Structural basis of FYCO1 and MAP1LC3A interaction reveals a novel binding mode for Atg8-family proteins - PubMed (original) (raw)

Structural basis of FYCO1 and MAP1LC3A interaction reveals a novel binding mode for Atg8-family proteins

Xiaofang Cheng et al. Autophagy. 2016.

Abstract

FYCO1 (FYVE and coiled-coil domain containing 1) functions as an autophagy adaptor in directly linking autophagosomes with the microtubule-based kinesin motor, and plays an essential role in the microtubule plus end-directed transport of autophagic vesicles. The specific association of FYCO1 with autophagosomes is mediated by its interaction with Atg8-family proteins decorated on the outer surface of autophagosome. However, the mechanistic basis governing the interaction between FYCO1 and Atg8-family proteins is largely unknown. Here, using biochemical and structural analyses, we demonstrated that FYCO1 contains a unique LC3-interacting region (LIR), which discriminately binds to mammalian Atg8 orthologs and preferentially binds to the MAP1LC3A and MAP1LC3B. In addition to uncovering the detailed molecular mechanism underlying the FYCO1 LIR and MAP1LC3A interaction, the determined FYCO1-LIR-MAP1LC3A complex structure also reveals a unique LIR binding mode for Atg8-family proteins, and demonstrates, first, the functional relevance of adjacent sequences C-terminal to the LIR core motif for binding to Atg8-family proteins. Taken together, our findings not only provide new mechanistic insight into FYCO1-mediated transport of autophagosomes, but also expand our understanding of the interaction modes between LIR motifs and Atg8-family proteins in general.

Keywords: Atg8-family proteins; FYCO1; LIR and MAP1LC3A interaction; LIR-binding mode; MAP1LC3A; autophagy adaptor.

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Figures

Figure 1.

Figure 1.

Biochemical characterizations of the specific interactions between FYCO1 LIR and Atg8-family proteins. (A) A schematic diagram showing the domain organization of FYCO1. In this drawing, the boundary of FYCO1 LIR motif used in this study is further indicated and boxed. (B) Analytical gel filtration chromatography analysis of the interaction between purified FYCO1 LIR and LC3A protein. (C) ITC-based measurement of the binding affinity of FYCO1 LIR with LC3A. (D) Superposition plot of the 1H-15N HSQC spectra of LC3A titrated with increasing molar ratios of the FYCO1 LIR. (E) The measured binding affinities between FYCO1 LIR and 6 Atg8-family proteins or their mutants by ITC-based binding assay. ‘N.D.’ stands for that the KD value is not detectable.

Figure 2.

Figure 2.

The overall structure of FYCO1 LIR in complex with LC3A. (A) The ribbon representation model showing the overall structure of FYCO1 LIR-LC3A complex. In this drawing, the LC3A is shown in forest green, FYCO1 LIR motif in orange. (B) The FO-FC map of the FYCO1 LIR showing that the densities of 14 LIR residues (DDAVFDIITDEELC) can be clearly assigned. The map is calculated by omitting a LIR peptide from the final PDB file and contoured at 1.8σ. (C) The surface representation showing the overall architecture of FYCO1 LIR-LC3A complex with the same color scheme as in panel (A).

Figure 3.

Figure 3.

Molecular detail of the FYCO1 LIR and LC3A interaction. (A) The combined surface charge potential representation (contoured at ±7 kT/eV; blue/red) and the ribbon-stick model showing the detailed interactions between FYCO1 LIR and LC3A. (B) Stereo view showing the detailed interactions between FYCO1 LIR and LC3A. The hydrogen bonds involved in the binding are shown as dotted lines.

Figure 4.

Figure 4.

Detailed structural comparisons of the FYCO1 LIR binding mode with currently known LIR-binding modes. (A) The combined surface representation and the ribbon-stick model showing the hydrophobic interaction interface between FYCO1 LIR and LC3A. In this presentation, the LC3A molecule is shown in the surface model and FYCO1 LIR in the ribbon-stick model. In particular, the hydrophobic amino acid residues in LC3A surface model are drawn in yellow, the positively charged residues in blue, the negatively charged residues in red, and the uncharged polar residues in gray. (B) Currently known canonical LIR binding modes observed in the ATG13 LIR-LC3A complex, the FAM134B LIR-LC3A complex, the SQSTM1 LIR-LC3B complex, the OPTN LIR-LC3B complex, the PLEKHM1 LIR-LC3B complex, the NBR1 LIR-GABARAPL1 complex, the ATG4B LIR-LC3B complex and the ALFY LIR-GABARAP complex. (C) The noncanonical LIR binding mode observed in the CALCOCO2 CLIR-LC3C complex. (D) A schematic cartoon diagram summarizing the 3 different binding modes of LIR motifs in binding to Atg8-family proteins.

Figure 5.

Figure 5.

The specific FYCO1 LIR and LC3A interaction is required for cellular colocalizations of FYCO1 and LC3A in the rapamycin-treated HeLa cells. (A) When coexpressed, FYCO1 colocalizes well with the LC3A clusters. (B and C) The R10E (B) and I66A (C) mutations of LC3A that weaken the FYCO1 LIR-LC3A interaction attenuate the colocalization of FYCO1 and LC3A. (D to G) Point mutations of key interface residues of LC3A or FYCO1 LIR that disrupted their interaction in vitro essentially eliminate the colocalization of FYCO1 and LC3A. (H) Statistical results related to the colocalizations of FYCO1 and LC3A in the rapamycin-treated HeLa cells shown as Pearson correlation. The Pearson correlation coefficient analysis was performed using the LAS X software based on a randomly selected region that roughly contains one cotransfected HeLa cell. The data represent mean±s .d. of >50 analyzed cells (selected regions) from 2 independent experiments. The unpaired Student t test (unequal variance) analysis was used to define a statistically significant difference, and the asterisks denote the significant differences between the indicated bars (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

References

    1. Klionsky DJ, Emr SD. Cell biology - Autophagy as a regulated pathway of cellular degradation. Science 2000; 290:1717-21; PMID:11099404; http://dx.doi.org/10.1126/science.290.5497.1717 - DOI - PMC - PubMed
    1. Mizushima N. Autophagy: process and function. Genes Dev 2007; 21:2861-73; PMID:18006683; http://dx.doi.org/10.1101/gad.1599207 - DOI - PubMed
    1. Kroemer G, Marino G, Levine B. Autophagy and the integrated stress response. Molecular cell 2010; 40:280-93; PMID:20965422; http://dx.doi.org/10.1016/j.molcel.2010.09.023 - DOI - PMC - PubMed
    1. Levine B, Mizushima N, Virgin HW. Autophagy in immunity and inflammation. Nature 2011; 469:323-35; PMID:21248839; http://dx.doi.org/10.1038/nature09782 - DOI - PMC - PubMed
    1. Gomes LC, Dikic I. Autophagy in antimicrobial immunity. Mol Cell 2014; 54:224-33; PMID:24766886; http://dx.doi.org/10.1016/j.molcel.2014.03.009 - DOI - PubMed

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