Human LC3 and GABARAP subfamily members achieve functional specificity via specific structural modulations

Abstract

Autophagy is a conserved adaptive cellular pathway essential to maintain a variety of physiological functions. Core components of this machinery are the six human Atg8 orthologs that initiate formation of appropriate protein complexes. While these proteins are routinely used as indicators of autophagic flux, it is presently not possible to discern their individual biological functions due to our inability to predict specific binding partners. In our attempts towards determining downstream effector functions, we developed a computational pipeline to define structural determinants of human Atg8 family members that dictate functional diversity. We found a clear evolutionary separation between human LC3 and GABARAP subfamilies and also defined a novel sequence motif responsible for their specificity. By analyzing known protein structures, we observed that functional modules or microclusters reveal a pattern of intramolecular network, including distinct hydrogen bonding of key residues (F52/Y49; a subset of HP2) that may directly modulate their interaction preferences. Multiple molecular dynamics simulations were performed to characterize how these proteins interact with a common protein binding partner, PLEKHM1. Our analysis showed remarkable differences in binding modes via intrinsic protein dynamics, with PLEKHM1-bound GABARAP complexes showing less fluctuations and higher number of contacts. We further mapped 373 genomic variations and demonstrated that distinct cancer-related mutations are likely to lead to significant structural changes. Our findings present a quantitative framework to establish factors underlying exquisite specificity of human Atg8 proteins, and thus facilitate the design of precise modulators.

A conserved ATG2‐GABARAP family interaction is critical for phagophore formation

“…This raises an ongoing question as to how specificity within the system is achieved, particularly in mammalian systems that are complicated by the expression of six LC3/GABARAP isoforms. We, and others [13,43,44], have attempted to decipher the code that dictates whether a protein with a particular LIR sequence will preferentially interact with LC3 over GABARAP.…”

The conformational and mutational landscape of the ubiquitin-like marker for the autophagosome formation in cancer

“…LC3 proteins have not been thoroughly explored by systematic biomolecular simulations or other computational structural methods. Among the simulations present in the literature, each one focused on specific aspects of LC3B dynamics using all-atom molecular dynamics (MD) simulations with a single force field (Jatana et al, 2019;Liu et al, 2013;Nuta et al, 2018;L. Zhao et al, 2017) or coarse-grained approaches (Thukral et al, 2015).…”

A conserved ATG2-GABARAP interaction is critical for phagophore closure

“…This raises an ongoing question as to how specificity within the system is achieved, particularly in mammalian systems that are complicated by the expression of six LC3/GABARAP isoforms. We, and others [13,43,44], have attempted to decipher the code that dictates whether a protein with a particular LIR sequence will preferentially interact with LC3 over GABARAP. Interestingly, ATG2A and ATG2B do not conform to the recently identified GABARAP interaction motif consensus sequence (W/F-I/V-X-I/V) [13] despite preferring GABARAP over LC3 in coimmunoprecipitation from cells ( Figure 1E; Figure 2B-C).…”