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Production, purification and characterization of EtpA protein with X-ray crystallography, AlphaFold and circular dichroism spectroscopy

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posted on 2024-05-22, 14:08 authored by Clifford NtuiClifford Ntui, Wolf-Dieter SchubertWolf-Dieter Schubert
Enterotoxigenic Escherichia coli (ETEC) is a diarrhoeal pathogen associated with severe morbidity and mortality among young children in developing countries. At present, there is no vaccine for ETEC. One candidate vaccine antigen, EtpA, is a conserved secreted adhesin that presumably binds to the tips of the E. coli flagellum to link ETEC to host intestinal glycans. EtpA is exported through a Gram-negative, two-partner secretion (TPS) system (type Vb) comprised of the secreted EtpA passenger (TpsA) protein and EtpB (TpsB) a transporter integrated into the outer bacterial membrane. TpsA proteins share a conserved, N-terminal domain followed by an extensive C-terminal domain with divergent sequence repeats. Three constructs of EtpA were prepared and analysed respectively including residues 67 to 447 (EtpA67-447), residues 1 to 606 (EtpA1-606) and residues 67-930 (EtpA67-930). The crystal structure of EtpA67-447 solved at 1.76 Å resolution revealed a right-handed parallel β-helix with two extra-helical hairpins and an N-terminal β-strand cap. Analyses by circular dichroism spectroscopy confirmed the β-helical fold and indicated high resistance to chemical and thermal denaturation as well as rapid refolding. A theoretical model of full-length EtpA by AlphaFold largely concurs with the crystal structure adding a long β-helical C-terminal domain after an interdomain kink. We propose that robust folding of the TPS domain upon secretion provides a template to extend the N-terminal β-helix into the C-terminal domains of TpsA proteins. Unexpectedly, size exclusion chromatography and molecular pulldown assays with the N-terminal domain of EtpA failed to show any interaction with flagellin implying that other factors may be involved.

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Biochemistry, Genetics and Microbiology

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