Research Article: High-resolution cryo-EM structures of the E. coli hemolysin ClyA oligomers

Date Published: May 2, 2019

Publisher: Public Library of Science

Author(s): Wei Peng, Marcela de Souza Santos, Yang Li, Diana R. Tomchick, Kim Orth, MD A. Motaleb.


Pore-forming proteins (PFPs) represent a functionally important protein family, that are found in organisms from viruses to humans. As a major branch of PFPs, bacteria pore-forming toxins (PFTs) permeabilize membranes and usually cause the death of target cells. E. coli hemolysin ClyA is the first member with the pore complex structure solved among α-PFTs, employing α-helices as transmembrane elements. ClyA is proposed to form pores composed of various numbers of protomers. With high-resolution cryo-EM structures, we observe that ClyA pore complexes can exist as newly confirmed oligomers of a tridecamer and a tetradecamer, at estimated resolutions of 3.2 Å and 4.3 Å, respectively. The 2.8 Å cryo-EM structure of a dodecamer dramatically improves the existing structural model. Structural analysis indicates that protomers from distinct oligomers resemble each other and neighboring protomers adopt a conserved interaction mode. We also show a stabilized intermediate state of ClyA during the transition process from soluble monomers to pore complexes. Unexpectedly, even without the formation of mature pore complexes, ClyA can permeabilize membranes and allow leakage of particles less than ~400 Daltons. In addition, we are the first to show that ClyA forms pore complexes in the presence of cholesterol within artificial liposomes. These findings provide new mechanistic insights into the dynamic process of pore assembly for the prototypical α-PFT ClyA.

Partial Text

As the largest family of bacterial toxins, pore-forming toxins (PFTs) represent important factors for bacterial virulence. The protein family is characterized by the pore-forming activity and these types of proteins exist in other kingdoms of life [1, 2]. Proteins exhibiting pore-forming activity, named as pore-forming proteins (PFPs), cause permeation of the target membrane, disruption of cellular homeostasis and death of target cells. With respect to protein structure, PFTs can be divided into two classes, α-PFTs, and β-PFTs, depending on the secondary structure of transmembrane elements of α-helices and β-barrels, respectively [1, 2].

PFTs are important bacterial virulence factors with pore-forming activity on target membranes [1, 2]. As a prototypical α-PFT, ClyA from E. coli assembles into pore structures on the membrane with a pore size of 40 Å [3, 12]. There has been controversy about the organization of ClyA pore complexes with low-resolution EM analysis [3, 13, 14]. In addition to the dodecamer which has been characterized by protein crystallography [3], here we report high-resolution cryo-EM structures of the tridecamer and the tetradecamer from a single dataset. The density maps allow accurate structural model building and elucidation of [25] the profile of ClyA pore complexes. Based on the cryo-EM structures, we eliminated artifacts such as the movement of the αG helix due to mercury atoms bound in the crystal structure [3]. The pore complex protomers from the tridecamer and the tetradecamer exhibit the same fold as observed with the dodecamer, indicating the rigidity of ClyA protomer when assembled into pore complexes. The protomer-protomer interaction paradigm in the dodecamer applies also to the tridecamer, and probably the tetradecamer.