AUTOPHAGY 2016, VOL. 12, NO. 8, 1330–1339 http://dx.doi.org/10.1080/15548627.2016.1185590

BASIC RESEARCH PAPER

Structural basis of FYCO1 and MAP1LC3A interaction reveals a novel binding mode for Atg8-family proteins Xiaofang Chenga,b, Yingli Wanga, Yukang Gonga, Faxiang Lia,b, Yujiao Guoa, Shichen Hua, Jianping Liua, and Lifeng Pana,c a State Key Laboratory of Biorganic and Natural Products Chemistry, Shanghai, China; bInterdisciplinary Research Center on Biology and Chemistry, Shanghai, China; cCollaborative Innovation Center of Chemistry for Life Sciences, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, China

ABSTRACT

ARTICLE HISTORY

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 Atg8family 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.

Received 3 August 2015 Revised 25 April 2016 Accepted 28 April 2016

Introduction Autophagy is a highly conserved and spatially regulated cellular catabolic process in eukaryotic cells, during which undesired cytosolic components including bulk protein aggregates, dysfunctional organelles and invasive microbes are targeted to the lysosome for degradation.1,2 Because of its fundamental roles in maintaining cellular homeostasis and adapting to various cellular stresses, autophagy is highly relevant for a number of physiologic processes such as embryogenesis, aging and immune response,3-5 and dysfunctions of autophagy are linked with numerous devastating human diseases such as cancer and neurodegenerative diseases.6-8 As the most prevalent subtype of autophagy, macroautophagy (hereafter referred to as autophagy) is characterized by the formation of the double-membrane vesicles termed autophagosomes, which engulf portions of the cytosol and deliver to lysosomes forming the degradative autolysosomes.9,10 The maturation of autolysosome relies on the tight coordination of autophagic vesicle trafficking.11 In particular, the intracellular autophagosomes need to be well transported along the cytoskeleton in order to encounter, and subsequently fuse with later endosomes or lysosomes.12,13 So far only a few proteins involved in the cytosolic transport of autophagic vesicles are identified, and little is known about their detailed working mechanism. The Atg8-ortholog and paralog mammalian proteins, which currently consist of the MAP1LC3 and GABARAP subfamilies

KEYWORDS

autophagy adaptor; Atg8family proteins; FYCO1; LIR and MAP1LC3A interaction; LIR-binding mode; MAP1LC3A

including 7 orthologs known as MAP1LC3A (LC3A), MAP1LC3B (LC3B), MAP1LC3B2 (LC3B2), MAP1LC3C (LC3C), GABARAP, GABARAPL1 and GABARAPL2 in mammals, are the only known ubiquitin-like proteins attached to the nascent preautophagosomal membrane named the phagophore by conjugation with a phosphatidylethanolamine (PE) lipid.14-16 The PE-conjugated Atg8 proteins are present both on the inner and outer membranes of the phagophore, and play vital roles in autophagosome biogenesis and cargoes recruitment during selective autophagy.13-15,17 In addition, Atg8-family proteins also directly engaged in the coordinated autophagic vesicles transportation by associating with proteins related to autophagic vesicle trafficking, such as FYCO118 and MAPK8IP1/JIP1,19 which coupled to the microtubule-based anterograde-directed kinesin and retrograde-directed dynein motors, respectively. Accumulating evidence showed that the specific interactions between Atg8-binding proteins and Atg8-family proteins were mainly mediated by a short motif named LC3interacting region (LIR).20-29 The canonical LIR motif has the consensus core sequence QxxG where Q being aromatic residues (W/F/Y), and G being bulk hydrophobic residues (L/I/ V).17,26 Additional acidic residues and/or serine/threonine phosphorylation sites preceding the hydrophobic LIR core sequence are also commonly found in LIR-containing proteins, and are demonstrated to regulate their specific interactions

CONTACT Lifeng Pan [email protected] Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, No.345 Lingling Road, Shanghai, China Supplemental data for this article can be accessed on the publisher’s website. © 2016 Taylor & Francis

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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 1 15 H- N 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.

with Atg8-family members.20,30-32 Recently, a noncanonical LIR motif, which solely comprises 3 consecutive hydrophobic residues (LVV), has been found in the autophagy receptor CALCOCO2/NDP52.33 This atypical LIR motif (termed as CLIR) exclusively interacts with the Atg8-family member, LC3C, and endows CALCOCO2 with a unique function in xenophagy.33 Although the LIR-mediated interactions of Atg8family proteins with LIR-containing proteins involved in autophagosome biogenesis and autophagic cargoes recruitment are well characterized, no relevant detailed information related to autophagic vesicle trafficking has been available yet. FYCO1 is an identified autophagy adaptor involved in the microtubule plus end-directed autophagosome transport.18 FYCO1 contains a N-terminal RUN domain, which is

implicated in the interaction with the small GTPases of the RAB and RAP families (Fig. 1A).34,35 In addition, the central region of FYCO1 is predicted to form several coiled-coil domains, which mediate the dimerization of FYCO1, and are also reported to interact with the microtubule plus end-directed kinesin motor KIF5B and the small GTPase RAB7.18,24,36 The C-terminal region of FYCO1 consists of a phosphatidylinositol-3-phosphate (PtdIns3P) lipid-binding FYVE domain, a LIR motif, and a following globular GOLD domain with unknown function. Depletion of FYCO1 in cells leads to the accumulation of autophagosomes in the perinuclear region, whereas overexpression of FYCO1 redistributes autophagic vesicles to microtubule plus ends at the cell periphery,18 indicating its crucial roles in the transport of autophagic vesicles. Interestingly,

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although FYCO1 could interact with the ubiquitin-like modifier Atg8-family proteins, in contrast to autophagy receptors such as SQSTM1/p62, NBR1 (neighbor of BRCA1 gene 1), OPTN (optineurin) and CALCOCO2/NDP52, which are sequestered into the autophagosome during the autophagy process, FYCO1 is exclusively recruited to the external membrane of the autophagosome.18,37 However, how and why FYCO1 only associates with Atg8-family members located at the outer surface of the autophagosome is currently not known, and the detailed binding mechanism of FYCO1 for Atg8-family proteins remains to be elucidated. In this study, we systematically characterized the interactions between FYCO1 LIR and Atg8-family proteins, and discovered that the unique FYCO1 LIR can interact with 6 mammalian Atg8 orthologs, but preferentially and strongly binds to the LC3A and LC3B members. Additionally, we determined the high-resolution crystal structure of FYCO1 LIR in complex with LC3A. The structure of the FYCO1 LIRLC3A complex not only provides mechanistic insights into the specific interaction between FYCO1 and LC3A, but also reveals a previously unknown interaction mode between LIRs and Atg8-family proteins, which, first, highlights the notion that extension sequences C-terminal to the LIR core motif are required for many LIR-containing proteins to specifically interact with Atg8-family proteins. In sum, our findings expand our knowledge on the detailed mechanism of LIRmediated target interactions and FYCO1-involved cytosolic transport of autophagosomes.

Results The LIR motif of FYCO1 discriminately binds to mammalian Atg8 orthologs The region between FYVE and GOLD domains of FYCO1 is reported to contain an LIR motif that can specifically bind to LC3B protein (Fig. 1A).18 Interestingly, detailed sequence alignment analysis showed that in addition to the canonical LIR core motif (“FDII”), the regions flanking this core motif also contained several highly conserved negatively charged and hydrophobic residues (Fig. S1A). Since adjacent acidic residues N-terminal of the canonical LIR core sequence had been demonstrated to participate in the Atg8-binding,22,31,38 we wondered whether these upstream and downstream regions of this core LIR motif might also contribute to the selective interactions between FYCO1 and different mammalian Atg8 orthologs. To test this hypothesis, we purified the FYCO1 LIR protein (residues 1273 to 1297) and investigated its interactions with 6 mammalian Atg8 orthologs. Using qualitative analytical gel filtration chromatography analysis, we found the FYCO1 LIR region could directly and specifically interact with all 6 Atg8 orthologs (Fig. 1B; Fig. S2). Further quantitative analyses of the interactions between FYCO1 LIR and different Atg8 orthologs using isothermal titration calorimetry (ITC) revealed that FYCO1 LIR bound to 6 Atg8 orthologs with distinct binding affinity KD values (Fig. 1C and E; Fig. S3). In particular, FYCO1 LIR preferentially bound to LC3A and LC3B with relatively strong KD values, 0.54 mM and 0.19 mM, respectively (Fig. 1C and E; Fig. S3A). Next, we used NMR spectroscopy to

further characterize the specific interaction between FYCO1 LIR and LC3A. Titration of the 15N-labeled LC3A (residues 1 to 121) with the FYCO1 LIR showed that a selected set of peaks in the 1H-15N HSQC spectrum underwent significant dosedependent peak-broadenings or chemical shift changes (Fig. 1D), and importantly a slow exchange pattern in the NMR experiment was observed before saturation, confirming the strong interaction between FYCO1 LIR and LC3A. Taken together, all these biochemical results demonstrated that FYCO1 LIR could specifically and discriminately bind to different mammalian Atg8 orthologs. Overall structure of the FYCO1 LIR in complex with LC3A To uncover the molecular basis governing this unique interaction between FYCO1 LIR and Atg8 proteins, we sought to determine their complex structures. Based on the aforementioned quantitative ITC result, the binding affinities of LC3A and LC3B toward FYCO1 LIR were much stronger than that of other Atg8 orthologs. Therefore, the initial crystal screen was carried out only using these 2 complexes. We failed to get any crystals using the LIR-LC3B complex, but fortunately using the LIR-LC3A complex we obtained good crystals that diffracted to  2.3-A resolution. The FYCO1 LIR-LC3A complex structure was solved using the molecular replacement method (Table S1). In the final refined structural model, each asymmetric unit contains 4 LIR-LC3A complex molecules, and each LIR-LC3A complex has a 1:1 stoichiometry (Fig. 2A). As expected, the LC3A in the complex structure adopts a typical Atg8 ortholog protein architecture consisting of 2 N-terminal a-helices and a following ubiquitin-like core, and is highly similar to the previously reported apo from the structure of LC3A (Fig. S4). The clearly defined LIR motif in the complex structure contains 14 highly conserved residues (DDAVFDIITDEELC) (Fig. 2B), and mainly forms a short C-terminal a-helix together with a preceding b-strand that directly augments the b2-strand of LC3A in a parallel manner (Fig. 2A). The entire LIR motif of FYCO1 packs extensively with a solvent-exposed elongated groove mainly formed by the a1-helix, a2-helix, b2-strand and a3-helix of LC3A, burying a  total surface area of »810 A2 (Fig. 2A and C), in line with the strong binding observed between FYCO1 LIR and LC3A using ITC analysis. Intriguingly, structural comparisons with currently known LIR-Atg8-family complexes revealed that the overall binding mode of FYCO1 LIR toward LC3A represented a novel interaction mode of LIR to Atg8 proteins (Fig. S5). The molecular interface of the FYCO1 LIR-LC3A complex Detailed structural analysis of the FYCO1 LIR-LC3A interface revealed that the specific interaction between FYCO1 LIR and LC3A was primarily mediated by hydrophobic and polar interactions (Fig. 3A and 3B). Particularly, the aromatic side chain of F1280 of FYCO1 LIR deeply inserted into a hydrophobic pocket (HP1) of LC3A formed by the hydrophobic side chains of V20, I23, P32, L53, F108 together with the aliphatic side chain of K51; the hydrophobic side chains of I1283 and L1288 from FYCO1 LIR occupied another hydrophobic pocket (HP2), which was situated at the b2/a3 groove and formed by

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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 1.8s. (C) The surface representation showing the overall architecture of FYCO1 LIR-LC3A complex with the same color scheme as in panel (A).

F52, V54, P55, V58, L63, and I66 residues of LC3A. In addition, the backbone oxygen of FYCO1 D1277 formed a strong hydrogen bond with the side chain of K51 located at the b2-strand of LC3A, and the backbone groups of D1281, I1283 residues located at the short b-strand of LIR formed 3 backbone hydrogen bonds with the K51, L53 residues of the paired LC3A b2-strand. The LIR-LC3A complex was further stabilized by 2 unique charge-charge interaction networks, one of which located at the N-terminal region of LIR-LC3A interface was mediated by the negatively charged D1277 of FYCO1 LIR and the positively charged R10, R11 residues of LC3A, while the other was formed between negatively charged D1281, E1287

residues of FYCO1 and positively charged K49, R69, R70 residues of LC3A. Importantly, all these key residues of FYCO1 LIR and LC3A involved in the binding interface were strictly conserved throughout evolution (Fig. S1). Using quantitative ITC analysis, we further verified the specific interaction between FYCO1 LIR and LC3A. Individual point mutations of the key residues involved in the complex interface either from FYCO1 or LC3A, such as the D1277A, L1288Q, F1280R, D1281R, E1287R mutations of FYCO1 LIR (Fig. S6A to E) or the R10E, I66A, R70E mutations of LC3A (Fig. S6F to H), all largely decreased or essentially abolished the specific interaction between FYCO1 LIR and LC3A.

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.

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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 LIRLC3A 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.

Intriguingly, further amino acid sequence alignment analysis showed that several key interface residues varied greatly among the 6 Atg8 orthologs (Fig. S7). For instance, the residue corresponding to the positively charged R10 in LC3A was a negatively charged Asp or Glu residue in the GABARAP subfamily, and the residue corresponding to the positively charged R69 in LC3A was a polar Ser residue in LC3C (Fig. S7). The identification of nonconserved interface residues among Atg8-family proteins not only provided a mechanistic explanation to the selective binding of FYCO1 LIR to different Atg8 orthologs, but also confirmed the notion that residues outside the LIRbinding hydrophobic pockets confer the binding selectivity to different Atg8 orthologs. Detailed comparisons of the FYCO1 LIR-binding mode with currently known LIR-binding modes The most striking feature of the FYCO1 LIR in binding to LC3A was that in addition to the canonical LIR core sequence (“FDII”) that mainly bound to the HP1 and HP2 hydrophobic pockets of LC3A as well as the N-terminal acidic residue D1277 that engaged in electrostatic interactions with positively charged R10 and R11 residues of LC3A, 2 adjacent residues C-terminal of the canonical LIR core motif, E1287 and L1288, also directly participated in the binding to LC3A (Fig. 4A). This extensive interaction between FYCO1 LIR and LC3A gave rise to an extremely strong binding affinity with a KD »0.54 mM, in contrast to the relatively weak KD value normally found for a typical LIR motif in binding to an Atg8-family protein.23,28,31 Intriguingly, further detailed structural comparisons

of FYCO1 LIR-LC3A complex with other solved LIR-Atg8family complexes revealed that all the currently known LIRmediated target interactions could be further categorized into 3 modes besides the conventional canonical and noncanonical LIR-binding modes classification (Fig. 4A to D). Mode I, which was the best characterized and observed in most LIR-Atg8-family protein complexes such as the ATG13 LIR-LC3A complex, the FAM134B LIR-LC3A complex, the SQSTM1 LIR-LC3B complex, the OPTN LIR-LC3B complex, the PLEKHM1 LIRLC3B complex, the NBR1 LIR-GABARAPL1 complex, and the atypical CALCOCO2 CLIR-LC3C complex (Fig. 4B and C), involved the synergic bindings of a LIR core motif to the canonical LIR docking site (mainly the hydrophobic HP1 and HP2 pockets) on an Atg8-family member and an acidic motif (either negatively charged residues or phosphorylated serine/ threonine sites) preceding the hydrophobic LIR core motif to some basic residues of an Atg8 protein (Fig. 4D). In Mode II, some LIR motifs lacked the N-terminal acidic motif (e.g. the LIR of ATG4B) but contained extension sequences immediate C-terminal to the hydrophobic LIR core motif, and these extension sequences synergized with the LIR core motif in binding to an Atg8-family protein (Fig. 4B and D). The interacting mode observed in FYCO1 LIR and ALFY LIR could be categorized as the Mode III that contained features from the first 2 modes for binding to Atg8-family proteins (Fig. 4A, B and D). Given the large number of LIR-containing proteins in mammalian genomes,24,25 it is likely that other LIR motifs may also interact with Atg8-family proteins using a similar binding mode that we have discovered for FYCO1 LIR. Indeed, further sequence analysis of the currently known 43

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LIR motifs revealed that the presence of a negatively charged Glu or Asp residue, which located at the 7th position C-terminal to the core aromatic residue (W/F/Y) corresponding to the E1287 residue of FYCO1 LIR, was highly prevalent (Fig. S8). Cellular colocalization of FYCO1 and LC3A required the specific interaction between FYCO1 LIR and LC3A We next investigated the role of the FYCO1 LIR-LC3A interaction in the cellular localizations of FYCO1 and LC3A in the transfected HeLa cells. Consistent with the data reported earlier,18 when cotransfected, the exogenously expressed mCherry-tagged FYCO1 showed a puncta staining pattern in the cytoplasm of transfected cells and colocalized well with the GFP-tagged LC3A puncta either with or without rapamycin treatment (Fig. 5A and H; Fig. S9A and H). In contrast, although the R10E and I66A mutants of LC3A still formed dense puncta in transfected cells, their abilities to colocalize with the FYCO1 puncta were largely compromised (Fig. 5B, C and H; Fig. S9B, C and H), in line with the in vitro ITC result that mutations of these 2 residues of LC3A would largely decrease but not completely disrupt the interaction between LC3A and FYCO1 LIR (Fig. S6F and G). To further evaluate the FYCO1 LIR-LC3A interaction in the cellular colocalization of FYCO1 with LC3A, we also tested the R70E mutant of LC3A and F1280R, D1281R, E1287R mutants of FYCO1, as mutations of these key interface residues would essentially abolish the FYCO1 LIR and LC3A interaction (Fig. 1E). When the 2 proteins were coexpressed, the colocalizations between FYCO1 and LC3A were almost completely eliminated (Fig. 5D to H; Fig. S9D to H), which demonstrated that the specific interaction between FYCO1 LIR and LC3A is crucial for the cellular colocalization of FYCO1 and LC3A in cotransfected cells. As control, the cellular localizations of exogenously expressed mCherry-FYCO1, GFP-LC3A and their mutants were not interfered by the cotransfected GFP tag or mCherry tag (data not shown).

Discussion Previously, the importance of extension sequences C-terminal to the hydrophobic LIR core motif in binding to Atg8-family proteins was almost completely ignored. Our study revealed that adjacent residues C-terminal to the hydrophobic LIR core motif of FYCO1 LIR also played a crucial role in binding to LC3A, especially the negatively charged E1287 residue, substitution of which with a positively charged Arg residue would essentially abolish the specific interaction between FYCO1 and LC3A in vitro and in cells (Fig. 1E; Fig. S6E; Fig. 5G). In the FYCO1 LIR-LC3A complex structure, the negatively charged E1287 residue formed strong charge-charge interactions with the positively charged R69 and R70 residues located at the a3helix of LC3A (Fig. 3). Importantly, a consensus sequence analysis of 43 known LIR motifs revealed that an acidic amino acid was highly prevalent at the position corresponding to the E1287 residue of FYCO1 LIR (Fig. S8), and meanwhile the positively charged residues that were critical for E1287-binding were highly conserved among Atg8-family proteins (Fig. S7). Therefore, it is highly conceivable that specific charge-charge

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interactions between extension sequences C-terminal to the LIR core motif and the positively charged residues located at the a3-helix of Atg8 proteins likely constitute a general mechanism for many LIR motifs in binding to Atg8-family proteins. Interestingly, a recent study showed that phosphorylation of a Ser residue C-terminal to the LIR core motif of BNIP3 (BCL2/ adenovirus E1B 19kDa interacting protein 3) could increase the bindings of BNIP3 with LC3B and GABARAPL2, and regulate the function of BNIP3 in mitophagy.39 Accordingly, post-translational modifications of residues located at the extension sequence C-terminal to the LIR core motif are also likely to regulate the interactions between LIR motifs and Atg8-family proteins. FYCO1 only interacts with Atg8-family proteins that resided on the external surface of autophagosomes.18 Given the strong interactions between FYCO1 LIR and the Atg8 members, LC3A and LC3B, which are also located at the inner surface of the phagophore, it is puzzling why FYCO1 only associated with the out surface of the autophagosome. Obviously, a regulation of FYCO1’s interaction with Atg8-family proteins is likely involved. In addition to the unique LIR motif, FYCO1 also contains a kinesin-binding region, a PtdIns3P-binding FYVE domain, and a protein-protein interaction GOLD domain (our unpublished data). Interestingly, previous study showed that the highly acidic connecting loop between the FYVE and GOLD domains, which includes the LIR motif, could regulate the PtdIns3P binding of FYCO1 in vitro and reduce the membrane recruitment of FYVE domain-containing FYCO1 mutants in cells.18 We speculated that the specific interaction between FYCO1 and Atg8-family proteins is likely well-tuned by these protein-lipid and protein-protein interactions. To clarify which regions of FYCO1 may provide the specificity for FYCO1 only interacting with Atg8-family proteins at the surface of the autophagosome, we generated several defective FYCO1 mutants including a LIR-swapping mutant with the FYCO1 LIR motif (DDAVFDIITDEELC) replaced by the LIR of SQSTM1/p62 (GDDDWTHLSSKE), a kinesin-binding deficient mutant by deleting its kinesin-binding region (residues 735 to 773) as well as a PtdIns3P-binding deficient mutant lacking its FYVE domain (residues 1166 to 1232), and assayed their localizations with exogenously expressed LC3A and LC3B in the cotransfected HeLa cells. The obtained image results together with subsequently quantitative analyses showed that the SQSTM1 LIR-swapping mutant of FYCO1 had a poor ability to colocalize with LC3A or LC3B in the cotransfected cells either with or without rapamycin treatment when compared with the wild-type FYCO1 (Fig. S10A to D; Fig. S11A to D), whereas the kinesin-binding deficient mutant and the FYVE deletion mutant could still colocalize well with LC3A or LC3B in the cotransfected cells (Fig. S10E to I; Fig. S11E to I). These data clearly indicated that the unique LIR motif, especially the newly identified stretch of amino acids C-terminal of the LIR, rather than its kinesin-binding region or the FYVE domain, is likely to determine that FYCO1 only interacts with Atg8 family proteins located at the cytoplasmic face of autophagosome. However, the precise underlying mechanism remains to be elucidated. In addition, when and how these Atg8-family proteins bound to FYCO1 are eventually released from the autophagosome is still unknown. Thus, further studies are required to

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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).

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elucidate the detailed molecular mechanism governing the temporal and spatial regulation of the interaction between FYCO1 and Atg8-family proteins. Although Atg8-family proteins in mammals have been implicated in autophagy, the precise functions of them in autophagy are still elusive. In this study, we demonstrated that 6 Atg8 orthologs discriminately bound to the LIR motif of FYCO1, and particularly the LC3A and LC3B members extremely strongly interacted with the FYCO1 LIR (Fig. 1E). Further detailed structural study elucidated that differences in the N-terminal 2 a-helices region as well as the region within the b2-strand and a3-helix of the 6 Atg8 orthologs led to their distinct abilities in binding to the FYCO1 LIR motif (Fig. 3; Fig. S7). Thus, our findings suggested that differences between the Atg8 proteins in those regions might reflect their distinct functions. As an autophagy adaptor, FYCO1 directly participated in the transport of autophagosomes, therefore a relatively stronger interaction between FYCO1 and Atg8 proteins is likely preferred. Although, in this study, we showed that the exogenously overexpressed LC3A and LC3B have highly similar cellular localization patterns with different FYCO1 variants in cotransfected cells (Fig. S10; Fig. S11), whether each endogenous Atg8-family protein has a specific role in FYCO1-mediated autophagic vesicular trafficking remains to be elucidated. In summary, the atomic structure of FYCO1 LIR in complex with LC3A determined in this study not only elucidated the detailed molecular mechanism underpinning the FYCO1 and LC3A complex formation, but also provided a mechanistic explanation to the selective binding of FYCO1 LIR to different Atg8 orthologs. Furthermore, the structure of the FYCO1 LIRLC3A complex also uncovered a previously ignored binding feature of LIR motifs, and highlighted the importance of extension sequences C-terminal to the hydrophobic LIR core motif in binding to Atg8-family proteins. In addition to providing new mechanistic insight into FYCO1-mediated transport of autophagosomes, the data presented in this work also expanded our knowledge on the interaction modes of LIR motifs in general.

Materials and methods Protein expression and purification The DNA fragment encoding human FYCO1 LIR (residues 1273 to 1297) was PCR amplified from the full-length human FYCO1 cDNA. The coding sequence of human LC3A (residues 1 to 121) was PCR-amplified from the full-length human MAP1LC3A. The fusion fragment of FYCO1 LIR-thrombinLC3A was cloned into a pET-32M vector (an in-house modified version of pET32a vector containing a N-terminal thioredoxin-tag and His6-tag) using the CloneExpress II One Step Cloning kit (Vazyme Biotech Co., C112-02). All point mutations of FYCO1 LIR and LC3A used in this study were created using the standard PCR-based mutagenesis method and further confirmed by DNA sequencing. Recombinant proteins were expressed in BL21 (DE3) E. coli cells at 16 C. The bacterial cells were lysed by the ultrahighpressure homogenizer FB-110XNANO homogenizer machine (Shanghai Litu Machinery Equipment Engineering Co.,

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Shanghai), then the lysis was spun down by centrifuge at 18000 rpm (39191 g) for 30 min to remove the pellets. His6tagged proteins were purified by Ni2C-NTA (nickel-charged nitrilotriacetic acid) agarose (GE Healthcare, 17-5318-03) affinity chromatography. The recombinant protein was further purified by size-exclusion chromatography. The terminal tag of each recombinant protein was cleaved by 3C protease (purified in house) and further removed by size-exclusion chromatography. The collected protein was then cleaved by thrombin and purified by size-exclusion chromatography. Uniformly 15Nlabeled LC3A protein was prepared by growing bacteria in M9 minimal medium using 15NH4Cl (Cambridge Isotope Laboratories Inc., NLM-467) as the sole nitrogen source.

NMR spectroscopy The protein samples for NMR studies were concentrated to »0.1 mM for titration experiments in 50 mM sodium phosphate buffer containing 50 mM NaCl, 1 mM EDTA, 1 mM DTT, pH 6.5. NMR spectra were acquired at 25 C on an Agilent 800 MHz spectrometer (Shanghai Institute of Organic Chemistry) equipped with an actively z-gradient shielded triple resonance cold probe.

Crystallography Crystals of FYCO1 LIR and LC3A complex were obtained by the hanging drop vapor diffusion technique at 16 C. Freshly purified complex protein (15 mg/ml in 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM DTT and 1 mM EDTA) was mixed with equal volumes of reservoir solution containing 1.2 M sodium citrate tribasic dihydrate, 0.1 M HEPES sodium (pH 7.3). Crystals were frozen using reservoir solution added 10%  glycerol as cryoprotectant. A 2.3 A resolution X-ray data set was collected at the beamline BL17U1 of the Shanghai Synchrotron Radiation Facility. The diffraction data were processed and scaled using HKL2000.40 The phase problem of the complex was solved by molecular replacement method using the crystal structure of LC3A (PDB id: 3WAL) as the search models with PHASER.41 The initial model was rebuilt manually using COOT,42 and then refined  using REFMAC43 and PHENIX44 against the 2.3 A resolution data set. The qualities of the final model were validated by MolProbity.45 The final refinement statistics are listed in Table S1. The structure figures were prepared using the program PyMOL (http://pymol.sourceforge.net/).

Analytical gel filtration chromatography Analytical gel filtration chromatography was carried out on an AKTA FPLC system (GE Healthcare, Shanghai Institute of Organic Chemistry). Proteins were loaded on to a Superose 12 10/300 GL column (GE Healthcare, 17-5173-01) equilibrated with a buffer containing 20 mM Tris-HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM DTT.

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Isothermal titration calorimetry assay ITC measurements were carried out on an ITC200 calorimeter (GE Healthcare, Shanghai Institute of Organic Chemistry) at 25 C. All protein samples were in the same buffer containing 50 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, 1 mM DTT. The concentrated 25 mM Atg8 proteins and 280 mM of FYCO1 LIR protein were loaded into the cell and the syringe, respectively. The titration processes were performed by injecting 40 ml aliquots of the FYCO1 LIR proteins into Atg8 proteins at time intervals of 2 min to ensure that the titration peak returned to the baseline. The titration data were analyzed using the program Origin7.0 from Micro Cal and fitted using the one-site binding model.

LC3 LC3A LC3B LC3C LIR NMR PtdIns3P

microtubule-associated protein 1 light chain 3 MAP1LC3A MAP1LC3B MAP1LC3C LC3-interacting region nuclear magnetic resonance phosphatidylinositol-3-phosphate

Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.

Acknowledgments Cell culture, transfection and fluorescence imaging HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, 11995–065) supplemented with 10% fetal bovine serum (Invitrogen, 10099–141). The wild-type FYCO1 gene from the human species (GenBank accession number XM_011534111) was cloned into the mCherry-C1 vector to generate the mCherry-FYCO1 plasmid, and human LC3A and LC3B were cloned into the pEGFP-C1 vector (Clontech Laboratories, 6084-1) for the GFP-LC3A and GFP-LC3B plasmids, respectively. Transfections of HeLa cells with 0.8 mg of each mCherry-FYCO1 or mutant plasmids, and 0.5 mg of each GFPLC3A, GFP-LC3B or mutant plasmids per well were performed using Lipofectamine 2000 (Invitrogen, 11668–019) according to the manufacturer’s instructions. After 20 h, transfected HeLa cells are treated with 5 mM rapamycin (dissolved in DMSO; Amresco, 0231–500 ml) or the same amount of DMSO for 3 h. Then, cells were fixed with 4% paraformaldehyde and punched with PBS buffer (MesGen Biotech, MG3150) containing 0.2% Triton X-100 (Sangon, T0694-500 ml). The cell images were captured and analyzed using the TCS SP5 confocal microscope equipped with LAS X software (Leica, Inc., Thornwood, NY, USA). Coordinates The atomic coordinate of the FYCO1 LIR and LC3A complex has been deposited in the Protein Data Bank under the accession codes 5CX3.

Abbreviations Atg8 BNIP3 CALCOCO2 DAPI DTT EDTA FYCO1 GABARAP HSQC ITC IPTG

autophagy-related protein 8 BCL2/adenovirus E1B 19kDa interacting protein 3 calcium-binding and coiled-coil domain 2 40 ,6-diamidino-2-phenylindole DL-dithiothreitol ethylene diamine tetraacetic acid FYVE and coiled-coil domain containing 1 GABA type A receptor-associated protein heteronuclear single-quantum coherence; isothermal titration calorimetry isopropyl-b-D-thiogalactopyranoside

We thank SSRF BL17U and NCPSS BL19U1 for X-ray beam time, Dr. Zhijie Lin, Dr. Shang Yuan and Dr. Jianchao Li for help in the X-ray diffraction data collection, Dr. Cong Liu for providing the ITC machine, Dr. Wenning Wang for the help in the confocal microscopy imaging, Dr. Jiahuai Han for the full length FYCO1 cDNA used in this study.

Funding This work was supported by grants from the National Natural Science Foundation of China 31470749, National Basic Research Program of China 2013CB836900, a Shanghai Rising Star Scholar award 13QA1404300, a “Thousand Talents Program” young investigator award, the start-up fund from State Key Laboratory of Bioorganic and Natural Products Chemistry and Chinese Academy of Sciences.

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