Characterisation of a mobile protein-binding epitope in the translocation domain of colicin E9

Macdonald, Colin J., Tozawa, Kaeko, Collins, Emily S., Penfold, C. N., James, Richard, Kleanthous, Colin, Clayden, Nigel J. and Moore, Geoffrey R. (2004) Characterisation of a mobile protein-binding epitope in the translocation domain of colicin E9. Journal of Biomolecular NMR, 30. pp. 81-96. ISSN 0925-2738

Full text not available from this repository. (Request a copy)

Abstract

The 61 kDa colicin E9 protein toxin enters the cytoplasm of susceptible cells by interacting with outer membrane and periplasmic helper proteins, and kills them by hydrolysing their DNA. The membrane translocation function is located in the N-terminal domain of the colicin, with a key signal sequence being a pentapeptide region that governs the interaction with the helper protein TolB ( the TolB box). Previous NMR studies ( Collins et al., 2002 J. Mol. Biol. 318, 787-804) have shown that the N-terminal 83 residues of colicin E9, which includes the TolB box, is largely unstructured and highly flexible. In order to further define the properties of this region we have studied a fusion protein containing residues 1-61 of colicin E9 connected to the N-terminus of the E9 DNase by an eight-residue linking sequence. 53 of the expected 58 backbone NH resonances for the first 61 residues and all of the expected 7 backbone NH resonances of the linking sequence were assigned with 3D H-1-C-13-N-15 NMR experiments, and the backbone dynamics of these regions investigated through measurement of H-1-N-15 relaxation properties. Reduced spectral density mapping, extended Lipari-Szabo modelling, and fitting backbone R-2 relaxation rates to a polymer dynamics model identifies three clusters of interacting residues, each containing a tryptophan. Each of these clusters is perturbed by TolB binding to the intact colicin, showing that the significant region for TolB binding extends beyond the recognized five amino acids of the TolB box and demonstrating that the binding epitope for TolB involves a considerable degree of order within an otherwise disordered and flexible domain.

Item Type:	Article
Faculty \ School:	Faculty of Science > School of Chemistry (former - to 2024)
UEA Research Groups:	Faculty of Science > Research Groups > Physical and Analytical Chemistry (former - to 2017) Faculty of Science > Research Groups > Biophysical Chemistry (former - to 2017)
Depositing User:	Rachel Smith
Date Deposited:	15 Jun 2011 11:18
Last Modified:	24 Sep 2024 10:05
URI:	https://ueaeprints.uea.ac.uk/id/eprint/32503
DOI:	10.1023/B:JNMR.0000042963.71790.19

Actions (login required)

View Item