大腸桿菌ClpYQ蛋白酶利用Methyl−π交互作用對基質SulA辨識與降解之研究

Abstract

ATP 依賴性蛋白酶ClpYQ為細菌細胞內重要的蛋白質降解系統之一，參與蛋白質品質管控與壓力反應等重要生理過程。先前研究已證實 ClpYQ 能有效辨識並降解細胞分裂抑制蛋白質SulA，且其初期辨識與抓握可能仰賴 π–π 或 cation–π 等非共價交互作用。然而，根據不同菌株的蛋白質序列分析，SulA中負責與ClpY作用的關鍵位點F143在部分菌種中為疏水性殘基Leucine，這暗示了π–π 或 cation–π 並非唯一的辨識模式，ClpY 與 SulA 間可能存在其他交互機制，其中 methyl–π 作用力的參與尚未被深入探討。為驗證此假設，本研究針對ClpY中的Y91以及SulA中的F143進行點突變，以具大型疏水性側鏈的胺基酸（如 Leu、Val、Met、Ile）取代，並結合in vivo與in vitro系統，從突變蛋白質之活性、生理功能、交互作用能力與降解效率等面向進行系統性分析，探討methyl–π交互作用是否為其中一種可能的機制。體內實驗結果顯示，ClpY Y91突變為疏水性胺基酸時，其蛋白酶活性完全喪失，無法辨識並降解SulA，顯示該位點需保留具有環狀結構的芳香族或咪唑類胺基酸（如 Tyr、Phe、Trp、His），以維持與SulA的有效辨識與交互作用。另一方面，當SulA F143位點若被大型疏水性胺基酸（如 Leu、Val、Met、Ile）取代時，這些突變蛋白質不僅保有原有的細胞抑制活性，其降解效率也與野生型相近，僅在半衰期上略有差異；而在體外免疫沉澱及降解試驗中，ClpY Y91以疏水性胺基酸取代時，無法與SulA形成有效的交互作用，也未觀察到明顯的降解現象。只有ClpY Y91F、Y91H和Y91W可有效與SulA及SulA F143L* 發生共沉澱，且在ClpQ與ATP存在下，這些ClpY突變蛋白酶均可有效降解SulA及SulA F143L*。此結果顯示，突變後的methyl基團仍能與ClpY形成有效的交互作用，進而啟動蛋白質降解機制。本研究結果支持 ClpY 與 SulA 之間可能透過選擇性 methyl–π 非共價作用進行辨識與結合的假設，並指出該作用模式需仰賴 ClpY 提供芳香環結構，由SulA 所提供之疏水性 methyl 基團形成交互作用界面。本研究結果，進一步釐清 ClpYQ 辨識基質的機制，並為探討蛋白酶與基質之間的非共價交互作用提供新的分子辨識觀點。

The ATP-dependent protease ClpYQ is one of the major protein degradation systems in bacterial cells, playing a critical role in protein quality control and stress responses. Previous studies have demonstrated that ClpYQ efficiently recognizes and degrades the cell division inhibitor SulA, with initial substrate recognition and engagement potentially mediated by non-covalent interactions such as π–π or cation–π interactions between 143rd-SulA and 91st-ClpY dual residues. However, sequence analyses of SulA homologs from different bacterial species reveal that the key ClpY-interacting residue F143 in SulA is replaced by a hydrophobic residue (e.g., Leucine) in some species. This suggests that π–π or cation–π interactions may not be the sole mode of recognition/gripping, and other mechanisms, such as methyl–π interactions, may also be involved. However, the role of methyl–π interactions remains largely unexplored. To test this hypothesis, this study employed site-directed mutagenesis targeting Tyr91 in ClpY and Phe143 in SulA, systematically substituting them with bulky hydrophobic amino acids (such as Leu, Val, Met, and Ile). Using both in vivo and in vitro experiment tests, we conducted comprehensive analyses of their physiological function, mutual interaction capability, and corresponding degradation efficiency to investigate whether methyl–π interactions contribute to substrate recognition/gripping. Our in vivo results revealed that when Tyr91 in ClpY was mutated to non-aromatic hydrophobic amino acids, its protease activity was completely abolished, and SulA was neither recognized nor degraded. This indicates that a ring-containing residue (such as Tyr, Phe, Trp, or His) maintain effective recognition and interaction with SulA. On the other hand, when Phe143 in SulA was replaced with bulky hydrophobic residues (e.g., Leu, Val, Met, or Ile), these SulA mutants not only retained most of their lethal activity in cells, but also rendered the efficient degradation efficiencies comparable to that of the wild-type, with only minor differences in half-life. As well, the in vitro pull-down and degradation assays, when Tyr91 in ClpY was substituted with hydrophobic amino acids, it failed to form effective interactions with SulA, and no significant degradation was observed. Only ClpY Y91F, ClpY Y91H and ClpY Y91W efficiently co-immunoprecipitated with the wild-type SulA and SulA F143L* and in the presence ClpQ and ATP, these ClpY variants were also able to efficiently degrade both substrates. These findings suggest that the substituted methyl groups of 143rd-SulA can still engage in effective interactions with the ring structure of 91st-ClpY to trigger an efficient degradation. Together, these results support the hypothesis that ClpY may recognize and bind SulA via selective methyl–π non-covalent interactions and highlight the requirement of an aromatic ring structure in ClpY to form an interaction interface with the hydrophobic methyl groups of SulA. This study further elucidates the substrate recognition mechanism of ClpYQ and offers a new perspective on the molecular basis of non-covalent interactions between proteases and their substrates.