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J Biol Chem. 2013 Feb 8;288(6):4048-55. doi: 10.1074/jbc.M112.395889. Epub 2012 Dec 11.

LcrH, a class II chaperone from the type three secretion system, has a highly flexible native structure.

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1
School of Biological and Chemical Sciences, Queen Mary, University of London, London, UK.

Abstract

The type three secretion system is a large and complex protein nano-machine that many Gram-negative pathogens employ to infect host cells. A key structure of this machine is a proteinaceous pore that inserts into the target membrane and forms a channel for bacterial toxins to flow from bacteria into the host cell. The pore is mainly formed from two large membrane proteins called "translocators." Importantly, effective secretion and thus pore formation of the translocators depend on their binding to and being transported by small specialized chaperones after synthesis in the bacterial cytosol. Recent crystal structures have shown these chaperones are formed from modular tetratricopeptide repeats. However, each crystal structure produced different homodimeric structures, suggesting flexibility in their topology that may be of importance to function. Given the crucial role of the translocator chaperones, we investigated the conformational stability of the chaperone LcrH (Yersinia pestis). Mutational analysis coupled with analytical ultracentrifugation and equilibrium denaturations showed that LcrH is a weak and thermodynamically unstable dimer (K(D) ≈15 μm, ΔG(H(2)O) = 7.4 kcal mol(-1)). The modular tetratricopeptide repeat structure of the dimer allows it to readily unfold in a noncooperative manner to a one-third unfolded dimeric intermediate (ΔG(H(2)O) = 1.7 kcal mol(-1)), before cooperatively unfolding to a monomeric denatured state (ΔG(H(2)O) = 5.7 kcal mol(-1)). Thus, under physiological conditions, the chaperone is able to populate C-terminally unraveled partially folded states, while being held together by its dimeric interface. Such ability suggests a "fly-casting" mechanism as a route to binding their far larger translocator cargo.

PMID:
23233673
PMCID:
PMC3567656
DOI:
10.1074/jbc.M112.395889
[Indexed for MEDLINE]
Free PMC Article
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