結構觀點探討細菌肽聚醣水解酶受到蛋白酶複合體的 調控機制

Abstract

細菌的生長與形態變化受限於肽聚醣的擴張。肽聚醣是由多糖鏈與短肽鏈組成的聚合物，為組成真細菌細胞壁的主要成分之一。肽聚醣的擴張需要肽聚醣水解酶的幫助，在大腸桿菌中，DD-內肽酶MepS功能為水解肽聚醣中肽橋 (cross-link)，打斷固有的鍵結後，插入新合成之結構單元並擴張肽聚醣囊膜。然而過多或過少的肽聚醣水解酶都會對細菌細胞壁造成致死性的損傷，顯然這些水解酶在細胞內被良好的調控。先前的研究發現，MepS在細胞內受到週質蛋白酶Prc以及脂蛋白NlpI的共同調控。NlpI作為連接蛋白分別與MepS及Prc結合，由Prc將MepS降解為小片段分子。在結構上，我們的合作者透過X射線蛋白質晶體繞射解出了解析度2.30 Å的NlpI-Prc複合物晶體結構。另外在先前研究中MepS截短後 (胺基酸37-162) 的結構透過核磁共振 (NMR) 被解出，其中截短的N端序列被認為是沒有結構的區域。除此之外目前沒有其他結構上的證據解釋MepS和NlpI-Prc複合物之間的交互作用。為了瞭解MepS在細胞中如何受到蛋白酶系統調控，進而說明細胞壁擴張的機制，我們使用生物物理和生物化學方法來研究蛋白質之間的交互作用。首先利用大腸桿菌大量表現重組蛋白，透過鎳離子親和性層析與膠體過濾層析等方式純化，將重組蛋白用於結構與功能上的分析。我們使用等溫滴定微量量熱法 （ITC） 檢測了NlpI，Prc和MepS之間的交互作用，發現MepS與NlpI結合作用之間具有放熱反應。但我們無法觀察到Meps與Prc之間的任何熱量變化。另外我們亦嘗試透過X射線蛋白質晶體繞射解析MepS-NlpI複合物的晶體結構，並成功培養出NlpI-Meps複合體的晶體，然而在該X射線繞射數據中由於電子密度過於破碎，無法計算出MepS的結構。基於合作者所建構的MepS分子對接模型，我們透過突變可能的結合位點胺基酸來驗證此結合模型。從等溫滴定微量量熱法實驗得知，MepS確實會透過N端螺旋區域與NlpI產生交互作用。另外為了探討N端非結構區域 (胺基酸1-36) 在NlpI結合中扮演的角色，我們將MepS之N端截短後，透過等溫滴定微量量熱法、核磁共振、表面電漿共振等生物物理實驗，偵測與NlpI之間的交互作用。相比於全長MepS，N端截短MepS與NlpI的親和力變得十分微弱，並且在細胞外降解實驗中較難受到NlpI-Prc複合物的降解。綜合以上結果，本篇論文透過突變後親和力分析確定了MepS和NlpI之間的交互作用區域，間接地證實MepS對接模型。另外也發現MepS之非結構N端區域在MepS的調控過程中扮演了重要的角色。

Bacterial growth and morpH ogenesis are intimately coupled to expansion of peptidoglycan (PG), a polymer consisting of sugars and amino acids and forming the cell wall. In Escherichia coli, expansion of PG requiring murein hydrolase MepS that cleaves the cross-links for insertion of new materials and resynthesis of cross-links. It is critical that such cleavage needs to be well regulated to avoid lethal damage of the PG sacculus. In recent study, it has showed that murein hydrolase MepS is specific modulated by the periplasmic protease Prc and the lipoprotein NlpI. NlpI acts as an adaptor to bring MepS and Prc together, and then Prc degrades MepS into small fragments. Our collaborator solved the crystal structure of NlpI-Prc complex at 2.30 Å resolution by X-ray Diffraction. The solution structure of N-terminal truncated MepS (residues 37-162) is determined by NMR (Nuclear Magnetic Resonance) spectroscopy while the first 36 N-terminal residues of MepS is significant disorder in the NMR spectral screening. However, there is no structural evidence to explain the interaction between MepS and NlpI-Prc complex. To understand how the NlpI–Prc complex regulates PG synthesis by altering the levels of MepS, we used biopH ysical and biochemical approaches to investigate the protein-protein interactions. First, the recombinant MepS, NlpI and Prc were expressed and purified by immobilized metal affinity chromatograpH y and gel filtration for structural and functional analysis. We detected the interaction between NlpI, Prc and MepS with isothermal titration calorimetry (ITC) and the results showed that the interaction between MepS and NlpI was an exothermic reaction. But we could not observe any heat changes for the interaction between MepS and Prc. We also tried to determine the crystal structure of NlpI-MepS complex by X-ray diffraction and successfully identified a condition that NlpI-MepS can be crystalized. However, it was difficult to identify the structure of MepS in our X-ray diffraction data due to broken density map. Based on a truncated MepS-docked model built by our collaborator, we examined this binding model by introducing mutations and the results showed that N-terminal helix of MepS is important for interacting to NlpI. To further explore the role of the first 36 N-terminal region of MepS, we detected the interaction between truncated MepS (residues 37-162) and NlpI by ITC, NMR and SPR (Surface Plasma Resonance). Compared to full-length MepS, truncated MepS showed much weak affinity with NlpI and the degradation efficiency was also affected in vitro. Taken together, our findings provide evidences for the MepS-docked model by mutation analyses and the unstructured N-terminal residues of MepS is important for the interaction with NlpI-Prc complex.