A Transient π–π or Cation–π Interaction between Degron and Degrader Dual Residues: A Key Step for the Substrate Recognition and Discrimination in the Processive Degradation of SulA by ClpYQ (HslUV) Protease in <i>Escherichia coli</i>
The <i>Escherichia coli</i> ATP-dependent ClpYQ protease constitutes ClpY ATPase/unfoldase and ClpQ peptidase. The Tyr<sup>91st</sup> residue within the central pore-I site of ClpY-hexamer is important for unfolding and translocating substrates into the catalytic site of ClpQ...
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MDPI AG
2023-12-01
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| Series: | International Journal of Molecular Sciences |
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| Online Access: | https://www.mdpi.com/1422-0067/24/24/17353 |
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| author | Chu-Hsuan Lin Chih-Hsuan Tsai Chun-Chi Chou Whei-Fen Wu |
| author_facet | Chu-Hsuan Lin Chih-Hsuan Tsai Chun-Chi Chou Whei-Fen Wu |
| author_sort | Chu-Hsuan Lin |
| collection | DOAJ |
| description | The <i>Escherichia coli</i> ATP-dependent ClpYQ protease constitutes ClpY ATPase/unfoldase and ClpQ peptidase. The Tyr<sup>91st</sup> residue within the central pore-I site of ClpY-hexamer is important for unfolding and translocating substrates into the catalytic site of ClpQ. We have identified the degron site (GFIMRP<sup>147th</sup>) of SulA, a cell-division inhibitor recognized by ClpYQ and that the Phe<sup>143rd</sup> residue in degron site is necessary for SulA native folded structure. However, the functional association of this degron site with the ClpYQ degrader is unknown. Here, we investigated the molecular insights into substrate recognition and discrimination by the ClpYQ protease. We found that the point mutants ClpY<sup>Y91F</sup>Q, ClpY<sup>Y91H</sup>Q, and ClpY<sup>Y91W</sup>Q, carrying a ring structure at the 91st residue of ClpY, efficiently degraded their natural substrates, evidenced by the suppressed bacterial methyl-methane-sulfonate (MMS) sensitivity, the reduced β-galactosidase activity of <i>cpsB::lacZ</i>, and the lowest amounts of MBP-SulA in both in vivo and in vitro degradation analyses. Alternatively, mimicking the wild-type SulA, SulA<sup>F143H</sup>, SulA<sup>F143K</sup> and SulA<sup>F143W</sup>, harboring a ring structure or a cation side-group in 143<sup>rd</sup> residue of SulA, were efficiently degraded by ClpYQ in the bacterial cells, also revealing shorter half-lives at 41 °C and higher binding affinities towards ClpY in pull-down assays. Finally, ClpY<sup>Y91F</sup>Q and ClpY<sup>Y91H</sup>Q, were capable of effectively degrading SulA<sup>F143H</sup> and SulA<sup>F143K</sup>, highlighting a correspondingly functional interaction between the SulA 143rd and ClpY 91st residues. According to the interchangeable substituted amino acids, our results uniquely indicate that a transient π–π or cation−π interaction between the SulA 143rd and ClpY 91st residues could be aptly gripped between the degron site of substrates and the pore site of proteases (degraders) for substrate recognition and discrimination of the processive degradation. |
| first_indexed | 2024-03-08T20:42:13Z |
| format | Article |
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| institution | Directory Open Access Journal |
| issn | 1661-6596 1422-0067 |
| language | English |
| last_indexed | 2024-03-08T20:42:13Z |
| publishDate | 2023-12-01 |
| publisher | MDPI AG |
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| series | International Journal of Molecular Sciences |
| spelling | doaj.art-ddd6d9169ca848a8b83727fb9874f8282023-12-22T14:14:09ZengMDPI AGInternational Journal of Molecular Sciences1661-65961422-00672023-12-0124241735310.3390/ijms242417353A Transient π–π or Cation–π Interaction between Degron and Degrader Dual Residues: A Key Step for the Substrate Recognition and Discrimination in the Processive Degradation of SulA by ClpYQ (HslUV) Protease in <i>Escherichia coli</i>Chu-Hsuan Lin0Chih-Hsuan Tsai1Chun-Chi Chou2Whei-Fen Wu3Department of Agricultural Chemistry, College of Bio-Resource and Agriculture, National Taiwan University, Taipei 10617, TaiwanDepartment of Microbiology and Immunology, College of Medicine, National Cheng Kung University, Tainan 701401, TaiwanDepartment of Agricultural Chemistry, College of Bio-Resource and Agriculture, National Taiwan University, Taipei 10617, TaiwanDepartment of Agricultural Chemistry, College of Bio-Resource and Agriculture, National Taiwan University, Taipei 10617, TaiwanThe <i>Escherichia coli</i> ATP-dependent ClpYQ protease constitutes ClpY ATPase/unfoldase and ClpQ peptidase. The Tyr<sup>91st</sup> residue within the central pore-I site of ClpY-hexamer is important for unfolding and translocating substrates into the catalytic site of ClpQ. We have identified the degron site (GFIMRP<sup>147th</sup>) of SulA, a cell-division inhibitor recognized by ClpYQ and that the Phe<sup>143rd</sup> residue in degron site is necessary for SulA native folded structure. However, the functional association of this degron site with the ClpYQ degrader is unknown. Here, we investigated the molecular insights into substrate recognition and discrimination by the ClpYQ protease. We found that the point mutants ClpY<sup>Y91F</sup>Q, ClpY<sup>Y91H</sup>Q, and ClpY<sup>Y91W</sup>Q, carrying a ring structure at the 91st residue of ClpY, efficiently degraded their natural substrates, evidenced by the suppressed bacterial methyl-methane-sulfonate (MMS) sensitivity, the reduced β-galactosidase activity of <i>cpsB::lacZ</i>, and the lowest amounts of MBP-SulA in both in vivo and in vitro degradation analyses. Alternatively, mimicking the wild-type SulA, SulA<sup>F143H</sup>, SulA<sup>F143K</sup> and SulA<sup>F143W</sup>, harboring a ring structure or a cation side-group in 143<sup>rd</sup> residue of SulA, were efficiently degraded by ClpYQ in the bacterial cells, also revealing shorter half-lives at 41 °C and higher binding affinities towards ClpY in pull-down assays. Finally, ClpY<sup>Y91F</sup>Q and ClpY<sup>Y91H</sup>Q, were capable of effectively degrading SulA<sup>F143H</sup> and SulA<sup>F143K</sup>, highlighting a correspondingly functional interaction between the SulA 143rd and ClpY 91st residues. According to the interchangeable substituted amino acids, our results uniquely indicate that a transient π–π or cation−π interaction between the SulA 143rd and ClpY 91st residues could be aptly gripped between the degron site of substrates and the pore site of proteases (degraders) for substrate recognition and discrimination of the processive degradation.https://www.mdpi.com/1422-0067/24/24/17353SulAClpYQ ATP-dependent proteasedegrondegraderπ–π interactioncation–π interaction |
| spellingShingle | Chu-Hsuan Lin Chih-Hsuan Tsai Chun-Chi Chou Whei-Fen Wu A Transient π–π or Cation–π Interaction between Degron and Degrader Dual Residues: A Key Step for the Substrate Recognition and Discrimination in the Processive Degradation of SulA by ClpYQ (HslUV) Protease in <i>Escherichia coli</i> International Journal of Molecular Sciences SulA ClpYQ ATP-dependent protease degron degrader π–π interaction cation–π interaction |
| title | A Transient π–π or Cation–π Interaction between Degron and Degrader Dual Residues: A Key Step for the Substrate Recognition and Discrimination in the Processive Degradation of SulA by ClpYQ (HslUV) Protease in <i>Escherichia coli</i> |
| title_full | A Transient π–π or Cation–π Interaction between Degron and Degrader Dual Residues: A Key Step for the Substrate Recognition and Discrimination in the Processive Degradation of SulA by ClpYQ (HslUV) Protease in <i>Escherichia coli</i> |
| title_fullStr | A Transient π–π or Cation–π Interaction between Degron and Degrader Dual Residues: A Key Step for the Substrate Recognition and Discrimination in the Processive Degradation of SulA by ClpYQ (HslUV) Protease in <i>Escherichia coli</i> |
| title_full_unstemmed | A Transient π–π or Cation–π Interaction between Degron and Degrader Dual Residues: A Key Step for the Substrate Recognition and Discrimination in the Processive Degradation of SulA by ClpYQ (HslUV) Protease in <i>Escherichia coli</i> |
| title_short | A Transient π–π or Cation–π Interaction between Degron and Degrader Dual Residues: A Key Step for the Substrate Recognition and Discrimination in the Processive Degradation of SulA by ClpYQ (HslUV) Protease in <i>Escherichia coli</i> |
| title_sort | transient π π or cation π interaction between degron and degrader dual residues a key step for the substrate recognition and discrimination in the processive degradation of sula by clpyq hsluv protease in i escherichia coli i |
| topic | SulA ClpYQ ATP-dependent protease degron degrader π–π interaction cation–π interaction |
| url | https://www.mdpi.com/1422-0067/24/24/17353 |
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