NSMB | Dong Haohao/Zhang Xing/Dong Changjiang collaborated to explain the function and structure of the protein machine LolCDE, systematically revealing the recognition and transport mechanism of the outer membrane lipoprotein of Gram-negative bacteria

Responsible Editor | xi At present, the infection of pathogenic microorganisms or super bacteria with multi-drug resistance has brought a major threat to human health.

Among them, gram-negative bacteria (Pseudomonas aeruginosa, Enterobacter, Acinetobacter baumannii) are clinically common and the most concentrated type of pathogenic bacteria resistant to bacteria.
Their unique outer membrane structure prevents antibiotics from entering The most important protective barrier for cells.

Lipoproteins located in the outer membrane of gram-negative bacteria are an important class of functional proteins that participate in many outer membrane components (including lipopolysaccharide [1], phospholipids [2], β-barrel protein [3] and lipoproteins [4] ] Etc.
) to maintain the asymmetry, integrity and various physiological functions of the lipid bilayer of the bacterial outer membrane.
It is the core of the formation of the entire outer membrane and plays an important role in the growth and survival of drug-resistant bacteria.
The role of.

The localization of lipoprotein LolCEDAB is responsible for transporting dozens of outer membrane lipoproteins from the bacterial inner membrane to the outer membrane.

The mislocalization of outer membrane lipoproteins in the inner cell membrane can lead to the death of bacteria.
Therefore, understanding the mechanism of outer membrane lipoprotein localization is of great significance for the development of potential new antibiotics.On March 29, 2021, the team of Professor Dong Haohao from the State Key Laboratory of Biotherapy of West China Hospital of Sichuan University/National Clinical Research Center for Geriatric Diseases, Professor Zhang Xing's team from the Cryoelectron Microscopy Center of Zhejiang University School of Medicine, and Dong Anglia University School of Medicine, UK Professor Changjiang’s team published a research paper titled "Structural basis for bacterial lipoprotein relocation by the transporter LolCDE" on Nature Structural & Molecular Biology, which analyzed the structure of the lipoprotein localization machine LolCDE for the first time, and systematically studied its recognition and transport at the same time.
The specific mechanism of the substrate lipoprotein has laid a solid foundation for the development of new antibacterial drugs based on this protein machine complex.

The lipoproteins of gram-negative bacteria (including inner membrane lipoproteins and outer membrane lipoproteins) are synthesized intracellularly and integrated into the inner membrane through the Sec pathway, and then processed and modified by a series of enzymes to form mature lipoproteins.
The protein machine LolCDE classifies whether it is an outer membrane lipoprotein by recognizing the transport signal at the N-terminus of lipoprotein, so that the target substrate lipoprotein is extracted from the inner membrane and transported to the transfer protein LolA in the periplasm.

In this process, the recognition and transport of lipoproteins are particularly important.
Although there have been some research reports in recent years, the mechanism is still not very clear, especially the specific role played by the protein machine LolCDE, which lacks complete evidence at the molecular level.
.

Figure 1.
Diagram of LolCDEAB transporting outer membrane lipoproteins and in vitro transport experiments are designed to deeply explore the specific mechanism of lipoprotein positioning machine LolCDE.
The research team used single particle cryo-electron microscopy technology to analyze the molecular structure of LolCDE responsible for this step for the first time.
The molecular details and interaction relationships between the functional domains of the protein complex and the ligand are revealed. By capturing different functional conformations of LolCDE in different transport stages, including apo-state, lipoprotein-binding state, ATP-bound dimerized state and binding to LolA Six high-resolution cryo-EM structures, including the LolA-bound pre-release state, show a complete set of dynamic changes in lipoprotein transport mechanisms.

This study confirmed that LolCDE is different from other bacterial ABC transporters in a symmetrical allosteric mechanism.
Its two transmembrane subunits, LolC and LolE, have different functions involved in lipoprotein transport.

LolE is responsible for recognizing and localizing lipoproteins, while LolC acts as a mechanical lever to conduct conformational changes from NBD (ATP-binding domains nucleotide-binding domains) to PD (periplasmic domains) before and after ATP binding and hydrolysis, by coordinating various functional domains of protein machinery In order to carry out the extraction and transportation of lipoproteins.

The structural homology between LolCDE and other type VII ABC transporters suggests that this mechanical transduction mechanism may be common in this type of transporter family.

Not only that, the study conducted a detailed analysis of the molecular details of the protein machine LolCDE bound to the substrate lipoprotein, and carried out site-directed single mutation and in vitro simulated transport experiments of phospholipid vesicles, and found that many inhibited bacterial growth by hindering its transport function These functional sites can be used as drug targets for the development of inhibitors.

In short, this study not only has important reference significance for understanding the mechanism of action of other type VII ABC transporters, but also has important guiding significance for the development of therapeutic drugs for multidrug-resistant Gram-negative bacteria.

 Dr.
Tang Xiaodi, Associate Professor of the State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, Dr.
Shenghai Chang, Engineer of the Cryoelectron Microscopy Center of Zhejiang University School of Medicine, Zhang Ke, an experimental assistant at the State Key Laboratory of Biotherapy, West China Hospital of Sichuan University, and Qinghua Luo, a Ph.
Co-first author. At the same time, the research was strongly supported by the team of Professor Wei Xiawei, Professor Zhu Xiaofeng of Sichuan University and Researcher Zhang Zhengyu of Wuhan University.

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doi.
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