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<p>Knotted Methyltransferases (MTase) from the SpoU-TrmD (SPOUT) family offer a unique opportunity to study a protein knot topology and enzymatic function. The knotted methyltransferase from <i>Staphylococcus aureus</i>, MTT<sub>SA</sub> (PDB: 1vh0, 4fak), is an ɑ/β-knotted 23s rRNA MTase containing only the minimal scaffold among the SPOUT family members. This dimeric enzyme is a minimalist model to study the deep + 3<sub>1</sub> knot. Here, we report the non-proline backbone assignments with 98.8% completion using TROSY-based NMR experiments on uniformly labeled <sup>2</sup>H/<sup>13</sup>C/<sup>15</sup>N-labeled protein. Complementary HBHA(CO)NH experiments on <sup>13</sup>C/<sup>15</sup>N-labeled protein were completed for Hα/Hβ sidechain assignments. Both backbone and sidechain assignments were deposited in the Biological Magnetic Resonance Bank (BMRB ID: 53391). Of 164 assigned residues, TALOS-N predictions provided 88.4% unambiguous assignments based on the H<sub>N</sub>, H<sub>α</sub>, C<sub>α</sub>, C<sub>β</sub> and N chemical shifts. In addition, the predicted secondary structure based on torsion angles and the ΔδC<sub>α</sub>-ΔδC<sub>β</sub> profile are in agreement with the crystal structures.</p>

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1H, 13C, 15N backbone assignment of the minimally tied trefoil knot, MTTSA, a 23s rRNA SPOUT methyltransferase

  • 期刊论文

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  • 发表日期：

    2026年4月10日

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  • Tiange Tao,
  • Dominique T. Capraro,
  • Patricia A. Jennings

摘要

Knotted Methyltransferases (MTase) from the SpoU-TrmD (SPOUT) family offer a unique opportunity to study a protein knot topology and enzymatic function. The knotted methyltransferase from Staphylococcus aureus, MTTSA (PDB: 1vh0, 4fak), is an ɑ/β-knotted 23s rRNA MTase containing only the minimal scaffold among the SPOUT family members. This dimeric enzyme is a minimalist model to study the deep + 31 knot. Here, we report the non-proline backbone assignments with 98.8% completion using TROSY-based NMR experiments on uniformly labeled 2H/13C/15N-labeled protein. Complementary HBHA(CO)NH experiments on 13C/15N-labeled protein were completed for Hα/Hβ sidechain assignments. Both backbone and sidechain assignments were deposited in the Biological Magnetic Resonance Bank (BMRB ID: 53391). Of 164 assigned residues, TALOS-N predictions provided 88.4% unambiguous assignments based on the HN, Hα, Cα, Cβ and N chemical shifts. In addition, the predicted secondary structure based on torsion angles and the ΔδCα-ΔδCβ profile are in agreement with the crystal structures.