Computational protocol: 1H, 15N, 13C backbone resonance assignments of human soluble catechol O-methyltransferase in complex with S-adenosyl-l-methionine and 3,5-dinitrocatechol

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[…] Backbone 1HN, 15N, 13Cα, 13Cβ and 13C’ chemical shifts were assigned for S-COMT in the S-COMT:SAM:DNC:Mg2+ complex using standard triple resonance methodology (Gardner and Kay ). Spectra were processed with TopSpin software version 3.2. Peak picking and frequency matching was performed within CCPNMR Analysis version 2.4 (Vranken et al. ) and the backbone assignments were checked independently using a simulated annealing algorithm employed by the “asstools” assignment program (Reed et al. ). The backbone 1H, 15N and 13C chemical shifts have been deposited in the BioMagResBank ( under the BMRB accession code 26848.Excluding the ten proline residues and the first eight residues of the N-terminal cloning tag from the 233-residue S-COMT protein sequence, 205 out of a total of 215 residues were assigned in the 1H-15N TROSY spectrum of the S-COMT:SAM:DNC:Mg2+ complex (Fig. ). In total, 97 % of all backbone resonances were assigned (95 % of 1HN, 95 % of 15N, 98 % of 13Cα, 97 % of 13Cβ and 98 % of 13C’ nuclei). There is evidence for exchange dynamics occurring on a slow NMR timescale due to the presence of duplicate spin systems in the 1H-15N TROSY and 3D correlation spectra. Cis–trans proline isomerisation at P221 is the most likely source of conformational dynamics responsible for spin system duplication at A219 and G220. There is also spin system duplication for Q1 and G2, where the cloning tag meets the S-COMT sequence. Fig. 1 There are ten residues that remain unassigned in the 1H-15N TROSY spectrum (D3, G43, D44, G47, V53, Q58, M76, G83, S187 and G214). From the crystal structure (PDB: 3BWM; (Rutherford et al. ), Fig. ), D3, Q58, G83, S187 and G214 are located at the surface of the protein, mostly in solvent exposed loops, and as a consequence the 1H-15N TROSY correlations are likely to be attenuated beyond detection by fast exchange with solvent. Several residues in the third α-helix (G43-Q58) and fourth α-helix (G70-R78) have 1H-15N TROSY peak intensities that are broadened by conformational exchange; specifically these are: K48, I49, D51, I54, E56, V74, R75 and A77. Such exchange behaviour points to dynamics occurring on the millisecond timescale in this region of the protein, which are the likely source of the broadening beyond detection of the 1H-15N TROSY correlations of G43, D44, G47, V53 and M76. An overlay of S-COMT crystal structures (PDB: 4PYI, 3A7E, 3BWM, 4PYQ, 4P7J) shows that the last turn of the second α-helix (C33-K36) and the first turn of the third α-helix (G43-K46) has positional heterogeneity resulting from the active site loop (E37-V42) occupying alternative conformations. One consequence of these conformational differences requires that the sidechain donors of R75 coordinate the sidechain acceptors of D44 and D51 differently, which might account for the exchange broadening behaviour observed for these residues, together with residues in their immediate vicinity. Fig. 2 The secondary structure content of S-COMT was predicted by uploading the backbone 1HN, 15N, 13Cα, 13Cβ and 13C’ chemical shifts of the S-COMT:SAM:DNC:Mg2+ complex to the TALOS+ webserver (Shen et al. ). Figure  compares the predicted secondary structure for the solution complex with the secondary structure observed in the crystal form of the complex. These data are in very good agreement, which indicates that the solution conformation is very similar to the protein structure observed in crystals, and provides confidence in the assignments of the S-COMT:SAM:DNC:Mg2+ complex. Fig. 3 […]

Pipeline specifications

Software tools CcpNmr, TALOS+
Databases BMRB
Applications NMR-based proteomics analysis, Protein structure analysis
Organisms Homo sapiens, Dipturus trachyderma
Diseases Parkinson Disease
Chemicals Hydrogen