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Escherichia coli cytolysin A (ClyA, also known as hemolysin E, HlyE) is a 34 kDa cytolytic α-helical pore-forming toxin. The crystal structure of soluble monomeric E. coli K-12 ClyA was previously solved at high resolution and this showed that ClyA had a novel structure that had not previously been seen in the data bank of proteins. Avian pathogenic E. coli (APEC), strain JM4660 ClyA is 75% sequence identical to E. coli K-12 ClyA and has many significant similarities. However, two significant differences between them are that JM4660 ClyA pores are more homogeneous when observed by electron microscopy (EM), and JM4660 ClyA is more thermostable than E. coli K-12 ClyA. Consequently, JM4660 ClyA could be a good model system to investigate ClyA membrane interaction. The expression and purification of JM4660 ClyA was successful. Crystals were grown of the pore form, but so far diffract to about 7 Å resolution. Bacillus cereus Nhe is a complex, pore-forming toxin consisting of three related proteins: NheA (43 kDa), NheB (39 kDa), and NheC (40 kDa). It is able to lyse mammalian cells from several organisms including humans in both Agar and liquid media and all three components of Nhe are necessary for toxicity. NheA has only 22% identity with NheB and NheC and its binding is the final stage of pore formation. Nhe proteins have sequence identity to B. cereus Hbl proteins. A previous structure solution of HblB revealed a structural resemblance to E. coli ClyA. To date, no crystal structure of any Nhe protein has been available in either the soluble form or in the pore form. Therefore, the project aimed to resolve the crystal structures of Nhe proteins in their water-soluble forms. The expression and purification of NheA was very successful. NheA was crystallized using PEG3350 as a precipitant by the sitting-drop vapour-diffusion method. The crystals belonged to space group C2, with unit-cell parameters a = 308 Å, b = 58 Å, c = 172.4 Å, α = γ = 90° and β =110° and were estimated to contain 8 protein molecules per asymmetric unit. The three-dimensional crystal structure was solved at 2.05 Å resolution using Multi-wavelength anomalous dispersion (MAD) data sets. NheA is a rod-shopped structure. The main body is formed by a bundle of four helices, each at least 70 Å long and near the end of C terminal there is a extra fifth helix known as αG. NheA has two subdomains: the tail domain and the head domain that contains amphipathic α/β hairpin. The structure of NheA reveals strong structure similarity to ClyA and HblB, which further suggests that Nhe, Hbl and ClyA belong to the some novel family of toxins. In addition, B. cereus NheB expressed in B. subtilis strain JH642, was purified and crystallized. This thesis also presents the initial electron microscopy studies involving NheA and NheB pore formation. |
