NDLI: Interactions of TolB with the Translocation Domain of Colicin E9 Require an Extended TolB Box

Content Provider	PubMed Central
Author	Hands, Sarah L. Holland, Lisa E. Vankemmelbeke, Mireille Fraser, Lauren Macdonald, Colin J. Moore, Geoffrey R. James, Richard Penfold, Christopher N.
Copyright Year	2005
Abstract	The mechanism by which enzymatic E colicins such as colicin E3 (ColE3) and ColE9 cross the outer membrane, periplasm, and cytoplasmic membrane to reach the cytoplasm and thus kill Escherichia coli cells is unique in prokaryotic biology but is poorly understood. This requires an interaction between TolB in the periplasm and three essential residues, D35, S37, and W39, of a pentapeptide sequence called the TolB box located in the N-terminal translocation domain of the enzymatic E colicins. Here we used site-directed mutagenesis to demonstrate that the TolB box sequence in ColE9 is actually larger than the pentapeptide and extends from residues 34 to 46. The affinity of the TolB box mutants for TolB was determined by surface plasmon resonance to confirm that the loss of biological activity in all except one (N44A) of the extended TolB box mutants correlates with a reduced affinity of binding to TolB. We used a PCR mutagenesis protocol to isolate residues that restored activity to the inactive ColE9 D35A, S37A, and W39A mutants. A serine residue at position 35, a threonine residue at position 37, and phenylalanine or tyrosine residues at position 39 restored biological activity of the mutant ColE9. The average area predicted to be buried upon folding (AABUF) was correlated with the activity of the variants at positions 35, 37, and 39 of the TolB box. All active variants had AABUF profiles that were similar to the wild-type residues at those positions and provided information on the size, stereochemistry, and potential folding pattern of the residues of the TolB Box.
Related Links	http://dx.doi.org/10.1128/jb.187.19.6733-6741.2005
Ending Page	6741
Page Count	9
Starting Page	6733
File Format	PDF
ISSN	00219193
e-ISSN	10985530
Journal	Journal of Bacteriology
Issue Number	19
Volume Number	187
Language	English
Publisher	American Society for Microbiology
Publisher Date	2005-10-01
Access Restriction	Open
Rights Holder	American Society for Microbiology
Subject Keyword	Molecular Biology Microbiology Research in Higher Education
Content Type	Text
Resource Type	Article
Subject	Molecular Biology Microbiology

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Competitive recruitment of the periplasmic translocation portal TolB by a natively disordered domain of colicin E9

The crystal structure of the TolB box of colicin A in complex with TolB reveals important differences in the recruitment of the common TolB translocation portal used by group A colicins

Destabilization of the Colicin E9 Endonuclease Domain by Interaction with Negatively Charged Phospholipids IMPLICATIONS FOR COLICIN TRANSLOCATION INTO BACTERIA

Investigating Early Events in Receptor Binding and Translocation of Colicin E9 Using Synchronized Cell Killing and Proteolytic Cleavage ▿

Flexibility in the Receptor-Binding Domain of the Enzymatic Colicin E9 Is Required for Toxicity against Escherichia coli Cells

Maturation and localization of the TolB protein required for colicin import.

Competitive recruitment of the periplasmic translocation portal TolB by a natively disordered domain of colicin E9

Enzymological characterization of the nuclease domain from the bacterial toxin colicin E9 from Escherichia coli.

NMR study of Ni2+ binding to the H-N-H endonuclease domain of colicin E9.

Interactions of TolB with the Translocation Domain of Colicin E9 Require an Extended TolB Box

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Competitive recruitment of the periplasmic translocation portal TolB by a natively disordered domain of colicin E9

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