Proteolysis of Rab32 by Salmonella GtgE induces an inactive GTPase conformation
Sergey Savitskiy,
Rudolf Wachtel,
Danial Pourjafar-Dehkordi,
Hyun-Seo Kang,
Vanessa Trauschke,
Don C. Lamb,
Michael Sattler,
Martin Zacharias,
Aymelt Itzen
Affiliations
Sergey Savitskiy
Department of Biochemistry and Signaltransduction, University Medical Centre Hamburg-Eppendorf (UKE), Martinistrasse 52, 20246 Hamburg, Germany; Center for Integrated Protein Science Munich (CIPSM), Department Chemistry, Technical University of Munich, Lichtenbergstrasse 4, 85748 Garching, Germany
Rudolf Wachtel
Center for Integrated Protein Science Munich (CIPSM), Department Chemistry, Technical University of Munich, Lichtenbergstrasse 4, 85748 Garching, Germany
Danial Pourjafar-Dehkordi
Physics Department T38, Technical University of Munich, James-Franck-Strasse 1, 85748 Garching, Germany
Hyun-Seo Kang
Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany; Chemistry Department, Biomolecular NMR and Center for Integrated Protein Science Munich, Technical University of Munich, 85748 Garching, Germany
Vanessa Trauschke
Department of Chemistry, Center for Nanoscience (CeNS), NanoSystems Initiative Munich (NIM) and Center for Integrated Protein Science Munich (CIPSM), Ludwig Maximilians-Universität München, Munich Germany
Don C. Lamb
Department of Chemistry, Center for Nanoscience (CeNS), NanoSystems Initiative Munich (NIM) and Center for Integrated Protein Science Munich (CIPSM), Ludwig Maximilians-Universität München, Munich Germany
Michael Sattler
Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany; Chemistry Department, Biomolecular NMR and Center for Integrated Protein Science Munich, Technical University of Munich, 85748 Garching, Germany
Martin Zacharias
Physics Department T38, Technical University of Munich, James-Franck-Strasse 1, 85748 Garching, Germany
Aymelt Itzen
Department of Biochemistry and Signaltransduction, University Medical Centre Hamburg-Eppendorf (UKE), Martinistrasse 52, 20246 Hamburg, Germany; Center for Integrated Protein Science Munich (CIPSM), Department Chemistry, Technical University of Munich, Lichtenbergstrasse 4, 85748 Garching, Germany; Centre for Structural Systems Biology (CSSB), University Medical Centre Hamburg-Eppendorf (UKE), Hamburg, Germany; Corresponding author
Summary: Rab GTPases are central regulators of intracellular vesicular trafficking. They are frequently targeted by bacterial pathogens through post-translational modifications. Salmonella typhimurium secretes the cysteine protease GtgE during infection, leading to a regioselective proteolytic cleavage of the regulatory switch I loop in the small GTPases of the Rab32 subfamily. Here, using a combination of biochemical methods, molecular dynamics simulations, NMR spectroscopy, and single-pair Förster resonance energy transfer, we demonstrate that the cleavage of Rab32 causes a local increase of conformational flexibility in both switch regions. Cleaved Rab32 maintains its ability to interact with the GDP dissociation inhibitor (GDI). Interestingly, the Rab32 cleavage enables GDI binding also with an active GTP-bound Rab32 in vitro. Furthermore, the Rab32 proteolysis provokes disturbance in the interaction with its downstream effector VARP. Thus, the proteolysis of Rab32 is not a globally degradative mechanism but affects various biochemical and structural properties of the GTPase in a diverse manner.
