Abstract
The Atg8 protein family comprises the GABA type A receptor-associated proteins (GABARAPs) and microtubule-associated protein 1 light chains 3 (MAP1LC3s) that are essential mediators of autophagy. The LC3-interacting region (LIR) motifs of autophagy receptors and adaptors bind Atg8 proteins to promote autophagosome formation, cargo recruitment, and autophagosome closure and fusion to lysosomes. A crystal structure of human GABARAPL2 has been published [PDB entry 4co7; Ma et al. (2015), Biochemistry, 54, 5469-5479]. This was crystallized in space group P21 with a monoclinic angle of 90° and shows a pseudomerohedral twinning pathology. This article reports a new, untwinned GABARAPL2 crystal form, also in space group P21, but with a 98° monoclinic angle. No major conformational differences were observed between the structures. In the structure described here, the C-terminal Phe117 binds into the LIR docking site (LDS) of a neighbouring molecule within the asymmetric unit, as observed in the previously reported structure. This crystal contact blocks the LDS for co-crystallization with ligands. Phe117 of GABARAPL2 is normally removed during biological processing by Atg4 family proteases. These data indicate that to establish interactions with the LIR, Phe117 should be removed to eliminate the crystal contact and liberate the LDS for co-crystallization with LIR peptides.
Highlights
Autophagy is a fundamental cellular degradation pathway that is required for the homeostatic recycling of cellular components and organelles (Mizushima, 2007)
A key step in autophagy is the formation of the isolation membrane, a double-membrane structure which upon sealing forms autophagosomes that fuse with lysosomes to degrade their contents
Crystals which formed in 0.15 M KBr, 30%(w/v) polyethylene glycol monomethyl ether diffracted to 1.9 Aresolution and the data were indexed, integrated and scaled using the Australian Synchrotron autoprocessing software with XDSME (Legrand, 2017), a Scicluna et al GABARAPL2 141
Summary
Autophagy is a fundamental cellular degradation pathway that is required for the homeostatic recycling of cellular components and organelles (Mizushima, 2007). Atg proteins can bind the LIR motifs of autophagy ‘adaptors’, which have functions beyond degradation, including autophagosome formation, transport and fusion with lysosomes (Wirth et al, 2019). Like other Atg proteins, GABARAPL2 is comprised of an N-terminal helical extension preceding four -sheets in a ubiquitin-like -grasp fold (Ma et al, 2015) This fold forms the LIR docking site (LDS), consisting of hydrophobic pocket 1 (HP1) and hydrophobic pocket 2 (HP2). The structure reported here shares the same P21 space group as the published structure, but showed a different monoclinic angle and is not twinned In these structures, the C-terminal Phe117 binds in the HP1 pocket of a neighbouring molecule, thereby blocking the LDS and providing a rationale for the absence of bound LIR peptide
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