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X-ray and Cryo-electron Microscopy Structures of Monalysin Pore-forming Toxin Reveal Multimerization of the Pro-form.

ABSTRACT: ?-Barrel pore-forming toxins (?-PFT), a large family of bacterial toxins, are generally secreted as water-soluble monomers and can form oligomeric pores in membranes following proteolytic cleavage and interaction with cell surface receptors. Monalysin has been recently identified as a ?-PFT that contributes to the virulence of Pseudomonas entomophila against Drosophila. It is secreted as a pro-protein that becomes active upon cleavage. Here we report the crystal and cryo-electron microscopy structure of the pro-form of Monalysin as well as the crystal structures of the cleaved form and of an inactive mutant lacking the membrane-spanning region. The overall structure of Monalysin displays an elongated shape, which resembles those of ?-pore-forming toxins, such as Aerolysin, but is devoid of a receptor-binding domain. X-ray crystallography, cryo-electron microscopy, and light-scattering studies show that pro-Monalysin forms a stable doughnut-like 18-mer complex composed of two disk-shaped nonamers held together by N-terminal swapping of the pro-peptides. This observation is in contrast with the monomeric pro-form of the other ?-PFTs that are receptor-dependent for membrane interaction. The membrane-spanning region of pro-Monalysin is fully buried in the center of the doughnut, suggesting that upon cleavage of pro-peptides, the two disk-shaped nonamers can, and have to, dissociate to leave the transmembrane segments free to deploy and lead to pore formation. In contrast with other toxins, the delivery of 18 subunits at once, nearby the cell surface, may be used to bypass the requirement of receptor-dependent concentration to reach the threshold for oligomerization into the pore-forming complex.

SUBMITTER: Leone P

PROVIDER: S-EPMC4505573 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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Project description:X-ray crystallography and cryo-electron microscopy (cryo-EM) are complementary techniques for structure determination. Crystallography usually reveals more detailed information, while cryo-EM is an extremely useful technique for studying large-sized macromolecules. As the gap between the resolution of crystallography and cryo-EM data narrows, the cryo-EM map of a macromolecule could serve as an initial model to solve the phase problem of crystal diffraction for high-resolution structure determination. FSEARCH is a procedure to utilize the low-resolution molecular shape for crystallographic phasing. The IPCAS (Iterative Protein Crystal structure Automatic Solution) pipeline is an automatic direct-methods-aided dual-space iterative phasing and model-building procedure. When only an electron-density map is available as the starting point, IPCAS is capable of generating a completed model from the phases of the input map automatically, without the requirement of an initial model. In this study, a hybrid method integrating X-ray crystallography with cryo-EM to help with structure determination is presented. With a cryo-EM map as the starting point, the workflow of the method involves three steps. (1) Cryo-EM map replacement: FSEARCH is utilized to find the correct translation and orientation of the cryo-EM map in the crystallographic unit cell and generates the initial low-resolution map. (2) Phase extension: the phases calculated from the correctly placed cryo-EM map are extended to high-resolution X-ray data by non-crystallographic symmetry averaging with phenix.resolve. (3) Model building: IPCAS is used to generate an initial model using the phase-extended map and perform model completion by iteration. Four cases (the lowest cryo-EM map resolution being 6.9 Å) have been tested for the general applicability of the hybrid method, and almost complete models have been generated for all test cases with reasonable Rwork/Rfree. The hybrid method therefore provides an automated tool for X-ray structure determination using a cryo-EM map as the starting point.

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X-ray and Cryo-electron Microscopy Structures of Monalysin Pore-forming Toxin Reveal Multimerization of the Pro-form.

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