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Structural basis of an endocytic checkpoint that primes the AP2 clathrin adaptor for cargo internalization

Abstract

Clathrin-mediated endocytosis (CME) is the main route of internalization from the plasma membrane. It is known that the heterotetrameric AP2 clathrin adaptor must open to simultaneously engage membrane and endocytic cargo, yet it is unclear how transmembrane cargos are captured to catalyze CME. Using cryogenic-electron microscopy, we discover a new way in which mouse AP2 can reorganize to expose membrane- and cargo-binding pockets, which is not observed in clathrin-coated structures. Instead, it is stimulated by endocytic pioneer proteins called muniscins, which do not enter vesicles. Muniscin-engaged AP2 is primed to rearrange into the vesicle-competent conformation on binding the tyrosine cargo internalization motif (YxxΦ). We propose adaptor priming as a checkpoint to ensure cargo internalization.

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Fig. 1: Cooperative engagement of AP2 by muniscins and membrane mimetic.
Fig. 2: The primed conformation of AP2.
Fig. 3: Cargo-binding pockets are exposed in the primed AP2 complex.
Fig. 4: YxxΦ-cargo-binding releases AP2 from muniscins.
Fig. 5: YxxΦ-cargo converts primed AP2 complexes to the open conformation on membranes.
Fig. 6: Primed AP2 stimulates assembly of cargo-containing clusters on membranes.
Fig. 7: Model for adaptor checkpoint to catalyze cargo incorporation.

Data availability

Cryo-EM data are deposited in the Electron Microscopy Data Bank (EMDB) under accession numbers EMBD-24710, EMBD-24711, EMBD-24712, EMBD-24713 and EMBD-24714. For each deposition, the unsharpened map, sharpened map, unfiltered half maps and refinement mask were provided. Associated atomic coordinates are deposited in the PDB under accession numbers: 7RW8, 7RW9, 7RWA, 7RWB and 7RWX. The following atomic coordinates from the PDB were used in this study: 2VGL, 1BXX, 2JKR and 2XA7. Source data are provided with this paper.

Code availability

We used a customized build of Relion, which is available at https://github.com/Rick-Baker/relion-recenter.

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Acknowledgements

We acknowledge J. Peck and J. Strauss of the UNC Cryo-EM Core Facility for technical assistance in this project. We thank A. Gladfelter from UNC Chapel Hill and J. Chappie and G. Beacham from Cornell University for use of reagents and equipment. We thank R. Cerione, S. Emr, C. Adler and C. Fromme from Cornell University for thoughtful suggestions to improve the manuscript. Funding was provided by National Institutes of Health grant no. R01GM127548 (G.H.)

Author information

Authors and Affiliations

Authors

Contributions

Conceptualization was done by E.A.P., G.H. and R.W.B. The methodology was devised by E.A.P., K.S.C., G.H. and R.W.B. The investigation was carried out by E.A.P., K.S.C., G.H. and R.W.B. The visualization was done by E.A.P., K.S.C. and R.W.B. Funding was acquired by G.H. and R.W.B. Project administration was done by G.H. Supervision was undertaken by G.H. and R.W.B. The original draft was written by E.A.P. Review and editing of the manuscript was done by E.A.P., K.S.C., G.H. and R.W.B.

Corresponding authors

Correspondence to Gunther Hollopeter or Richard W. Baker.

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Competing interests

The authors declare no competing interests.

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Nature Structural & Molecular Biology thanks Sandra Schmid, Reza Paraan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Florian Ullrich was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

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Extended data

Extended Data Fig. 1 Supporting data for Fig. 1.

(a) Alignment of muniscin APA domains from four model organisms. Vertebrates (H. sapiens, D. rerio) contain two homologs of FCHo and the neuronal SGIP1. D. Melanogaster and C. elegans each contain a single muniscin. Black boxes: 100% identity, full color: 100% similarity, partial color: >75% similarity. (b) Confocal slices of AP2core bound to lipid-coated beads alone or pre-seeded with muniscin. Both proteins are added at 50 nM concentration. Scale bar 5 µm. FCHoBAR and FCHoBAR+APA images are also used in Fig. 1f. (c) Maximum intensity projections of AP2core (blue) and FCHoBAR+APA (green) bound to beads. Both proteins are added at 50 nM concentration. (d) Representative images of three assay conditions: AP2 alone, AP2 + FCHoBAR, and AP2 + FCHoBAR+APA. (e) Puncta formation of AP2core in the presence of soluble SGIPAPA at three concentrations. AP2 is held at 2 µM concentration. (f) Representative images of AP2 binding to SLBs +/- SGIPMP+APA, which includes the membrane phospholipid-binding domain (MP) and APA domain of SGIP. (g) Quantification of SLBs showing that AP2 binding is increased by the addition of 50 nM SGIPMP+APA. (h) Quantification of SLBs showing that the number of AP2 puncta per bead is increased by the addition of 50 nM SGIPMP+APA. (G-H) Data are mean +/- sd of triplicate measurements. ****P < 0.0001 for unpaired t-test (i) Representative pulldown of AP2 by either HT:SGIPAPA, HT:SGIPMP+APA, or HT:FCHo2BAR+APA in the presence of 5 µM heparin. HT: HaloTag. (j) Representative full SDS-PAGE gels of the protease sensitivity experiments quantified in Fig. 1g. Data for graphs in g,h are available as source data.

Source data

Extended Data Fig. 2 Data collection, processing, and model building for the AP2core-heparin complex.

(a) SDS-PAGE gel of the AP2core purification (left) and a schematic of the components used to make cryo-EM grids (right) (b) 2D class averages of the ‘clean’ dataset that was used for ab initio model generation. Two classes were found for further processing. (c) Final cryo-EM map of the ‘closed’ AP2-heparin complex colored by local resolution. Standard FSC plots (d) and directional FSC plots (e) are shown. (f) Final cryo-EM map of the ‘bowl’ AP2-heparin complex colored by local resolution. Note the µ2-CTD has been ejected from the complex and is not resolved in this map. Standard FSC plots (g) and directional FSC plots (h) are shown. (i) Pseudo-difference density map for the AP2 bowl complex made in Chimera and colored red to show density that cannot be accounted for by AP2. A region of low-resolution density is seen on the surface of α, corresponding to a known PIP2 binding site. Additional density is found at the α/σ2 interface. (j) Same analysis as done in (I), but for the closed AP2-heparin complex. (k) The N-terminus of ß2 packs into the [D/E]xxL[L/I] binding pocket on σ2 (yellow) (l) The structures of the closed AP2 (2VGL.pdb) and [D/E]xxL[L/I]-bound unlatched AP2 (2JKR.pdb) are shown aligned to the model for AP2-heparin. The closed heparin structure shows an intermediate conformation between the closed apo and [D/E]xxL[L/I]-cargo bound states.

Extended Data Fig. 3 Cryo-EM structure determination of AP2-heparin-YxxΦ-cargo.

(a) SDS-PAGE gel of the AP2core purification (left) and a schematic of the components used to make cryo-EM grids (right) (b) Representative cryo-EM micrograph and particle picking workflow. (c) 2D class averages (middle, right) of the ‘clean’ dataset that was used for ab initio model generation (left). (d) Final cryo-EM map of the AP2-heparin-YxxΦ complex colored by local resolution. (e) Gold standard FSC plot. (f) Particle distribution plot for 3D refinement. (g) Directional FSC plot. (h) The sharpened map is shown with potential heparin density colored red. Two copies of AP2-YxxΦ are shown docked into the cryo-EM map (right). (i) The refined model (grey) is overlaid with the ‘open’ AP2-YxxΦ structure (2xa7.pdb).

Extended Data Fig. 4 Data collection and processing workflow for the AP2core-heparin-SGIPAPA complex.

(a) SDS-PAGE gel of the AP2core purification (left) and a schematic of the components used to make cryo-EM grids (right). (b) 2D class averages of the ‘clean’ dataset that was used for ab initio model generation. Two classes were found for further processing. (c) Final cryo-EM map of the dimeric AP2core-heparin-SGIPAPA complex colored by local resolution Standard FSC plots (d) and directional FSC plots (e) are shown. (f) FSC plot for the monomer class 3D refinement. (g) Refined monomer map is shown with opaque and transparent surface with a docked model of AP2 that was manually segmented and re-arranged in UCSF Chimera, showing a preliminary assignment of density to AP2 subunits. (h) The AP2core-heparin-SGIPAPA dimeric cryo-EM map is shown (left) and with a transparent surface and docked molecular models (middle). A pseudo difference map was generated in Chimera and is shown colored pink. (i) Density along the dimeric interface is shown. This density is lined by known PIP2 binding residues (colored red), so we assign this density to heparin.

Extended Data Fig. 5 Assigning the conformation of the µ2-CTD in the dimeric AP2core-heparin-SGIPAPA structure.

(a) The AP2core-heparin-SGIPAPA cryo-EM map (left) contains a symmetry-related dimer of two complexes assembled on the same heparin molecule. The resolution of our structure in combination with the flexibility of the linker connecting the µ2-NTD and the µ2-CTD precludes us from unambiguously identifying which ‘bowl’ contributes which µ2-CTD, resulting in 2 candidate monomers (center and right). (b) Surface representation of candidate monomers with PIP2-binding residues shaded red and YxxΦ cargo-binding residues shaded orange. (c) Surface representation of candidate monomers along with surface representations of the ‘bowl’ and µ-CTD for each. Magenta represents buried surface area, totaling 499 Å2 for candidate 1 (left), and 199 Å2 for candidate 2 (right).

Extended Data Fig. 6 AP2core-heparin-SGIPAPA symmetry expansion and SGIPAPA model building.

(a) Density that is not accounted for by AP2 subunits in the AP2core-heparin-SGIPAPA structure is shown in pink. Density on the surface of β2 we assign to SGIPAPA as it is not observed in AP2-heparin structures and all residues at the α and β2 termini are accounted for. (b) Masks used for partial signal subtraction and manual symmetry expansion. (c) AP2-heparin-SGIPAPA ‘monomer’ refinement using signal subtracted particles. A map filtered by local resolution is shown colored by local resolution (left). The sharpened map is shown (grey) along with the FSC plot. (d) The poly-ALA model of SGIPAPA is shown in the density between β2 helices 25 and 27.

Extended Data Fig. 7 AP2 pull-down assays in the presence of cargo peptide.

(a) Representative pulldown of AP2 by HT:SGIPAPA in the presence of 5 µM heparin and either YxxΦ or ExxxLL cargo peptide. HT: HaloTag. (b) Representative pulldown of AP2 by either HT:SGIPAPA or HT:FCHo2BAR+APA in the presence of 5 µM heparin and YxxΦ-cargo peptide. HT: HaloTag. Uncropped image for b is available as source data.

Source data

Extended Data Fig. 8 Engineered disulfide crosslinks to probe AP2 conformation.

(a) Cartoon representation of the closed, primed, and open structures showing the location and Cß-Cß distances of all three engineered disulfides. (b) Representative uncropped Western blots for µ2 showing the primed-conformation disulfide experiment quantified in Fig. 3e.

Extended Data Fig. 9 Uncropped gels from disulfide crosslinking experiments.

Gels showing the technical replicates quantified in Fig. 5 are shown for the primed-specific disulfide (a), the closed-specific disulfide (b), and the open-specific disulfide (c). All gels are ordered as shown in (A).

Extended Data Fig. 10 Examples of three-color fluorescence imaging.

(a) Five lipid-coated beads are shown in three separate imaging channels and merged. Beads contain 2% YxxΦ-cargo and were incubated with 1 µM FCHoBAR and 1 µM AP2core. (b) Five example beads are shown as in (A), but were incubated with 1 µM FCHoBAR+APA. (c) and (d) show the same experiment in (A) and (B), but using SGPMP+APA. (e) Quantification of beads containing YxxΦ-cargo puncta with or without SGIPMP+APA Data are mean +/- sd of duplicate measurements. ****P < 0.0001 for unpaired t-test (f) Quantification of the AP2 to lipid ratio in cargo puncta with or without SGIPMP+APA. Data are mean +/- sd of triplicate measurements. ****P < 0.0001 for unpaired t-test. Data for graphs in e,f are available as source data. Scale bar: 5 µm.

Source data

Supplementary information

Source data

Source Data Fig. 1

Unprocessed gels.

Source Data Fig. 1

Numerical data for graphs.

Source Data Fig. 4

Unprocessed gels.

Source Data Fig. 4

Numerical data for graphs.

Source Data Fig. 5

Numerical data for graphs.

Source Data Fig. 6

Numerical data for graphs.

Source Data Extended Data Fig. 1

Numerical data for graphs.

Source Data Extended Data Fig. 7

Unprocessed gels.

Source Data Extended Data Fig. 10

Numerical data for graphs.

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Partlow, E.A., Cannon, K.S., Hollopeter, G. et al. Structural basis of an endocytic checkpoint that primes the AP2 clathrin adaptor for cargo internalization. Nat Struct Mol Biol 29, 339–347 (2022). https://doi.org/10.1038/s41594-022-00749-z

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