Non-canonical Shedding of TNFα by SPPL2a Is Determined by the Conformational Flexibility of Its Transmembrane Helix
Charlotte Spitz,
Christine Schlosser,
Nadja Guschtschin-Schmidt,
Walter Stelzer,
Simon Menig,
Alexander Götz,
Martina Haug-Kröper,
Christina Scharnagl,
Dieter Langosch,
Claudia Muhle-Goll,
Regina Fluhrer
Affiliations
Charlotte Spitz
Biochemistry and Molecular Biology, Institute of Theoretical Medicine, Medical Faculty, University of Augsburg, Universitätsstrasse 2, 86159 Augsburg, Germany
Christine Schlosser
Biochemistry and Molecular Biology, Institute of Theoretical Medicine, Medical Faculty, University of Augsburg, Universitätsstrasse 2, 86159 Augsburg, Germany
Nadja Guschtschin-Schmidt
Karlsruhe Institute of Technology, Institute for Biological Interfaces 4, 76344 Eggenstein- Leopoldshafen, Germany and Karlsruhe Institute of Technology, Institute of Organic Chemistry, 76131 Karlsruhe, Germany
Walter Stelzer
Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany
Simon Menig
Physics of Synthetic Biological Systems, Technische Universität München, Maximus-von-Imhof Forum 4, 85340 Freising, Germany
Alexander Götz
Present Address: Leibniz Supercomputing Centre, Boltzmannstr. 1, 85748 Garching, Germany
Martina Haug-Kröper
Biochemistry and Molecular Biology, Institute of Theoretical Medicine, Medical Faculty, University of Augsburg, Universitätsstrasse 2, 86159 Augsburg, Germany
Christina Scharnagl
Physics of Synthetic Biological Systems, Technische Universität München, Maximus-von-Imhof Forum 4, 85340 Freising, Germany
Dieter Langosch
Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Weihenstephaner Berg 3, 85354 Freising, Germany
Claudia Muhle-Goll
Karlsruhe Institute of Technology, Institute for Biological Interfaces 4, 76344 Eggenstein- Leopoldshafen, Germany and Karlsruhe Institute of Technology, Institute of Organic Chemistry, 76131 Karlsruhe, Germany
Regina Fluhrer
Biochemistry and Molecular Biology, Institute of Theoretical Medicine, Medical Faculty, University of Augsburg, Universitätsstrasse 2, 86159 Augsburg, Germany; DZNE – German Center for Neurodegenerative Diseases, Feodor-Lynen-Str 17, 81377 Munich, Germany; Corresponding author
Summary: Ectodomain (EC) shedding defines the proteolytic removal of a membrane protein EC and acts as an important molecular switch in signaling and other cellular processes. Using tumor necrosis factor (TNF)α as a model substrate, we identify a non-canonical shedding activity of SPPL2a, an intramembrane cleaving aspartyl protease of the GxGD type. Proline insertions in the TNFα transmembrane (TM) helix strongly increased SPPL2a non-canonical shedding, while leucine mutations decreased this cleavage. Using biophysical and structural analysis, as well as molecular dynamic simulations, we identified a flexible region in the center of the TNFα wildtype TM domain, which plays an important role in the processing of TNFα by SPPL2a. This study combines molecular biology, biochemistry, and biophysics to provide insights into the dynamic architecture of a substrate's TM helix and its impact on non-canonical shedding. Thus, these data will provide the basis to identify further physiological substrates of non-canonical shedding in the future.
