Solution structure of the autophagy-related protein LC3C reveals a polyproline II motif on a mobile tether with phosphorylation site

Carsten Krichel,Oliver Schillinger,Pitter F Huesgen,Christina Möckel,Heinrich Sticht,Dieter Willbold,Birgit Strodel,Philipp Neudecker,Oliver H Weiergräber

doi:10.1038/s41598-019-48155-8

Abstract

(Macro-)autophagy is a compartmental degradation pathway conserved from yeast to mammals. The yeast protein Atg8 mediates membrane tethering/hemifusion and cargo recruitment and is essential for autophagy. The human MAP1LC3/GABARAP family proteins show high sequence identity with Atg8, but MAP1LC3C is distinguished by a conspicuous amino-terminal extension with unknown functional significance. We have determined the high-resolution three-dimensional structure and measured the backbone dynamics of MAP1LC3C by NMR spectroscopy. From Ser18 to Ala120, MAP1LC3C forms an α-helix followed by the ubiquitin-like tertiary fold with two hydrophobic binding pockets used by MAP1LC3/GABARAP proteins to recognize targets presenting LC3-interacting regions (LIRs). Unlike other MAP1LC3/GABARAP proteins, the amino-terminal region of MAP1LC3C does not form a stable helix α1 but a “sticky arm” consisting of a polyproline II motif on a flexible linker. Ser18 at the interface between this linker and the structural core can be phosphorylated in vitro by protein kinase A, which causes additional conformational heterogeneity as monitored by NMR spectroscopy and molecular dynamics simulations, including changes in the LIR-binding interface. Based on these results we propose that the amino-terminal polyproline II motif mediates specific interactions with the microtubule cytoskeleton and that Ser18 phosphorylation modulates the interplay of MAP1LC3C with its various target proteins.

Highlights

Macroautophagy - hereafter termed autophagy - is an intracellular lysosomal degradation pathway conserved in eukaryotes[1]
In contrast to other LC3 proteins, LC3-interacting regions (LIRs) binding to LC3C in selective autophagy can be mediated by a non-canonical LIR motif (CLIR)[15]
High-resolution structure determination by NMR spectroscopy confirms that the well-ordered core of the autophagy-related protein MAP1LC3C adopts virtually the same tertiary structure in solution as in X-ray crystallography, extensive exchange line broadening reveals the existence of alternate, low-populated conformations

Summary

Introduction

Macroautophagy - hereafter termed autophagy - is an intracellular lysosomal degradation pathway conserved in eukaryotes[1]. The human MAP1LC3 (or LC3) proteins differ most significantly in the amino-terminal region (NTR) preceding this α-helical domain, which is considerably longer in LC3C as compared to LC3A and LC3B (Fig. 1). While for the latter two proteins a role of the NTR in membrane tethering[2,16] or in recognition of mitochondrial phospholipids during mitophagy has been reported[17], the functional implications of the longer NTR found in LC3C are still poorly understood. LC3C shares the PKA phosphorylation site mentioned above (Ser[18] in this case) with LC3A and LC3B22, and, unlike other LC3-proteins, contains a second PKC phosphorylation site at Ser[9] as predicted

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Results

Conclusion