRNA (E116-G37), and the protein D169 residue for interaction with G37 in the tRNA (D169-G37) in the wild-type and G55A mutant structure. The data show that the G55-G27 interaction is reduced to 40% in the mutant relative to the wild-type enzyme. Error bars are s.d. (n =3 independent experiments).

Supplementary Figure 6 Simulations of TrmD dimers.

(a) Correlation of experimentally and computationally determined binding energy of AdoMet. The computational values were obtained using MMPBSA, while the experimental values were obtained from kinetic analysis shown in Supplementary Table 3. Error bars for both experimental values (in X axis) and computational values (in Y axis) are s.d. (n = 3 independent experiments). The trend line shows linear correlation of these values between the two sets of data (r = 0.96 for site A). (b) The sum of the first 5 eigenvalues with respect to the structural regions of TrmD. Comparison of 3 structures: TrmD without any substrates, TrmD with two AdoMets, and TrmD with two AdoMets and one molecule of tRNA. Blue refers to the region of TrmD before the knot (residues 1-82), orange refers to the knotted region (residues 83-144), and yellow refers to the region after the knot (residues 145-246). Error bars are s.d. (n =3 independent experiments). (c) Solvent Accessible Surface Area (SASA) of the two active sites. Active site A consists of the knot from chain A and the CTD of chain B. (d) Root Mean Square Deviation (r.m.s.d.) of AdoMet in each chain. Error bars are s.d. (n =3 independent experiments).

Supplementary Figure 7 Dimer structure of EcTrmD mutants.

(a) Size exclusion analysis of EcTrmD mutants through a Superdex 75 column. Calibration of the column with marker proteins is shown on the top and determination of the molecular weights of EcTrmD mutants based on retention time is shown at the bottom. All EcTrmD mutants exhibited an apparent molecular weight of ~60 kDa, the predicted mass of a TrmD dimer based on the natural size of 274 amino acids for each monomer with the addition of an N-terminal His tag. (b) Native gel analysis of EcTrmD mutants, all of which co-migrated with the wild-type (wt) enzyme to a position corresponding to an apparent molecular weight of ~60 kDa. The small variations in migration among EcTrmD mutants suggested the possibility of different globular shapes caused by individual mutations.

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Supplementary Figures 1–7, Supplementary Tables 1–6 and Supplementary Note (PDF 1884 kb)

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Christian, T., Sakaguchi, R., Perlinska, A. et al. Methyl transfer by substrate signaling from a knotted protein fold. Nat Struct Mol Biol 23, 941–948 (2016).

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