探討細菌蛋白NlpI與肽聚醣水解酶MepS形成複合體之結構機轉

Abstract

肽聚醣是一個位於細菌細胞壁的外骨骼結構，其骨架由多糖鏈串連著短肽鏈所組成。肽聚醣囊袋的結構包括線性的多糖鏈串，其骨架是由兩種糖衍生物：N-乙醯葡萄糖胺 （NAG）和N-乙醯包壁酸（NAM），這些多醣鏈透過短肽鏈相互交聯形成肽聚醣。裂解肽聚醣的交聯網絡是細菌細胞增生時細胞壁擴展所必需的。NlpI是一種嵌和於外膜的脂質蛋白，在革蘭氏陰性細菌中廣泛存在並分佈在細胞外膜上。NlpI利用四肽重複序列（TPRs）促進蛋白質間的交互作用，使其參與各種細胞功能，如細胞分裂、細胞壁代謝、毒性以及與宿主細胞的相互作用。作為一個接合蛋白，NlpI會與各種肽聚醣水解酶進行交互作用，並與肽聚醣合成裝置協同作用。這種接合蛋白NlpI促進了多種肽聚醣內切酶（endopeptidase）,如MepS、MepH、MepM、MepK、PBP4和PBP7, 的定位，同時也影響了MepM和MepS在體外的內切酶活性。此外，MepS在細胞生長的指數生長期表現量相當高，但在穩定期時急劇下降。MepS的蛋白表現量受到外膜蛋白PDZ-蛋白酶Prc以及接合蛋白NlpI的直接調控。過去的研究還顯示，在缺乏NlpI的情況下，Prc難以有效降解MepS，突顯了NlpI在MepS招募中的關鍵角色。然而，有關NlpI是如何調節並影響這些肽聚醣水解酶活性的相關機制，以及NlpI是如何與Prc蛋白酶調節特定PG水解酶的蛋白表現量仍有待進一步研究。在本研究中，我們揭示了包含MepS內切酶和Prc蛋白酶的兩個與接合蛋白結合的複合體晶體結構。我們發現了MepS內切酶的內生性無序區域（IDR）在異質複合體形成中的意外角色，以及此複合體在細菌形態生成中的生理意義。我們的生物物理實驗結果顯示，MepS 的IDR參與NlpI-MepS複合體的形成，缺乏IDR將會導致MepS無法與NlpI形成穩定的複合體。我們的NlpI-MepS結構顯示，MepS中的IDR在與NlpI結合時發生線團至螺旋的轉變（coil to helix transition），誘導MepS不對稱二聚體的形成。由NlpI引起的MepS的這種局部區域濃縮效應暗示NlpI對MepS活性的正向調節作用。此外，我們通過結構分析和功能突變完整地解釋MepS是如何被NlpI-Prc複合體所快速降解的機制。NlpI-Prc-MepS複合體的形成同樣倚仗於MepS IDR，而這也經歷了線團至螺旋的轉變，最終產生的成對MepS座落於由NlpI-Prc複合體形成的空間中。IDR的突變顯著影響了MepS與NlpI-Prc複合物之間的相互結合，並阻礙了有效的MepS降解。重要的是，在體內（in vivo）的定性和形態實驗顯示IDR短肽鏈足以影響NlpI-Prc-MepS的複合體形成，進而抑制MepS被NlpI-Prc降解的效率，最終導致細菌外觀異常並損害細菌外膜的完整性。總結，我們的研究結果顯示NlpI是如何促進MepS二聚體的共定位（co-localization）以執行有效率的肽聚醣水解以及MepS後續由Prc蛋白酶降解的複雜機制。我們期許本篇研究能成為革蘭氏陰性病原體的肽聚醣生合成調控進一步建立更完備的解釋模型。這些見解將對於針對細胞壁生合成過程的抗生素研發有著相當重要的影響。

Bacteria have a protective exoskeleton called peptidoglycan (PG), which is composed of glycan strands linked by short peptides. The structure of the PG sacculus consists of linear glycan strands alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) linked to short peptide chains. Cleavage peptide crosslinks are necessary for the expansion of PG during cell growth. NlpI, an outer membrane-anchored lipoprotein, is widely present in gram-negative bacteria and distributed across the cell envelope. It utilizes tetratricopeptide repeats (TPRs) to facilitate protein-protein interactions, enabling its involvement in various cellular functions, such as cellular division, cell wall metabolism, pathogenicity and host cell interaction. As an adaptor protein, NlpI interacts with various hydrolases and associates with peptidoglycan (PG) synthetic machinery. This adaptor, NlpI, facilitates the localization of several PG endopeptidases such as MepS, MepH, MepM, MepK, PBP4, and PBP7, while also influencing the endopeptidase activity of MepM and MepS in vitro. Furthermore, the protein level of MepS is highly ample during the logarithmic phase of cell growth. However, its level experience a significant decrease as the cells transition into the stationary phase. This decline in MepS protein concentration is closely regulated by the adaptor NlpI in complex with the periplasmic PDZ-protease Prc, known as tail-specific protease (tsp). Prior studies also suggest that, in the absence of NlpI, Prc struggles to efficiently degrade MepS, underscoring the crucial role of NlpI in MepS recruitment. However, the mechanism by which NlpI regulates these PG hydrolases potentially affects their activities and how NlpI modulates the protein levels of specific PG hydrolases in the presence of Prc protease remains unclear. In this study, we revealed the crystal structures of two adaptor complexes containing MepS endopeptidase and Prc protease. Our research revealed an unexpected function of the intrinsically disordered region (IDR) of MepS in the formation of heterocomplexes, highlighting its physiological significance in bacterial morphogenesis. Our biophysical analyses indicated the involvement of MepS IDR in the formation of the NlpI-MepS complex, with the absence of the IDR resulting in the inability to form a stable complex with NlpI, characterized by size exclusion chromatography (SEC) and pull-down assays. Our adaptor-endopeptidase structure revealed that IDR in MepS undergoes a coil-to-helix transition upon NlpI binding, inducing asymmetric MepS dimerization. This locally concentrated effect of MepS induced by NlpI implies a positive regulatory role of NlpI on MepS activity. Furthermore, we elucidated the mechanism about how MepS undergoes rapid proteolysis by the NlpI-Prc complex using structural analysis and functional mutagenesis. The formation of the adaptor-endopeptidase-protease complex relies on MepS IDR, which also undergoes a disorder-to-order transition, while the resulting paired MepS dock into the cradle formed by the NlpI-Prc complex. IDR mutations dramatically compromised the interaction between MepS and the NlpI-Prc complex and hindered efficient MepS degradation. Importantly, in vivo quantitative and morphological analyses shed light on how the IDR peptide competitively inhibits NlpI-enhanced proteolysis of MepS, which in turn results in abnormal bacterial appearance and impairs cell envelope integrity. In summary, our work unveils the complex mechanism through which NlpI facilitates the colocalization of dual MepS endopeptidases for efficient PG hydrolysis and subsequent degradation by the Prc protease. We anticipate that our research will pave the way for more sophisticated models of multi-enzyme complexes involved in PG biosynthesis regulation in gram-negative pathogens.