title: Structural and Functional Analysis of a Novel Interaction Motif within UFM1-activating Enzyme 5 (UBA5) Required for Binding to Ubiquitin-like Proteins and Ufmylation.
abstract: The covalent conjugation of ubiquitin-fold modifier 1 (UFM1) to proteins generates a signal that regulates transcription, response to cell stress, and differentiation. Ufmylation is initiated by ubiquitin-like modifier activating enzyme 5 (UBA5), which activates and transfers UFM1 to ubiquitin-fold modifier-conjugating enzyme 1 (UFC1). The details of the interaction between UFM1 and UBA5 required for UFM1 activation and its downstream transfer are however unclear. In this study, we described and characterized a combined linear LC3-interacting region/UFM1-interacting motif (LIR/UFIM) within the C terminus of UBA5. This single motif ensures that UBA5 binds both UFM1 and light chain 3/γ-aminobutyric acid receptor-associated proteins (LC3/GABARAP), two ubiquitin (Ub)-like proteins. We demonstrated that LIR/UFIM is required for the full biological activity of UBA5 and for the effective transfer of UFM1 onto UFC1 and a downstream protein substrate both in vitro and in cells. Taken together, our study provides important structural and functional insights into the interaction between UBA5 and Ub-like modifiers, improving the understanding of the biology of the ufmylation pathway.
authors.affiliation: From Translational Innovation Platform Oncology, Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany, the Institute of Biochemistry II, Goethe University School of Medicine, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany, the BMLS Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Riedberg Campus, Max-von-Laue-Strasse 15, 60438 Frankfurt am Main, Germany.
authors.forename: Sabrina
authors.initials: S
authors.lastname: Habisov
authors.affiliation: the Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main, Germany.
authors.forename: Jessica
authors.initials: J
authors.lastname: Huber
authors.affiliation: the Department of Biochemistry, School of Medicine, Niigata University, Chuo-ku, Niigata 951-8510, Japan.
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Ichimura
authors.affiliation: the Institute of Biochemistry II, Goethe University School of Medicine, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany, the BMLS Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Riedberg Campus, Max-von-Laue-Strasse 15, 60438 Frankfurt am Main, Germany.
authors.forename: Masato
authors.initials: M
authors.lastname: Akutsu
authors.affiliation: the Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main, Germany.
authors.forename: Natalia
authors.initials: N
authors.lastname: Rogova
authors.affiliation: the Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main, Germany.
authors.forename: Frank
authors.initials: F
authors.lastname: Loehr
authors.affiliation: the Institute of Biochemistry II, Goethe University School of Medicine, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany, the Division of Cell Signaling and Immunology, School of Life Sciences, University of Dundee, Dundee, DD1 4HN United Kingdom, and.
authors.forename: David G
authors.initials: DG
authors.lastname: McEwan
authors.affiliation: the Molecular Cancer Research Group, Institute of Medical Biology, University of Tromsø-The Arctic University of Norway, 9037 Tromsø, Norway.
authors.forename: Terje
authors.initials: T
authors.lastname: Johansen
authors.affiliation: the Institute of Biochemistry II, Goethe University School of Medicine, Theodor-Stern-Kai 7, 60590 Frankfurt am Main, Germany, the BMLS Buchmann Institute for Molecular Life Sciences, Goethe University Frankfurt, Riedberg Campus, Max-von-Laue-Strasse 15, 60438 Frankfurt am Main, Germany.
authors.forename: Ivan
authors.initials: I
authors.lastname: Dikic
authors.affiliation: the Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main, Germany.
authors.forename: Volker
authors.initials: V
authors.lastname: Doetsch
authors.affiliation: the Department of Biochemistry, School of Medicine, Niigata University, Chuo-ku, Niigata 951-8510, Japan.
authors.forename: Masaaki
authors.initials: M
authors.lastname: Komatsu
authors.affiliation: the Institute of Biophysical Chemistry and Center for Biomolecular Magnetic Resonance, Goethe University, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main, Germany, rogov@bpc.uni-frankfurt.de.
authors.forename: Vladimir V
authors.initials: VV
authors.lastname: Rogov
authors.affiliation: From Translational Innovation Platform Oncology, Merck KGaA, Frankfurter Strasse 250, 64293 Darmstadt, Germany, vladimir.kirkin@merckgroup.com.
authors.forename: Vladimir
authors.initials: V
authors.lastname: Kirkin
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020816:Amino Acid Motifs; D051017:Apoptosis Regulatory Proteins; D057809:HEK293 Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship; D044764:Ubiquitin-Activating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D051017:Apoptosis Regulatory Proteins; C116741:GABARAP protein, human; D008869:Microtubule-Associated Proteins; D011506:Proteins; C502095:UBA5 protein, human; C502096:UFC1 protein, human; C502094:UFM1 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D044764:Ubiquitin-Activating Enzymes
keywords: LC3/GABARAP; LIR; UBA5; UFIM; UFM1; isothermal titration calorimetry (ITC); nuclear magnetic resonance (NMR); protein motif; signal transduction; x-ray crystallography