AI Insight
Researchers developed a new method called GENTLE (Gradient Enrichment of Native Targets from Lipid Environments) to determine high-resolution structures of outer membrane proteins directly from native Campylobacter jejuni bacterial membranes without traditional purification. Using cryo-electron microscopy, they solved structures of three critical proteins—PorA, OMP50, and Cj0034c—that are essential for bacterial invasion and infection of host cells. The study revealed OMP50's unique two-domain architecture and showed that Cj0034c assembles as a nine-unit channel structure, while PorA contains flexible surface loops potentially important for virulence.
Why it matters
This methodology enables structural studies of membrane proteins in their native environment, potentially accelerating drug development against bacterial pathogens like Campylobacter jejuni, a leading cause of food-borne gastroenteritis. The structural information obtained could inform development of targeted therapies and vaccines by revealing how these proteins facilitate bacterial infection and interact with host cells.
Understand the Science
by Zhemin Zhang, William D. Gregor, Muslum Ilgu, Yue Yin, Philip A. Klenotic, Qijing Zhang, Edward W. Yu
Bacterial outer membrane proteins (OMPs) are critical players in host–pathogen interactions and environmental adaptation. Here we describe the newly developed “Gradient Enrichment of Native Targets from Lipid Environments” (GENTLE) methodology and use this approach to elucidate the structures of Campylobacter jejuni OMPs directly from native, detergent-solubilized crude membranes. We identify and solve high-resolution cryo-EM structures of PorA, OMP50, and Cj0034c from C. jejuni membranes, all of which are required for Campylobacter invasion, adhesion, and initiation of host infection. Notably, our results provide the first structural information of OMP50, revealing a two-domain architecture constructed with an all β-stranded transmembrane domain and an all α-helical periplasmic domain. This structure depicts that all tyrosine residues, many of which are expected to be critical for phosphorylation and host–pathogen interaction, are localized to the outer membrane of C. jejuni. Our studies also led to the first structure of the full-length Cj0034c protein, which assembles as a nonamer with each protomer containing a single-spanning transmembrane helix and a large periplasmic domain. The nine protomers stack side-by-side to form a channel that spans the entire lipid bilayer. However, whether Cj0034c spans the outer membrane (OM) or inner membrane (IM) of C. jejuni must await further experimental studies. In addition, we observed that the surface-exposed extracellular loop L4 of PorA is very flexible, which may be critical for the virulence of this porin. Collectively, this work provides novel structural information for functionally important OMPs and sheds light on how they assemble in native bacterial membranes. These findings further demonstrate that it is possible to obtain high-resolution structural information for targeted membrane proteins from crude native membranes without their overexpression and purification.