Solution Structure of the Autophagy-Related Protein LC3C

Autophagy is a major lysosomal catabolic pathway essential for cellular homeostasis in eukaryotes. Cytoplasmatic cargo destined for autophagic degradation may comprise single proteins or protein aggregates, invading pathogens, and even entire organelles. In macroautophagy, the most intensely studied autophagic pathway, cargo is engulfed by a cup-shaped, growing double-membrane structure termed phagophore. Elongation of this autophagosomal membrane and sequestering of autophagic cargo is assisted by Atg8-like proteins. The lastly discovered and least well characterised member of the human LC3-like protein subfamily, LC3C, was the focus of this work. Integral to a comprehensive understanding of a protein’s function is knowledge of its three-dimensional structure. Therefore, the solution structure of LC3C was determined by liquid-state NMR spectroscopy. To this end, a cloning procedure, expression and purification schemes were established to procure pure and isotopically 15N- and / or 13C-labelled LC3C. Additionally, protein buffer, and NMR measurement conditions had to be established and optimised to generate highly concentrated (up to 760 μM), stable LC3C protein samples for data acquisition in multidimensional, heteronuclear NMR experiments. Subsequently, backbone and side-chain resonance assignments were determined and deposited in the BMRB (Krichel et al., 2016; BMRB accession ID: 26603). The determination of LC3C’s three-dimensional structure followed acquisition of NOESY data, which was translated into distance restraints in conjunction with residue-specific chemical shift assignments. The overall fold of LC3C is in accordance with other LC3-like paralogues, consisting of an ubiquitin-like core structure and an additional amino- terminal α-helix. Of special interest were the residues preceding this α-helix, which are-compared to other LC3-like proteins–in a state of higher structural dynamics or disorder as was shown through relaxation and heteronuclear NOE experiments. Similiar results in regard to its dynamic behaviour were obtained for LC3C’s carboxy-terminus. While LC3C is structurally closely related to other LC3 paralogues, its cellular activity may be decisively modulated by post-translational modifications. Therefore, the phosphorylation of LC3C by PKA was studied. To this end, in-vitro phosphorylation experiments were optimised and phosphorylation of LC3C’s PKA target residue (S18) observed by [1H-15N] HSQC NMR spectroscopy. This residue is also involved in the N-capping motif of the amino-terminal helix α2. Upon phosphorylation of S18, S18 to Q23, residues A31 to F33 (connecting the α2-helix to the ubiquitin-like core), and residues D110 to D112 (establishing electrostatic interactions to said α2-helix) experienced significant chemical shift changes. This might indicate an increased structural mobility of LC3C’s amino-terminal helix α2 upon phosphorylation of S18. Overall, these findings provide the first indication of a post-translational phosphorylation modulating the structure of an LC3-like protein.