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Tetratricopeptide repeats in the type III secretion chaperone, LcrH: their role in substrate binding and secretion.
Umeå University, Faculty of Medicine, Molecular Biology (Faculty of Medicine). (Francis)
Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). (Forsberg)
Umeå University, Faculty of Science and Technology, Molecular Biology (Faculty of Science and Technology). (Forsberg)
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2006 (English)In: Molecular Microbiology, ISSN 0950-382X, Vol. 59, no 1, 31-44 p.Article in journal (Refereed) Published
Abstract [en]

Non-flagellar type III secretion systems (T3SSs) transport proteins across the bacterial cell and into eukaryotic cells. Targeting of proteins into host cells requires a dedicated translocation apparatus. Efficient secretion of the translocator proteins that make up this apparatus depends on molecular chaperones. Chaperones of the translocators (also called class-II chaperones) are characterized by the possession of three tandem tetratricopeptide repeats (TPRs). We wished to dissect the relations between chaperone structure and function and to validate a structural model using site-directed mutagenesis. Drawing on a number of experimental approaches and focusing on LcrH, a class-II chaperone from the Yersinia Ysc-Yop T3SS, we examined the contributions of different residues, residue classes and regions of the protein to chaperone stability, chaperone-substrate binding, substrate stability and secretion and regulation of Yop protein synthesis. We confirmed the expected role of the conserved canonical residues from the TPRs to chaperone stability and function. Eleven mutations specifically abrogated YopB binding or secretion while three mutations led to a specific loss of YopD secretion. These are the first mutations described for any class-II chaperone that allow interactions with one translocator to be dissociated from interactions with the other. Strikingly, all mutations affecting the interaction with YopB mapped to residues with side chains projecting from the inner, concave surface of the modelled TPR structure, defining a YopB interaction site. Conversely, all mutations preventing YopD secretion affect residues that lie on the outer, convex surface of the triple-TPR cluster in our model, suggesting that this region of the molecule represents a distinct interaction site for YopD. Intriguingly, one of the LcrH double mutants, Y40A/F44A, was able to maintain stable substrates inside bacteria, but unable to secrete them, suggesting that these two residues might influence delivery of substrates to the secretion apparatus.

Place, publisher, year, edition, pages
2006. Vol. 59, no 1, 31-44 p.
Keyword [en]
Amino Acid Sequence, Bacterial Outer Membrane Proteins/chemistry/metabolism, Bacterial Proteins/chemistry/*genetics/*metabolism, Binding Sites, Humans, Models; Molecular, Molecular Chaperones/chemistry/*genetics/*metabolism, Molecular Sequence Data, Mutagenesis; Site-Directed, Phenotype, Protein Binding, Protein Conformation, Repetitive Sequences; Nucleic Acid, Sequence Alignment, Two-Hybrid System Techniques, Yersinia/genetics/metabolism
URN: urn:nbn:se:umu:diva-16670DOI: 10.1111/j.1365-2958.2005.04923.xPubMedID: 16359316OAI: diva2:156343
Available from: 2007-10-08 Created: 2007-10-08 Last updated: 2010-03-03Bibliographically approved
In thesis
1. Multiple twists in the molecular tales of YopD and LcrH in type III secretion by Yersinia pseudotuberculosis
Open this publication in new window or tab >>Multiple twists in the molecular tales of YopD and LcrH in type III secretion by Yersinia pseudotuberculosis
2007 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The type III secretion system (T3SS) is a highly conserved secretion system among Gram negative bacteria that translocates anti-host proteins directly into the infected cells to overcome the host immune system and establish a bacterial infection. Yersinia pseudotuberculosis is one of three pathogenic Yersinia spp. that use a plasmid encoded T3SS to establish an infection. This complex multi-component Ysc-Yop system is tightly regulated in time and space. The T3SS is induced upon target cell contact and by growth in the absence of calcium. There are two kinds of substrates for the secretion apparatus, the translocator proteins that make up the pore in the eukaryotic target cell membrane, and the translocated effector proteins, that presumably pass through this pore en route to the eukaryotic cell interior.

The essential YopD translocator protein is involved in several important steps during effector translocation, such as pore formation, effector translocation. Moreover, in complex with its cognate chaperone LcrH, it maintains regulatory control of yop gene expression. To understand the molecular mechanism of YopD function, we made sequential in-frame deletions throughout the entire protein and identified discrete functional domains that made it possible to separate the role of YopD in translocation from its role in pore formation and regulation, really supporting translocation to be a multi-step process. Further site-directed mutagenesis of the YopD C-terminus, a region important for these functions, revealed no function for amino acids in the coiled-coil domain, while hydrophobic residues within the alpha-helical amphipathic domain are functionally significant for regulation, pore formation and translocation of effectors.

Unique to the T3SSs are the chaperones which are required for efficient type III protein secretion. The translocator-class chaperone LcrH binds two translocator proteins, YopB and YopD, which is necessary for their pre-secretory stabilization and their efficient secretion. We have shown that LcrH interacts with each translocator at a unique binding-site established by the folding of its three tandem tetratricopeptide repeats (TPRs). Beside the regulatory LcrH-YopD complex, LcrH complexes with YscY, a component of the Ysc-Yop T3SS, that is also essential for regulatory control. Interestingly the roles for LcrH do not end here, because it also appears to function in fine tuning the amount of effector translocation into target cells upon cell contact. Moreover, LcrH’s role in pre-secretory stability appears to be an in vitro phenomenon, since upon bacteria-host cell contact we found accumulated levels of YopB and YopD inside the bacteria in absence of a LcrH chaperone. This suggests the true function of LcrH is seen during target cell contact. In addition, these stable YopB and YopD are secreted in a Ysc-Yop independent manner in absence of a functional LcrH. We propose a role for LcrH in conferring substrate secretion pathway specificity, guiding its substrate to the cognate Ysc-Yop T3SS to secure subsequent effector translocation.

Together, this work has sought to better understand the key functions of LcrH and YopD in Yersinia pathogenicity. Using an approach based heavily on recombinant DNA technology and tissue culture infections, the complex molecular cross-talk between chaperone and its substrate, and the effect this has on the Yersinia lifestyle, are now being discovered.

Place, publisher, year, edition, pages
Umeå: Molekylärbiologi (Teknat- och Medfak), 2007. 88 p.
Umeå University medical dissertations, ISSN 0346-6612 ; 1074
Yersinia pseudotuberculosis, T3SS, YopD, translocation process, LcrH, class II chaperone, substrate secretion pathway specificity
National Category
Biochemistry and Molecular Biology
urn:nbn:se:umu:diva-985 (URN)91-7264-231-9 (ISBN)
Public defence
2007-02-16, Major Groove, 6 L NUS, Umeå Universitet, Umeå, 09:00 (English)
Available from: 2007-01-31 Created: 2007-01-31 Last updated: 2009-05-15Bibliographically approved

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Francis, Matthew S
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Molecular Biology (Faculty of Medicine)Molecular Biology (Faculty of Science and Technology)Umeå Centre for Microbial Research (UCMR)
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