ABSTRACT
Selective autophagy is mediated by cargo receptors that link the cargo to the isolation membrane via interactions with Atg8 proteins. Atg8 proteins are localized to the membrane in an ubiquitin-like conjugation reaction, but how this conjugation is coupled to the presence of the cargo is unclear. Here we show that the S. cerevisiae Atg19, Atg34 and the human p62, Optineurin and NDP52 cargo receptors interact with the E3-like enzyme Atg12~Atg5-Atg16, which stimulates Atg8 conjugation. The interaction of Atg19 with the Atg12~Atg5-Atg16 complex is mediated by its Atg8-interacting motifs (AIMs). We identify the AIM-binding sites in the Atg5 subunit and mutation of these sites impairs selective autophagy. In a reconstituted system the recruitment of the E3 to the prApe1 cargo is sufficient to drive accumulation of conjugated Atg8 at the cargo. The interaction of the Atg12~Atg5-Atg16 complex and Atg8 with Atg19 is mutually exclusive, which may confer directionality to the system.
Subject(s)
Autophagy-Related Protein 5/chemistry , Autophagy-Related Protein 8 Family/chemistry , Autophagy-Related Proteins/chemistry , Autophagy/genetics , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae/genetics , Amino Acid Sequence , Autophagy-Related Protein 12/genetics , Autophagy-Related Protein 12/metabolism , Autophagy-Related Protein 5/genetics , Autophagy-Related Protein 5/metabolism , Autophagy-Related Protein 8 Family/genetics , Autophagy-Related Protein 8 Family/metabolism , Autophagy-Related Proteins/genetics , Autophagy-Related Proteins/metabolism , Binding Sites , Biological Transport , Cell Cycle Proteins , Cloning, Molecular , Escherichia coli/genetics , Escherichia coli/metabolism , Gene Expression Regulation , HeLa Cells , Humans , Membrane Transport Proteins , Molecular Docking Simulation , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Structure, Secondary , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Sequestosome-1 Protein/genetics , Sequestosome-1 Protein/metabolism , Signal Transduction , Transcription Factor TFIIIA/genetics , Transcription Factor TFIIIA/metabolism , Vesicular Transport Proteins/genetics , Vesicular Transport Proteins/metabolismABSTRACT
Thermodynamic integration (TI) is one of the most commonly used free-energy calculation methods. The derivative of the Hamiltonian with respect to lambda, ⟨∂H/∂λ⟩, is determined at multiple λ-points. Because a numerical integration step is necessary, high curvature regions require simulations at densely spaced λ-points. Here, the principle of extended TI is introduced, where ⟨∂H/∂λ⟩ values are predicted at nonsimulated λ-points. On the basis of three model systems, it is shown that extended TI requires significantly fewer λ-points than regular TI to obtain similar accuracy.
ABSTRACT
A central intermediate in purine catabolism, the inosine nucleobase hypoxanthine is also one of the most abundant modified nucleobases in RNA and plays key roles in the regulation of gene expression and determination of cell fate. It is known that hypoxanthine acts as guanine when interacting with other nucleobases and base pairs most favorably with cytosine. However, its preferences when it comes to interactions with amino acids remain unknown. Here we present for the first time the absolute binding free energies and the associated interaction modes between hypoxanthine and all standard, non-glycyl/non-prolyl amino acid side chain analogs as derived from molecular dynamics simulations and umbrella sampling in high- and low-dielectric environments. We illustrate the biological relevance of the derived affinities by providing a quantitative explanation for the specificity of hypoxanthine-guanine phosphoribosyltransferase, a key enzyme in the purine salvage pathway. Our results demonstrate that in its affinities for protein side chains, hypoxanthine closely matches guanine, much more so than its precursor adenine.
