Supplemental materials for

"Utilization of lysine 13C-methylation NMR for protein–protein interaction studies"

Y. Hattori et al.

Two tables and 13 figures:

Table S1. ITC parameters of non-methylated ubiquitin or methylated ubiquitin titration of YUH1.

Table S2. Intermolecular distances between lysine side-chain Nζ or backbone N atoms of ubiquitin and the nearest heavy atoms of YUH1 or Dsk2UBA.

Figure S1. Reaction scheme of the 13C-methylation of the amino group.

Figure S2. Matrix-assisted laser desorption/ionization (MALDI) MS spectra of non-methylated ubiquitin and methylated ubiquitin.

Figure S3. 1H-13C HSQC spectra of fully or partially di-methylated ubiquitin.

Figure S4. 1H-13C HSQC spectra of mutated or N-terminal-tag digested methylated ubiquitin.

Figure S5. ITC experiments of non-methylated ubiquitin or methylated ubiquitin titrating YUH1.

Figure S6. 1H and 15N chemical shift changes of non-methylated ubiquitin and methylated ubiquitin in the presence of YUH1.

Figure S7. NMR titration experiments of methylated ubiquitin with YUH, Dsk2UBA and p62UBA monitored by the 1H-13C HSQC spectra of the methylated-lysine methyl groups.

Figure S8. 1H and 15N chemical shift changes of methylated ubiquitin in the presence of YUH1, Dsk2UBA and p62UBA.

Figure S9. NMR titration curves of methylated ubiquitin assuming 1:1 binding stoichiometry with p62UBA.

Figure S10. Methylated K6 in ubiquitin - Dsk2UBA complex structure.

Figure S11. Sensitivity comparison among three different 1H-13C correlation methods.

Figure S12. 1H-13C HSQC spectra of 0.2 μM methylated ubiquitin.

Figure S13. 1H and 15N chemical shift changes induced by methylation.

Table S1. ITC parameters of non-methylated ubiquitin or methylated ubiquitin titration of YUH1#

#The experimental data of ITC are shown in Figure S5. N, Kd, ΔH and ΔS are the stoichiometry, dissociation constant, enthalpy change and entropy change, respectively.

Table S2. Intermolecular distances between the lysine side-chain Nζ or backbone N atoms of ubiquitin to the nearest heavy atoms of YUH1 or Dsk2UBA, calculated from the complex structure#

#For the YUH1 and Dsk2UBA complexes, crystal (PDB ID: 1CMX) and NMR (PDB ID: 1WR1) structures are available, respectively. For the NMR structure, the distances are calculated for 20 coordinates each with standard deviations.

Figure S1. Reaction scheme of the 13C-methylation of the amino group. One or two 13C-methyl groups are conjugated to the amino group in the presence of 13C-labeled formaldehyde in reduced conditions.

Figure S2. MALDI-MS spectra of non-methylated ubiquitin (black) and methylated ubiquitin (red). The increment of ubiquitin molecular mass by methylation is about 240, corresponding to the addition of 16 13C-methyl groups {(+16) × 16 = 256} and the elimination of 16 protons {(-1) × 16 = -16}.

Figure S3. 1H-13C HSQC spectra of fully (a) or partially (b) di-methylated ubiquitin. Ubiquitin is methylated using 200-fold (a) or 10-fold (b) of 13C-formaldehyde. Methyl signals of the mono-methylated and di-methylated lysine are observed around 35 and 45 ppm in the 13C dimension, respectively. * indicates the minor peak from N-terminus.

Figure S4. 1H-13C HSQC spectra of mutated or N-terminal-tag digested methylated ubiquitin. The peak positions disappeared in the presence of mutations or after the N-terminal-tag digestion. These are indicated by the dotted-line boxes.

Figure S5. ITC experiments of non-methylated ubiquitin or methylated ubiquitin titration of YUH1. The thermograms of non-methylated ubiquitin (a) and methylated ubiquitin (b) are obtained by titrating YUH1 at 298K.

Figure S6. 1H and 15N chemical shift changes of non-methylated ubiquitin and methylated ubiquitin in the presence of YUH1. (a) The averaged 1H and 15N chemical shift changes of non-methylated ubiquitin (red) and methylated ubiquitin (black) in the presence of YUH1. * the letter P indicates the unassigned and proline residues, respectively. (b) The correlation plots of the chemical shift changes of non-methylated ubiquitin and methylated ubiquitin. The best-fit line is given.

Figure S7. NMR titration experiments of methylated ubiquitin with YUH (a), Dsk2UBA (b) and p62UBA (c) monitored by the 1H-13C HSQC spectra of the methylated-lysine methyl groups. The numbers in left top indicate the molar ratios of the titrated proteins, and their colors correspond to the colors of the spectra.

Figure S8. 1H and 15N chemical shift changes of methylated ubiquitin in the presence of YUH1 (a), Dsk2UBA (b) and p62UBA (c). * the letter P indicates the unassigned and proline residues, respectively.

Figure S9. NMR titration curves of methylated ubiquitin assuming 1:1 binding stoichiometry with p62UBA. Titration curves for K6, K27b and K48 are shown. The best-fit lines are given assuming 1:1 binding stoichiometry without considering the dimer formation of p62UBA.

Figure S10. Enlarged view of space-filling model of the ubiquitin-Dsk2UBA complex (PDBID: 1WR1). Side chains of K6 and Q338 are drawn by stick model. Ubiquitin and Dsk2UBA are colored white and blue, respectively. Two free amino protons of ubiquitin K6 are replaced with methyl groups. These two methyl groups are exposed to the solvent, and no steric clash arises.

Figure S11. Peak intensity ratios (a, b), signal-to-noise (S/N) values (c-e), and the ratios of S/N values (f, g) of 1H-13C correlation spectra of 200 µM methylated ubiquitin in D2O measured by 500 MHz NMR at 303 K. (a) Peak intensity ratios of HSQC with States-TPPI scheme (straight HSQC) against HSQC with gradient sensitivity enhancement scheme (HSQC-SE). (b) Peak intensity ratios of HMQC with States-TPPI scheme (straight HMQC) against HSQC with gradient sensitivity enhancement scheme. (c) Histograms of S/N ratios of HSQC-SE, (d) straight HSQC, and (e) straight HMQC. (f) The ratios of S/N values of straight HSQC against HSQC-SE. (g) The ratios of S/N values of straight HMQC against HSQC-SE. S/N values are calculated using the following equation, S/N = 2.5 × P / NPP, where P = peak intensity and NPP = height-to-height noise. The spectral region within 6-7 ppm in 1H and 40-50 ppm in 13C was selected as noise area. Straight HSQC/HMQC gives the higher S/N ratios than HSQC-SE (~ 20%).

Figure S12. 1H-13C HSQC spectrum of 0.2 μM methylated ubiquitin. 2D spectrum (top) and horizontal slices (bottom). The NMR spectra are acquired at 950 MHz.

Figure S13. 1H and 15N chemical shift changes induced by methylation. (a) The averaged 1H and 15N chemical shift differences between non-methylated ubiquitin and methylated ubiquitin. The letter P indicates the proline residue. (b) Ubiquitin is shown by the ribbon representation. Backbone N atoms with large chemical shift changes are shown by colored spheres. Chemical shift changes within 0.2-0.4 ppm and larger than 0.4 ppm are colored orange and red, respectively. All lysine residues are drawn by stick representation.

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