Conformational Polymorphism in Autophagy-Related Protein GATE-16

Abstract

Autophagy is a fundamental homeostatic process in eukaryotic organisms, fulfilling essential roles in development and adaptation to stress. Among other factors, formation of autophagosomes critically depends on proteins of the Atg8 (autophagy-related protein 8) family, which are reversibly conjugated to membrane lipids. We have applied X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics simulations to study the conformational dynamics of Atg8-type proteins, using GATE-16 (Golgi-associated ATPase enhancer of 16 kDa), also known as GABARAPL2, as a model system. This combination of complementary approaches provides new insight into a structural transition centered on the C-terminus, which is crucial for the biological activity of these proteins.

Structure and Dynamics in the ATG8 Family From Experimental to Computational Techniques

“…These data indicate a disordered N-terminal region and the presence of slow conformational exchange, involving the helices α1 and α2, and the loop α4-β4 (Stangler et al, 2002;Krichel et al, 2019). X-ray crystallography also suggested the existence of alternative conformations in the N-terminal region, as shown for the GABARAP subfamily (Coyle et al, 2002;Ma et al, 2015). Here, the changes are likely to be associated with rearrangements in the α1-α2 loop in the proximity of proline P10 of GABARAP (Coyle et al, 2002).…”

“…The C-terminal region of ATG8 proteins (i.e., the tract after the β4 strand) is also highly flexible and with a propensity to disorder, as indicated by NMR relaxation analysis and NOE measurements (Krichel et al, 2019). NMR relaxation measurements of GATE-16 also support the notion of a disordered C-terminal tract (residue 112-117), which populates different conformations in solution, from extended and solventaccessible "open" states to "closed" conformations, forming interactions with the LIR binding surface (Ma et al, 2015). To gain atom-level details on these conformational changes, the authors used an enhanced sampling approach based on a combination of Hamiltonian Replica-Exchange and conventional MD using the AMBER ff99SB-ILDN (Lindorff-Larsen et al, 2010) and CHARMM27 (Bjelkmar et al, 2010) force fields.…”

“…It should be noted that the major biological activities of ATG8 proteins depend on their membrane-bound state, as detailed in section "Interaction of ATG8 Family Members With Autophagic Membranes." The dramatic decrease in solubility of lipidconjugated ATG8 proteins challenges experimental techniques, so the majority of the biophysical studies in solution has been carried out on the non-lipidated forms (Stangler et al, 2002;Kouno et al, 2005;Kumeta et al, 2010;Schwarten et al, 2010;Klionsky et al, 2012;Rogov et al, 2013;Ma et al, 2015;Krichel et al, 2019). This is also true for the studies performed in silico with modeling and simulations (Ma et al, 2015;Di Rita et al, 2018;Holdgaard et al, 2019;Jatana et al, 2019).…”

“…To gain atom-level details on these conformational changes, the authors used an enhanced sampling approach based on a combination of Hamiltonian Replica-Exchange and conventional MD using the AMBER ff99SB-ILDN (Lindorff-Larsen et al, 2010) and CHARMM27 (Bjelkmar et al, 2010) force fields. The combination of NMR and MD allowed the authors to identify several conformational states in dynamic equilibrium for the C-terminal region (Ma et al, 2015). The simulation data suggest a 'swing-out' movement depending on the F115 anchoring residue (Ma et al, 2015).…”

The Atg8 Family of Proteins—Modulating Shape and Functionality of Autophagic Membranes

“…Recently, it was shown for yeast Atg4 that in addition to the LIR a so-called Atg8-PE association region (APEAR) close to the catalytic center is required for specific binding of membrane-bound Atg8 proteins, thus allowing for efficient recycling ( Abreu et al, 2017 ). Both experimental evidence and molecular dynamics simulations indicate that the Atg8 C-terminus needs to adopt an extended conformation to access the active site of the protease ( Satoo et al, 2009 ; Ma et al, 2015 ). In the next step, Atg8 gets activated by the E1-like enzyme Atg7 under consumption of ATP, yielding an adenylated intermediate which reacts to form a thioester with a cysteine residue of Atg7.…”