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Negatively charged lipid membranes promote a disorder-order transition in the Yersinia YscU protein
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Medicine, Department of Molecular Biology (Faculty of Medicine). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Umeå Centre for Microbial Research (UCMR).
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Chemistry.
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2014 (English)In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 107, no 8, 1950-1961 p.Article in journal (Refereed) Published
Abstract [en]

The inner membrane of Gram-negative bacteria is negatively charged, rendering positively charged cytoplasmic proteins in close proximity likely candidates for protein-membrane interactions. YscU is a Yersinia pseudotuberculosis type III secretion system protein crucial for bacterial pathogenesis. The protein contains a highly conserved positively charged linker sequence that separates membrane-spanning and cytoplasmic (YscUC) domains. Although disordered in solution, inspection of the primary sequence of the linker reveals that positively charged residues are separated with a typical helical periodicity. Here, we demonstrate that the linker sequence of YscU undergoes a largely electrostatically driven coil-to-helix transition upon binding to negatively charged membrane interfaces. Using membrane-mimicking sodium dodecyl sulfate micelles, an NMR derived structural model reveals the induction of three helical segments in the linker. The overall linker placement in sodium dodecyl sulfate micelles was identified by NMR experiments including paramagnetic relaxation enhancements. Partitioning of individual residues agrees with their hydrophobicity and supports an interfacial positioning of the helices. Replacement of positively charged linker residues with alanine resulted in YscUC variants displaying attenuated membrane-binding affinities, suggesting that the membrane interaction depends on positive charges within the linker. In vivo experiments with bacteria expressing these YscU replacements resulted in phenotypes displaying significantly reduced effector protein secretion levels. Taken together, our data identify a previously unknown membrane-interacting surface of YscUC that, when perturbed by mutations, disrupts the function of the pathogenic machinery in Yersinia.

Place, publisher, year, edition, pages
Cell Press , 2014. Vol. 107, no 8, 1950-1961 p.
National Category
Biophysics
Identifiers
URN: urn:nbn:se:umu:diva-95192DOI: 10.1016/j.bpj.2014.09.005ISI: 000343682700021PubMedID: 25418176OAI: oai:DiVA.org:umu-95192DiVA: diva2:757880
Available from: 2014-10-23 Created: 2014-10-23 Last updated: 2017-12-05Bibliographically approved
In thesis
1. Regulation of the multi-functional protein YscU in assembly of the Yersinia type III secretion injectisome
Open this publication in new window or tab >>Regulation of the multi-functional protein YscU in assembly of the Yersinia type III secretion injectisome
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Alternative title[sv]
Reglering av det multifunktionella proteinet YscU i sammansättningen av Yersinias typ III-sekretionssystem
Abstract [en]

Yersinia pseudotuberculosis is a Gram-negative zoonotic pathogenic bacterium causing gastroenteritis in human and animals. It shares a conserved virulence plasmid encoding for a needle-like secretion machinery, or type III secretion system, which can be found in other pathogenic Gram-negative bacteria. The type III secretion system (T3SS) is a macromolecular assembly that enables pathogenic effector proteins (or Yersinia outer proteins, Yops) to be transported into eukaryotic host cells. This export machinery is assembled in a highly ordered stepwise mechanism. The activation of T3SS is also dependent on calcium concentration, temperature, and pH of the growth media as mimic factors for host cell’s contact. The T3SS-associated inner-membrane protein, YscU, of Yersinia is proposed to function as a substrate specificity switch protein and forms basal structure of T3SS. YscU has four α helical transmembrane domain and a soluble cytoplasmic domain YscUC which undergoes auto-proteolysis at a conserved N↑PTH motif. The auto-proteolysis process, which is required for the assembly of the injectisome and secretion of Yops, results in a 10-kDa C-terminal polypeptide fragment, denoted YscUCC and 6-kDa N-terminal fragment YscUCN. In this thesis, we showed that YscUC dissociation was important for Yops secretion and resulted in unfolded YscUCN and oligomeric YscUCC. By combination in vivo and in vitro methods, growth media conditions as calcium, temperature, and pH were indicated to control secretion by regulation of YscUC dissociation. The calcium-binding isotherm to YscUC was fit best with a one-site binding model resulting in Kd 800 µM, which is identical to calcium level that blocks secretion in vivo. YscU is also the key protein for the T3SS pH dependence, demonstrated by thermal unfolding profile and secondary structure of protein were altered between pH 7.4 and 6.0. In addition, bacterial inner membrane was proposed to assist the YscUCN folding, monitored by using lipid bilayer as a mimic environment in nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. This binding is important for Yops secretion and YscUC is anchored to bacterial membrane upon dissociation. The other substrate specificity switch protein YscP has function as a “molecular ruler” controlling length of the secretion needle. Previous genetic experiments have suggested that YscP and YscU interact physically, when mutation at defined residues on yscU (suppressor mutants) rescued Yops secretion in null-yscP mutant. In this research, direct binding of YscU and YscP was proved as weak but important interaction with Kd 430 mM by application of NMR and the binding interface of YscP was centred on the last helix of YscUC. Furthermore, we found that the YscP interaction could inhibit YscU auto-proteolysis. Studying the dissociation kinetic of suppressor YscUC variants at temperature 30 and 37oC provides strong support to a model where YscU is a temperature sensor for T3SS and YscUC dissociation is required for Yops secretion. Interestingly, the NPTH motif is conserved through most of YscU family members, meaning that role of dissociation may be conserved also in other bacterial injectisomes. To this end, the dissociation of YscU can be used as a therapeutic target in drug discovery. We attempted to identify the small-molecules that can hinder YscU dissociation. The small compound methyl(5-methyl-2-phenyl-1,3-thiazolidin-4-yl)acetate was found to be able to inhibit dissociation and to crystalize full YscUC, which has never been successfully done before. Finally, we found that the inner-rod protein YscI is binding to YscUC with a 1:1 stoichiometry as shown with pull-down assays and isothermal titration calorimetry. Taken together we have made several discoveries that expand the functional palette of YscU and all these functions were shown to have biological relevance with Yops secretion levels. In light of the strong sequence conservation between T3SS utilizing pathogenic bacteria the findings are likely to be general characters.

Place, publisher, year, edition, pages
Umeå: Umeå University, 2017. 64 p.
Keyword
Type III secretion system (T3SS), injectisome, Yersinia pseudotuberculosis, inhibitors, Yops, YscU, YscP, YscI, disorder-to-order transition, nuclear magnetic resonance (NMR), circular dichroism (CD), isothermal titration calorimetry (ITC)
National Category
Chemical Sciences
Research subject
biological chemistry
Identifiers
urn:nbn:se:umu:diva-130134 (URN)978-91-7601-613-8 (ISBN)
Public defence
2017-02-10, KB3B1, Stora Hörsalen, KBC huset, Umeå, 10:00 (English)
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Available from: 2017-01-20 Created: 2017-01-12 Last updated: 2017-01-19Bibliographically approved

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Weise, Christoph FLogin, Frédéric HHo, OanhGröbner, GerhardWolf-Watz, HansWolf-Watz, Magnus
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Department of ChemistryDepartment of Molecular Biology (Faculty of Medicine)Molecular Infection Medicine Sweden (MIMS)Umeå Centre for Microbial Research (UCMR)
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