Atg8-Family Proteins-Structural Features and Molecular Interactions in Autophagy and Beyond

Abstract

Autophagy is a common name for a number of catabolic processes, which keep the cellular homeostasis by removing damaged and dysfunctional intracellular components. Impairment or misbalance of autophagy can lead to various diseases, such as neurodegeneration, infection diseases, and cancer. A central axis of autophagy is formed along the interactions of autophagy modifiers (Atg8-family proteins) with a variety of their cellular counter partners. Besides autophagy, Atg8-proteins participate in many other pathways, among which membrane trafficking and neuronal signaling are the most known. Despite the fact that autophagy modifiers are well-studied, as the small globular proteins show similarity to ubiquitin on a structural level, the mechanism of their interactions are still not completely understood. A thorough analysis and classification of all known mechanisms of Atg8-protein interactions could shed light on their functioning and connect the pathways involving Atg8-proteins. In this review, we present our views of the key features of the Atg8-proteins and describe the basic principles of their recognition and binding by interaction partners. We discuss affinity and selectivity of their interactions as well as provide perspectives for discovery of new Atg8-interacting proteins and therapeutic approaches to tackle major human diseases.

Keywords: Atg8; GABARAP; LC3; LIR motif; SAR; UBL; autophagy.

Figure 1

Structural features of ubiquitin and Atg8/LC3/GABARAP proteins. Ribbon diagrams of the (A) ubiquitin and (B) LC3B structures aligned to the common ubiquitin core (left plot in B). The right plot was generated by rotation of the LC3B structure by −70° around the Y-axis. The secondary structure elements in both proteins are colored in rainbow color-code from α-helix α1 in LC3B (red-orange-green-cyan-blue), all β-strands are colored yellow. The PDB ID codes of each structure are presented under the protein names. (C) LC3B structure with C-terminal α-helix α5 (the same orientation as the right plot in (B)). (D) Structural alignment of yeast Atg8 and human LC3A, LC3B, LC3C, GABARAP, GABARAPL1, and GABARAPL2 (rainbow color-code) proteins shown as ribbon diagrams (the same orientation as the left plot in (B)). (E) Left plot: surface representation of LC3B structure (the same orientation as the left plot in (B)), showing the main interacting sites—HP1 (magenta) and HP2 (light green), which form the LC3 docking site (LDS). Position of additional interacting sites, like HP0 [50], Y-site [51], etc., are indicated by arrows. The alternative interacting area, the UIM docking site (UDS), is located on the opposite side of the LC3B molecule (right plot). The most relevant residues are colored dark red, additional hydrophobic residues around it are colored yellow.

Figure 2

Sequence alignment of Atg8/LC3/GABARAP proteins. Sequence alignment of the Atg8-family members from six model species—yeast (Saccharomyces cerevisiae), Nematoda (Caenorhabditis elegans), insect (Drosophila melanogaster), fish (Danio rerio), human (Homo sapiens), and plant (Arabidopsis thaliana). Secondary structure elements from the human LC3B (PDB ID 2ZJD) are shown on top (color-code as in Figure 1B,C). Every tenth residue in each sequence is marked bold/underlined, the catalytic Gly is marked green. The identity scores (asterix, * , for identical residues; colon, : , for very similar residues; dot, . , for analogous residues; space, , for residues without any similarity; dash, - , for gaps) are presented below each group of the Atg8/LC3/GABARAP. The residues (or their absence) separating GABARAP/Atg8 and LC3 protein subtypes are marked red and blue, respectively. The consensus string for all 28 proteins is presented at the bottom of alignment. The residues showed conservation are grouped within the following classes: residues participating in the protein folding (grey); residues forming HP1 (magenta); residues forming HP2 (light green); and residues forming UDS (yellow).

Figure 3

Structural differences between the LC3 and GABARAP proteins. (A) Specific structural difference between GABARAP/Atg8 and LC3 protein subtypes. Intramolecular contacts within LC3 (top) and GABARAP (bottom) proteins; color code as in Figure 1D. Involved residues are presented as sticks; the shortest distance is given for specific protein residues (indicated at the plot). (B) Orientation of H27 and K30 sidechains in LC3B (top) and corresponding Y25 and R28 sidechains in GABARAPs (bottom). Cation-π interactions (the non-covalent electrostatic interaction between an electron-reach face of aromatic rings and adjacent cations), stabilizing the specific orientation of Y25/R28 sidechains in GABARAPs are shown as dashed lines.

Figure 4

Atg8/LC3/GABARAP interactions with their partners. (A,B) The LIR concept. Structure of p62/SQSTM1-LIR:LC3B complex (PDB ID 2ZJD) (A). LC3B is shown as a semi-transparent surface with the structural elements (α-helices and β-strands) visible. p62/SQSTM1-LIR is shown as a main chain (cyan) with sidechains of core LIR residues (W340 and L343, blue) as sticks. Two hydrophobic pockets of LC3B, accommodating W340 and L343 sidechains, are shown on LC3B surface (HP1—magenta, HP2—light green). (B) Alignment of canonical (upper section) and non-canonical (lower section) LIR motifs with positions of residues indicated on top (from −6 to +7). Negatively charged residues (red), polar residues (orange), and phosphorylatable residues (green) are indicated over the LIR sequences. The phosphorylatable residues confirmed to be phosphorylated are marked bold/underlined. The underlined characters within core LIR sequences indicate residues whose sidechains are accommodated by HP1 and HP2. (C–H) Types of Atg8/LC3/GABARAP-interacting motifs and elements are shown as examples of known structures. For all plots, LC3B surface in orientations as in Figure 1E is shown. The interacting elements are given as ribbon diagrams for the known structures; in the case of Ubx5, the putative position of the helical ubiquitin-interacting motif (UIM) is indicated by a gray cylinder. Within the interacting elements, residues contributed with sidechains into HP1 and HP2 are shown in sticks and colored blue, residues within the close LDS contacts are colored cyan, and residues with other contacts are yellow. For each type, the names of the known interactors are indicated, as well as their functional roles sorted in groups of “core autophagy machinery,” “selective autophagy receptors,” and “other proteins.”

Figure 5

Emerging types of Atg8/LC3/GABARAP interacting motifs and elements: the antiparallel β-strand (A), and displacing α-helical structure (B). The interacting elements shown as grey arrows or cylinders on LC3B ribbon diagram (top) and on LC3B surface (bottom, with HP1 and HP2 indicated). (C) Superbinder construction. Combination of the known/hypothetical binding elements (shown as red cylinders/arrows for each type), complete polypeptide with all the elements, and the resulting position of the “superbinder” on the surface of LC3B.