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Structural dynamics of the receptor-binding domain of colicin E9
Ruth Boetzel1, Emily S Collins, Nigel J Clayden
1School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
Abstract:
Colicin E9 is a 61 kDa antibacterial protein secreted by E. coli. In order for it to enter the cytoplasm of susceptible bacteria and kill them by hydrolysing their DNA, the colicin must first interact with an outer membrane receptor on the target cell, BtuB, and a translocation pathway involving Tol proteins. The receptor binding, translocation and DNase functions of colicin E9 are housed in discrete structural domains, which have been independently expressed and characterized. The minimal receptor-binding domain is a 76 amino acid protein (min-R). X-ray structure determination of a related colicin shows its receptor-binding-domain to have a helical hairpin structure (S. Soelaiman, K. Jakes, N. Wu, C. Li and M. Shoham, Molecular Cell. 2001, 8, 1053). Our solution NMR studies of min-R have confirmed it has a helical hairpin structure, and shown it has multiple slowly interchanging conformers and a flexible inter-helix loop. A plausible interpretation of these data is that in solution the helical hairpin can adopt a variety of structures differing in the spatial relationship of the two helices. A possible biological role for this involves the hairpin opening during translocation into bacteria.
Insights
Colicin E9 uses a helical hairpin structure to bind bacterial receptors. This structure exhibits flexibility, potentially aiding its entry into target cells for DNA hydrolysis and bacterial killing.
Area of Science:
- Bacteriology
- Structural Biology
- Molecular Biology
Background:
- Colicin E9 is an antibacterial protein from E. coli that kills susceptible bacteria by DNA hydrolysis.
- Entry into target bacteria requires interaction with the outer membrane receptor BtuB and the Tol protein translocation pathway.
- Colicin E9's functions are localized in discrete structural domains.
Purpose of the Study:
- To characterize the structure and dynamics of the minimal receptor-binding domain (min-R) of Colicin E9.
- To investigate the solution structure of min-R using NMR spectroscopy.
- To explore the potential biological implications of min-R's structural flexibility.
Main Methods:
- Expression and characterization of the minimal receptor-binding domain (min-R) of Colicin E9.
- Solution Nuclear Magnetic Resonance (NMR) spectroscopy to determine the structure and dynamics of min-R.
- Comparison with existing X-ray crystallography data of related colicins.
Main Results:
- Solution NMR studies confirmed that min-R possesses a helical hairpin structure.
- Multiple slowly interchanging conformers and a flexible inter-helix loop were identified in min-R.
- The helical hairpin structure of min-R appears to be dynamic in solution, with varying spatial relationships between the helices.
Conclusions:
- The minimal receptor-binding domain of Colicin E9 has a flexible helical hairpin structure in solution.
- This structural flexibility, including a dynamic inter-helix loop, may be crucial for the colicin's translocation process.
- The hairpin may undergo conformational changes, such as opening, to facilitate entry into the bacterial cytoplasm.