Dissection of an ABC transporter LolCDE function analyzed by photo-crosslinking

Area of Science:

• Bacterial cell envelope biogenesis
• Protein trafficking mechanisms
• Outer membrane protein assembly

Background:

• Escherichia coli's outer membrane contains ~100 lipoproteins, crucial for cell structure and function.
• The lipoprotein localization (Lol) system, comprising five Lol proteins, mediates lipoprotein transport to the outer membrane.
• While LolCDE structure is known, the mechanism of lipoprotein transfer to LolA remains unclear.

Purpose of the Study:

• To elucidate the interaction mechanism between the LolCDE complex and outer membrane lipoproteins.
• To identify specific domains involved in lipoprotein binding and transfer within the LolCDE system.

Main Methods:

• Site-specific photo-crosslinking introduced photo-crosslinkable amino acids into LolCDE helices.
• In vivo crosslinking identified domains interacting with peptidoglycan-associated lipoprotein (Pal).
• An in vitro system was developed to analyze lipoprotein binding to LolCDE.

Main Results:

• Photo-crosslinking identified specific LolCDE domains interacting with lipoproteins in vivo.
• The LolCDE inhibitor, compound 2, did not block lipoprotein binding.
• Compound 2 was observed to promote the dissociation of lipoproteins from LolCDE.

Conclusions:

• This study provides novel insights into the molecular mechanisms of lipoprotein trafficking in E. coli.
• The findings suggest LolCDE's role in lipoprotein release rather than solely binding.
• Understanding these mechanisms can inform strategies for targeting bacterial cell envelope integrity.