Methyl transfer by substrate signaling from a knotted protein fold

Journal name:
Nature Structural & Molecular Biology
Volume:
23,
Pages:
941–948
Year published:
DOI:
doi:10.1038/nsmb.3282
Received
Accepted
Published online

Abstract

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.

At a glance

Figures

  1. Ternary crystal structure and dynamics of the TrmD-tRNA-SFN complex.
    Figure 1: Ternary crystal structure and dynamics of the TrmD–tRNA–SFN complex.

    (a) The TrmD dimer binds one tRNA and two SFNs. Amino acid residues 1–160 are in the NTD, 169–246 are in the CTD, and 161–168 are in the flexible linker. (b) Ribbon diagram of the trefoil knot in chain A, showing β-strands in green (except for β3 in red), α-helices in orange, and SFN in stick representation. The r.m.s.d. of the superposition of the knot with the knot in an AdoMet-bound binary complex is 0.32 Å for 64 Cα atoms. (c) Ribbon diagram of the interdomain linker (residues 160–169) of chain B, which becomes organized after binding of G37-tRNA. The r.m.s.d. of superposition of the CTD containing this linker deviates from the corresponding region in the binary complex by 0.94 Å for 85 Cα atoms outside of the linker. Structure is based on PDB 4YVI (ref. 31). (d) Mean SASA of the active site A and active site B of HiTrmD (4.2 versus 4.8 nm2) and MjTrm5 (8.2 nm2). Purple, active site A; teal, active site B. Error bars, s.d. (n = 3 independent simulations). (e) R.m.s.d. of AdoMet A and AdoMet B in the active site of HiTrmD (1.3 and 3.3 Å) and MjTrm5 (2.0 Å). Purple, AdoMet A; teal, AdoMet B. Error bars, s.d. (n = 3 independent simulations). (f) Superposition of AdoMet conformations throughout a course of molecular dynamics simulations. The convergence of simulations was confirmed through independent and multiple rounds of calculations. Simulations were based on the crystal structure31 of HiTrmD (PDB 1UAK) and the crystal structure29 of MjTrm5 (PDB 2ZZN).

  2. Molecular simulations of the bent versus open shape of AdoMet.
    Figure 2: Molecular simulations of the bent versus open shape of AdoMet.

    (a,b) The shape of AdoMet, as shown by sticks (a) and by van der Waals surface (b) in the TrmD knot in the bent and the open conformation. (c,d) Positions of TrmD catalytic residues required for methyl transfer from AdoMet in the bent (c) and open (d) conformations. (e,f) Accommodation of the bent shape in the knotted TrmD structure31 (PDB 4YVG) (e) and in the unknotted TrmD structure37 (PDB 1OY5) (f). (g) AdoMet in the knotted TrmD structure has the option to adopt the bent conformation or the alternative open conformation31 (PDB 4YVG). (h) AdoMet in the unknotted TrmD structure37 (PDB 1OY5) can adopt only the extended open conformation but not the bent conformation. Simulations in g and h were performed with SiteMap software.

  3. Signaling strengths from individual protein-ligand contacts mapped to the ternary structure of HiTrmD.
    Figure 3: Signaling strengths from individual protein-ligand contacts mapped to the ternary structure of HiTrmD.

    (a) Surface representation of HiTrmD (blue), showing quantitative effects of individual alanine substitutions (from white to pink to red) on protein-AdoMet binding. (b) Surface representation of HiTrmD, showing quantitative effects of individual alanine substitutions on protein-tRNA binding. (c) Surface representation of HiTrmD, showing quantitative effects of individual alanine substitutions on the kchem (kobs, max) of methyl transfer. The scale at the bottom of each panel calibrates ΔΔG0 = −RT ln[(kchemwild type/kchemmutant)], where T = 310 K, and R = 1.987 kcal/mol.

  4. Mutations leading to fold changes in kinetic parameters of TrmD methyl transfer.
    Figure 4: Mutations leading to fold changes in kinetic parameters of TrmD methyl transfer.

    Values from enzyme titration of tRNA were used for free-energy analysis. Blue bars, fold changes in Kd (AdoMet); red bars, fold changes in Kd (tRNA); green bars, fold changes in kchem (methyl transfer). (a,b) Structures and residues tested by alanine substitutions of the Ado pocket (a) and the fold change relative to the wild-type enzyme for each substitution (b). WT, wild type. (c,d) Structures and residues tested by alanine substitutions of the Met pocket (c) and the fold change relative to the wild-type enzyme for each substitution (d). (e,f) Structures and residues tested by alanine substitutions of the G37 pocket (e) and the fold change relative to the wild-type enzyme for each substitution (f). X marks on axis indicate mutants not tested. Error bars in b, d and f, s.d. (n = 5 independent measurements).

  5. Effects of the Y115A mutation.
    Figure 5: Effects of the Y115A mutation.

    (a) Simulation of the active site of chain A (magenta), chain B (cyan), and tRNA (orange), showing residues with the most notable changes in structure and dynamics as sticks in the Y115A-substitution mutant. (b) The mean occurrence of hydrogen bonds in the wild-type (left) and Y115A (right) structures. Blue, hydrogen bond between D177* and AdoMet A; orange, hydrogen bond between D177* and AdoMet B; yellow, hydrogen bond between Glu116 and nucleotide G37 in tRNA; green, hydrogen bond between Arg145 and nucleotide G37 in tRNA; burgundy, hydrogen bond between Asp50 and nucleotide G36 in tRNA. Error bars, s.d. (n = 3 independent trajectories). (c) Projection of the knots of the wild-type and Y115A-mutant structures along the first and second principal component (PC) in the free-energy landscape. The wild-type structure is based on HiTrmD31 (PDB 4YVI).

  6. The active sites and the ligand binding stoichiometries of the wild-type and Y115A structures of TrmD.
    Figure 6: The active sites and the ligand binding stoichiometries of the wild-type and Y115A structures of TrmD.

    (a) Mean SASA of active site A (light gray) and active site B (dark gray). In active site A, the knot is from chain A, and the CTD is from chain B, whereas in active site B, the knot is from chain B, and the CTD is from chain A. (b) Mean r.m.s.d. of AdoMet A (light gray) and AdoMet B (dark gray). Error bars, s.d. (n = 3 independent simulations). (c,d) Binding stoichiometry of tRNA to TrmD dimers. Determination of binding stoichiometry by monitoring the quenching of TrmD's intrinsic tryptophan fluorescence with increasing amounts of tRNA, for the wild-type dimer (c) and the Y115A dimer (d). (e,f) Biding stoichiometry of AdoMet to TrmD dimers. Determination of binding stoichiometry by monitoring the quenching of TrmD's intrinsic tryptophan fluorescence with increasing amounts of AdoMet, for the wild-type dimer (e) and the Y115A dimer (f). Error bars, s.d. (n = 3 independent measurements).

  7. Diagram of substrate signaling in TrmD.
    Figure 7: Diagram of substrate signaling in TrmD.

    This signaling starts with Ado binding to the trefoil knot. Met then binds, and active site assembly occurs through G37 binding, which stabilizes tRNA binding. Methyl transfer then occurs at the active site.

  8. Conformations of AdoMet in TrmD and Trm5.
    Supplementary Fig. 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.

  9. R.m.s.d. of C[alpha] atoms of both chains in wild-type and mutated structures of TrmD.
    Supplementary Fig. 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).

  10. Analysis of r.m.s.f. across TrmD or Trm5.
    Supplementary Fig. 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.

  11. Mixing controls for determination of kobs.
    Supplementary Fig. 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).

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

  13. Simulations of TrmD dimers.
    Supplementary Fig. 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).

  14. Dimer structure of EcTrmD mutants.
    Supplementary Fig. 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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Referenced accessions

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Author information

  1. These authors contributed equally to this work.

    • Thomas Christian,
    • Reiko Sakaguchi &
    • Agata P Perlinska

Affiliations

  1. Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania, USA.

    • Thomas Christian,
    • Reiko Sakaguchi,
    • Georges Lahoud &
    • Ya-Ming Hou
  2. Center of New Technologies, University of Warsaw, Warsaw, Poland.

    • Agata P Perlinska &
    • Joanna I Sulkowska
  3. Inter-Faculty Individual Studies in Mathematics and Natural Sciences, University of Warsaw, Warsaw, Poland.

    • Agata P Perlinska
  4. RIKEN Systems and Structural Biology Center, Yokohama, Japan.

    • Takuhiro Ito &
    • Shigeyuki Yokoyama
  5. Graduate School of Science, University of Tokyo, Tokyo, Japan.

    • Takuhiro Ito &
    • Shigeyuki Yokoyama
  6. RIKEN Center for Life Science Technologies, Yokohama, Japan.

    • Takuhiro Ito
  7. Department of Chemistry, Wesleyan University, Middletown, Connecticut, USA.

    • Erika A Taylor
  8. RIKEN Structural Biology Laboratory, Yokohama, Japan.

    • Shigeyuki Yokoyama
  9. Department of Chemistry, University of Warsaw, Warsaw, Poland.

    • Joanna I Sulkowska

Contributions

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.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Conformations of AdoMet in TrmD and Trm5. (629 KB)

    (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.

  2. Supplementary Figure 2: R.m.s.d. of Cα atoms of both chains in wild-type and mutated structures of TrmD. (271 KB)

    (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).

  3. Supplementary Figure 3: Analysis of r.m.s.f. across TrmD or Trm5. (413 KB)

    (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.

  4. Supplementary Figure 4: Mixing controls for determination of kobs. (88 KB)

    (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).

  5. 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. (246 KB)

    (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).

  6. Supplementary Figure 6: Simulations of TrmD dimers. (113 KB)

    (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).

  7. Supplementary Figure 7: Dimer structure of EcTrmD mutants. (78 KB)

    (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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  1. Supplementary Text and Figures (1,929 KB)

    Supplementary Figures 1–7, Supplementary Tables 1–6 and Supplementary Note

Additional data