title: Structural dynamics of the membrane translocation domain of colicin E9 and its interaction with TolB.
abstract: In order for the 61 kDa colicin E9 protein toxin to enter the cytoplasm of susceptible cells and kill them by hydrolysing their DNA, the colicin must interact with the outer membrane BtuB receptor and Tol translocation pathway of target cells. The translocation function is located in the N-terminal domain of the colicin molecule. (1)H, (1)H-(1)H-(15)N and (1)H-(13)C-(15)N NMR studies of intact colicin E9, its DNase domain, minimal receptor-binding domain and two N-terminal constructs containing the translocation domain showed that the region of the translocation domain that governs the interaction of colicin E9 with TolB is largely unstructured and highly flexible. Of the expected 80 backbone NH resonances of the first 83 residues of intact colicin E9, 61 were identified, with 43 of them being assigned specifically. The absence of secondary structure for these was shown through chemical shift analyses and the lack of long-range NOEs in (1)H-(1)H-(15)N NOESY spectra (tau(m)=200 ms). The enhanced flexibility of the region of the translocation domain containing the TolB box compared to the overall tumbling rate of the protein was identified from the relatively large values of backbone and tryptophan indole (15)N spin-spin relaxation times, and from the negative (1)H-(15)N NOEs of the backbone NH resonances. Variable flexibility of the N-terminal region was revealed by the (15)N T(1)/T(2) ratios, which showed that the C-terminal end of the TolB box and the region immediately following it was motionally constrained compared to other parts of the N terminus. This, together with the observation of inter-residue NOEs involving Ile54, indicated that there was some structural ordering, resulting most probably from the interactions of side-chains. Conformational heterogeneity of parts of the translocation domain was evident from a multiplicity of signals for some of the residues. Im9 binding to colicin E9 had no effect on the chemical shifts or other NMR characteristics of the region of colicin E9 containing the TolB recognition sequence, though the interaction of TolB with intact colicin E9 bound to Im9 did affect resonances from this region. The flexibility of the translocation domain of colicin E9 may be connected with its need to recognise protein partners that assist it in crossing the outer membrane and in the translocation event itself.
authors.affiliation: School of Chemical Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
authors.forename: Emily S
authors.initials: ES
authors.lastname: Collins
authors.forename: Sara B-M
authors.initials: SB
authors.lastname: Whittaker
authors.forename: Kaeko
authors.initials: K
authors.lastname: Tozawa
authors.forename: Colin
authors.initials: C
authors.lastname: MacDonald
authors.forename: Ruth
authors.initials: R
authors.lastname: Boetzel
authors.forename: Christopher N
authors.initials: CN
authors.lastname: Penfold
authors.forename: Ann
authors.initials: A
authors.lastname: Reilly
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Clayden
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Osborne
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Hemmings
authors.forename: Colin
authors.initials: C
authors.lastname: Kleanthous
authors.forename: Richard
authors.initials: R
authors.lastname: James
authors.forename: Geoffrey R
authors.initials: GR
authors.lastname: Moore
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001693:Biological Transport, Active; D003087:Colicins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D033903:Periplasmic Proteins; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013816:Thermodynamics
chemical_list: D001426:Bacterial Proteins; D003087:Colicins; D029968:Escherichia coli Proteins; D033903:Periplasmic Proteins; C007104:colicin immunity proteins; C488859:immE9 protein, E coli; C062094:tolB protein, E coli