1. Phosphorylation of the mitochondrial autophagy receptor Nix enhances its interaction with LC3 proteins
- Author
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Hironori Suzuki, Anne Hamacher-Brady, Matilda Šprung, Masato Kawasaki, Soichi Wakatsuki, Ryuichi Kato, Natalia Yu. Rogova, Nathan R. Brady, Verena Lang, Vladimir V. Rogov, Ivan Dikic, Maja Buljubašić, Mija Marinković, Volker Dötsch, and Ivana Novak
- Subjects
Models, Molecular ,0301 basic medicine ,Autophagosome ,Magnetic Resonance Spectroscopy ,Protein Conformation ,GABARAP ,Science ,Plasma protein binding ,Calorimetry ,Crystallography, X-Ray ,Article ,Serine ,Nix ,LIR phosphorylation ,LC3 ,mitophagy ,03 medical and health sciences ,Protein structure ,Proto-Oncogene Proteins ,ddc:570 ,Mitophagy ,Autophagy ,Humans ,Phosphorylation ,Multidisciplinary ,Chemistry ,Tumor Suppressor Proteins ,Membrane Proteins ,Mitochondria ,Cell biology ,030104 developmental biology ,ddc:540 ,Medicine ,Microtubule-Associated Proteins ,Protein Processing, Post-Translational ,HeLa Cells ,Protein Binding - Abstract
The mitophagy receptor Nix interacts with LC3/GABARAP proteins, targeting mitochondria into autophagosomes for degradation. Here we present evidence for phosphorylation-driven regulation of the Nix:LC3B interaction. Isothermal titration calorimetry and NMR indicate a ~100 fold enhanced affinity of the serine 34/35-phosphorylated Nix LC3-interacting region (LIR) to LC3B and formation of a very rigid complex compared to the non-phosphorylated sequence. Moreover, the crystal structure of LC3B in complex with the Nix LIR peptide containing glutamic acids as phosphomimetic residues and NMR experiments revealed that LIR phosphorylation stabilizes the Nix:LC3B complex via formation of two additional hydrogen bonds between phosphorylated serines of Nix LIR and Arg11, Lys49 and Lys51 in LC3B. Substitution of Lys51 to Ala in LC3B abrogates binding of a phosphomimetic Nix mutant. Functionally, serine 34/35 phosphorylation enhances autophagosome recruitment to mitochondria in HeLa cells. Together, this study provides cellular, biochemical and biophysical evidence that phosphorylation of the LIR domain of Nix enhances mitophagy receptor engagement.
- Published
- 2017
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