umu.sePublications
Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Characterization of the Ruler Protein Interaction Interface on the Substrate Specificity SwitchProtein in the Yersinia Type III Secretion System
Umeå University, Faculty of Science and Technology, Department of Chemistry. (Magnus Wolf-Watz)
Umeå University, Faculty of Science and Technology, Department of Chemistry.
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
Umeå University, Faculty of Science and Technology, Department of Molecular Biology (Faculty of Science and Technology). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
Show others and affiliations
2017 (English)In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, no 8, 3299-3311 p.Article, review/survey (Refereed) Published
Abstract [en]

Many pathogenic Gram-negative bacteria use the type III secretion system (T3SS) to deliver effector proteins into eukaryotic host cells. In Yersinia the switch to secretion of effector proteins is induced first after that intimate contact between the bacterium and its eukaryotic targetcell has been established and the T3SS proteins YscP and YscU are playing a central role in thisprocess. Here we identify the molecular details of the YscP binding site on YscU by means o fnuclear magnetic resonance (NMR) spectroscopy. The binding interface is centeredon the C-terminal domain of YscU. Disruptingthe YscU/YscP interaction by introducing point mutations at the interaction interface significantly reduced the secretion of effector proteins and HeLa cell cytotoxicity. Interestingly, the bindingof YscP to the slowly self-cleaving YscU variantP264A conferred significant protection againstauto-proteolysis. The YscP mediated inhibition of YscU auto-proteolysis suggest that the cleavage event may act as a timing switch in the regulationof early vs. late T3SS substrates. We also show that YscUC binds to the inner-rod protein YscI with a Kd of 3.8 μM and with one-to-one stoichiometry. The significant similarity between different members of the YscU, YscP, YscI families suggests that the protein-protein interactions discussed in this study are alsorelevant for other T3SS-containing Gram-negative bacteria.

Place, publisher, year, edition, pages
2017. Vol. 292, no 8, 3299-3311 p.
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:umu:diva-130048DOI: 10.1074/jbc.M116.770255ISI: 000395538800021OAI: oai:DiVA.org:umu-130048DiVA: diva2:1064042
Available from: 2017-01-11 Created: 2017-01-11 Last updated: 2017-05-20Bibliographically 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)
Opponent
Supervisors
Available from: 2017-01-20 Created: 2017-01-12 Last updated: 2017-01-19Bibliographically approved

Open Access in DiVA

No full text

Other links

Publisher's full text

Search in DiVA

By author/editor
Ho, OanhRogne, PerEdgren, TomasWolf-Watz, HansLogin, FrédericWolf-Watz, Magnus
By organisation
Department of ChemistryDepartment of Molecular Biology (Faculty of Science and Technology)Molecular Infection Medicine Sweden (MIMS)
In the same journal
Journal of Biological Chemistry
Biochemistry and Molecular Biology

Search outside of DiVA

GoogleGoogle Scholar

Altmetric score

Total: 173 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf