透過親和力富集質譜平台比較大腸桿菌中肽聚醣合成複合物的相互作用組分析

Comparative interactome analysis of the peptidoglycan synthesizing complexes in Escherichia coli

柏樂(Bolor Buyanbadrakh)

指導教授 ： 林俊宏

共同指導教授 ： 梁素雲 ； 馬徹(Che Ma)

國立臺灣大學/生命科學院/生化科學研究所/博士(2021年)

https://doi.org/10.6342/NTU202103656

摘要

肽聚醣 (peptidoglycan, PG) 細胞壁是所有細菌中必不可少的重要組成部分，其合成是通過動態或是瞬時的多蛋白複合物進行的。革蘭氏陰性菌之大腸桿菌(Escherichia coli)具有獨特的 PG 合成複合物，包括延長體、分裂體和兩種 A 類的青黴素結合蛋白（penicillin binding proteins, PBPs）。這些生化實體催化基本相同的反應，近期的研究已經確定它們的特殊功能，主要是基於波動的環境條件下，對其突變表型的分析。儘管 PG 合成複合物重要的動態特性已被廣泛接受，但尚未有系統地研究複合物與主成分之間的相互作用。在這裡，我們開發了一種強親和力的富集質譜實驗平台，將複合物保留在其天然的膜結合環境中，且 PG 合成複合物 PBP1a、PBP1b、PBP2、MreB和 FtsZ 分別與弱/瞬態夥伴進行結合。為了將複合物保留在其天然條件下，我們設計大腸桿菌菌株，透過內源性表達之誘餌蛋白和親和標籤相融合。將化學交聯劑應用於活細菌樣品和蛋白質之間的交聯作用，並使用苯乙烯馬來酸 (SMA) 在膜結合的天然環境中提取蛋白質複合物。純化的複合物用於質譜分析以進行蛋白質鑑定。我們在上述五種誘餌蛋白的相互作用組中鑑定了 971 個獨特的結合夥伴。豐富的功能性分析允許識別其不同功能間的關聯性。用定量數據集探索其誘餌蛋白的相對特異性，並確定其潛在的誘餌特異性蛋白之相互作用物。此處介紹的主要 PG 合成和調節蛋白間的相互作用組，並針對功能冗餘蛋白提供獨特的見解，作為其保守且實質重要的細菌系統中不同的相互作用組模塊的藍圖。

關鍵字

肽聚醣合成 ； 相互作用組 ； 親和力富集質譜

並列摘要

A bacterial cell wall is an essential component of nearly all bacteria. The biosynthesis is carried out by dynamic and possibly transient multiprotein complexes. Gram negative bacteria, Escherichia coli, has distinct PG synthesizing complexes including elongasome, divisome, and two class-A PBPs (penicillin binding proteins). These biochemical entities catalyze essentially the same reactions and knowledge of their specialized function has been identified by recent studies mostly based on mutant phenotype analysis under fluctuating environmental conditions. Although the importance of the dynamic nature of PG synthesizing complexes is well accepted, the interactomes associated with the main components of the complexes has not yet been systematically studied. Here we developed an affinity enrichment mass spectrometry experimental platform to retain the complexes in their native membrane-bound environment and with weak/transient binding partners of the components of the PG synthesizing complexes, PBP1a, PBP1b, PBP2, MreB and FtsZ. To retain the complexes in their native condition, we engineered the Escherichia coli strains to endogenously express the bait proteins with affinity-tag fusion. Chemical cross-linkers were applied to live bacterial samples to crosslink proteins and styrene maleic acid (SMA) was used to extract the protein complex in the membrane-bound native environment. The purified complexes were then applied to mass spectrometry analysis for protein identification. We identified 971 unique binding partners in the interactomes of the aforementioned five proteins. Functional enrichment analysis allowed us to identify the distinct functional associations. In total, 153 biological process gene ontology terms were enriched significantly, from which 16 terms matched exactly to the reported GO terms of the PG synthesizing baits as ‘known’ and 79 terms were clustered with the bait GO terms as ‘related’. The remaining 74 GO terms could not be associated with the GO terms of the PG synthesizing baits and could have a ‘novel’ functional association. The specificities of the five bait proteins were explored with quantitative datasets and identified potential bait specific protein interactors. The interactomes of the main PG synthesizing and regulatory proteins presented here offer insights into specificity of the functionally redundant proteins, serving as the blueprint of the distinct interactome modules of this conserved and practically important bacterial system.

並列關鍵字

Peptidoglycan synthesis ； interactome ； affinity enrichment mass spectrometry

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