細菌分裂體FtsB-FtsL-FtsQ的異三聚體膜蛋白 複合物結構與分子機轉

Abstract

細胞分裂是細菌生存的基本過程，包含協調細胞的收縮、細胞壁合成和細胞的分隔。大腸桿菌內的這個過程由分裂體執行，包含至少12種核心蛋白質，它們在分裂早期和晚期依序被招募到細胞中央，中間有一段時間差。由三種穿膜蛋白質組成的複合物，即FtsB、FtsL和FtsQ，在細菌細胞分裂中扮演至關重要的作用。FtsB-FtsL-FtsQ (FtsBLQ)充當了早期和晚期分裂體組成之間的連接器，並作為下游分隔肽聚醣（PG）合成的調節器。然而，FtsBLQ調控PG合成酶活性的機制仍然不清楚。長久以來這個膜蛋白複合物的結構一直無法獲得，制約了這個研究領域的進展。在本博士論文研究中，我們首次使用X光結晶學成功地揭示了FtsBLQ異源三聚體的結構，包括其穿膜區域。我們觀察到了這個複合物出乎意料的V形結構，其頂部的異聚三聚體β-片狀折疊之間有廣泛的相互作用。有趣的是，兩個V形分支的長度差異表明這個複合物插入細胞膜時可能出現傾斜的方向。這種構型進一步提供與其結合夥伴（包括FtsK、FtsW-FtsI和PBP1b）的相互作用以及FtsBL穿膜螺旋的縮短的合理解釋。此外，我們還發現了在FtsBL協同螺旋中的插入和非常規的親水胺基酸殘基，解決了以前關於協同螺旋結構的爭議。此外、FtsBLQ複合物的關鍵功能區域的位置也首次被確定，主要位於協同螺旋的C末端周圍，並且大部分分佈在螺旋的兩側。我們還進行了關於FtsBLQ關鍵胺基酸殘基的結構，生化和遺傳研究，為分裂體介導的PG合成調節提供了見解。

Bacterial cell division is a fundamental process that orchestrates cell constriction, cell wall biosynthesis, and cell septation. The machinery in Escherichia coli, called divisome, contains at least twelve core proteins which are recruited hierarchically to the mid-cell during the early and late stages, with a delay in between. A complex consisting of three transmembrane proteins, namely FtsB, FtsL and FtsQ, plays a crucial role in bacterial cell division. FtsB-FtsL-FtsQ (FtsBLQ) serves as a connector between the early and late-stage components, and functions as a regulator for the downstream septal peptidoglycan (PG) synthesis. However, the mechanism by which FtsBLQ regulates the activity of PG synthases remains unclear. The structure of this protein complex remained unavailable, hindering progress in this area of study. In this study, using X-ray crystallography we have successfully revealed the structure of the heterotrimer FtsBLQ, including its transmembrane regions, for the first time. We observed an unexpected V-shape architecture of this complex with an extensive interaction between heterotrimeric β-sheets at the apex. Interestingly, the difference in length of two V-shape branches suggests a tilted orientation when this complex inserts into the membrane. This conformation is further strengthened by the interactions with its binding partners including FtsK, FtsW-FtsI and PBP1b, as well as the shortening of FtsBL transmembrane helices. Additionally, we discovered a stammer insertion, and unconventional core hydrophilic residues within the heterodimeric FtsBL coiled coil, resolving all previous controversies regarding the coiled coil structure. Furthermore, the location of key functional domains of FtsBLQ complex were also newly revealed, which are primarily situated around, but mostly at two sides of the coiled coil C-terminus. Structural, biochemical, and genetic studies of the FtsBLQ key residues were performed, providing insight into the regulation of a divisome-mediated PG synthesis.