Translating Membrane Geometry into Protein Function: Multifaceted Membrane Interactions of Human Atg3 Promote LC3-Phosphatidylethanolamine Conjugation during Autophagy

Abstract

Autophagosome formation is the hallmark of macroautophagy (herein referred to as autophagy) and requires the covalent conjugation of LC3 proteins (or Atg8 in yeast) to the amino headgroup of PE (phosphatidylethanolamine) lipids. Atg3 is an enzyme that catalyzes the final step of this reaction by transferring LC3 from an LC3-Atg3 intermediate to PEs in targeted membranes. Here, we determine the solution structure of human Atg3 (hAtg3) and demonstrate that the catalytically important regions of hAtg3 are conformationally dynamic. Furthermore, we reveal that these regions and hAtg3′s N-terminal membrane curvature-sensing amphipathic helix concurrently interact with the membrane. These structural studies indicate that hAtg3 exploits a multifaceted membrane-association mechanism to position its catalytic center at the membrane surface and to bring the reaction substrates of LC3 and PE lipids to proximity for effective LC3-PE conjugation. In addition, our studies demonstrate that the interaction of the His266 residue with the membrane is primarily responsible for hAtg3′s pH-dependent activity. Our investigations advance an emerging concept that the interactions of Atg3 with the highly curved membrane rims of the phagophore spatially regulate autophagosome biogenesis.

Three-step docking by WIPI2, ATG16L1, and ATG3 delivers LC3 to the phagophore

“…We found neutral His 266 to stabilize a catalytically competent structure of the active site, consistent with retention of full lipidation activity by the H266A mutant. In contrast, protonation of His 266 disrupted the active site in our MD simulations, consistent with a role of His 266 as pH sensor ( 35 ). While uncharged His 266 serves to stabilize the catalytic loop conformation, our data point to active participation of His 262 in the initiation of the LC3 lipidation reaction.…”

“…S1B). The core of the human ATG3 structure is architecturally similar to the yeast and Arabidopsis Atg3 proteins, as has been previously reported ( 35 ), with an intrinsically disordered region ( 36 ) forming a ~100-residue loop that contains the ATG12-binding sequence ( 21 ) (Fig. 1A) as well as a region predicted to participate in β sheet formation in the presence of LC3 (fig.…”

“…His 262 and Cys 264 of human ATG3 form part of the HPC motif that is conserved across orthologs of ATG3 as well as ATG10, an E2-like autophagic enzyme that catalyzes ATG12 conjugation to ATG5 (6). Combined with previous (35,51) and present evidence of a critical role of His 262 for ATG3 conjugase activity, our simulation results are suggestive of a plausible reaction mechanism in which the His 262 imidazole ring would deprotonate the PE amine group for nucleophilic attack on the Gly 120 carbonyl of LC3 (Fig. 6C).…”

“…His 266 , the second of the two conserved histidine residues described above, has been implicated in the pH-dependent conjugase activity of ATG3 in a recent study (35). To assess the effect of altering the protonation state of His 266 , we performed additional MD simulations of the ATG3-LC3 conjugate in which the His 266 imidazole ring was doubly protonated.…”

Three-step docking by WIPI2, ATG16L1 and ATG3 delivers LC3 to the phagophore

“…S1B). The core of the human ATG3 structure is architecturally similar to the yeast and Arabidopsis Atg3 proteins, as has been previously reported (35), with an intrinsically disordered region (36) forming a ~100residue loop that contains the ATG12-binding sequence (21) (Fig. 1A) as well as a region predicted to participate in β sheet formation in the presence of LC3 (fig.…”