Photon Factory Activity Report 2005 #23 Part B (2006)
Biological Science

NW12/2004G343

High-resolution Crystal Structure of a Truncated ColE7 Translocation Domain:Implications for Colicin Transport Across Membranes
Yi-Sheng Cheng1, Zhonghao Shi1,2, Lyudmila G. Doudeva1,Wei-Zen Yang1, Kin-Fu Chak2 and Hanna S. Yuan1,2*1Institute of Molecular BiologyAcademia Sinica, Taipei, Taiwan ROC
2
Institute of Biochemistry National Yang-Ming UniversityTaipei, Taiwan, ROC
ColE7 is a nuclease-type colicin released fromEscherichia coli to kill sensitive bacterial cells bydegrading the nucleic acid molecules in their cytoplasm.
ColE7 is classified as one of the group A colicins, since
the N-terminal translocation domain (T-domain) of thenuclease-type colicins interact with specific membranebound or periplasmic Tol proteins during protein import.
Here, we show that if the N-terminal tail of ColE7 isdeleted, ColE7 (residues 63–576) loses its bactericidal
activity against E. coli. Moreover, TolB protein interacts
directly with the T-domain of ColE7 (residues 1–316),
but not with the N-terminal deleted T-domain (residues60–316), as detected by co-immunoprecipitation
experiments, confirming that the N-terminal tail isrequired for ColE7 interactions with TolB. The crystal
structure of the N-terminal tail deleted ColE7 T-domain
was determined by the multi-wavelength anomalous
dispersion method at a resolution of 1.7 A °. The structureof the ColE7 T-domain superimposes well with the Tdomain of ColE3 and TR-domain of ColB, a group A
Tol-dependent colicin and a group B TonB-dependent
colicin, respectively. The structural resemblance of group
A and B colicins implies that the two groups of colicins
may share a mechanistic connection during cellular
import.

FIGURE 1
Structures of the ColE7 T-domain and its superposition onto
ColE3 and ColB. (a) The crystal structure of the ColE7 Tdomain contains three central layers of b-sheets (in magenta, redand gold) flanked by three a-helices. The N-terminal end(residues 60–75) and a loop region from residues 120–123(displayed in a broken line) are disordered and not seen in thestructure.

Crystal Structure of ColE7 Translocation Domain

TAABLE 1T-domain was then produced and crystallized. Three setsof X-ray diffraction data were collected using synchrotron
radiation at different wavelengths at beamline NW12 in
the KEK Photon Factory, Tsukuba, Japan. The structure
was solved by the MAD method using anomalous signals
from four Se-atoms.

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Users' Report

FIGURE 2
Structural model of ColE7/Im7 complex. (a) The crystal
structure of the ColE7 T-domain was docked with thecrystal structure of the C-domain/Im7 complex (PDBentry, 1MZ8) by ZDOCK.28 The full-length ColE7/Im7model was constructed using the crystal structure ofColE3 (PDB entry, 1JCH) as a template to assemble theR-domain with T/C domains by Modeller 8.1.41 (b) The
molecular surface of the ColE7 T-domain is mapped withelectrostatic potential (electropositive charges in blue andnegative in red). This view is rotatedw1808 verticallyfrom the one in (a) to show the convex surface, which isfacing outward, not blocked by the C-domain and Im7.
The left region of the T-domain in this view is positively
charged and this surface is highly conserved among
ColE2, E3, E6, E7 and E9. This conserved basic region issuggested to be tested for a role in protein or membraneinteractions during colicin import.