9C59

Human AP-3 dimer bound to myristoylated Arf1 (Q71L) and LAMP1 cargo on a lipid nanodisc

Summary for 9C59

• Entry DOI: 10.2210/pdb9c59/pdb
• EMDB information: 45208
• Descriptor: AP-3 complex subunit delta-1, AP-3 complex subunit beta-1, AP-3 complex subunit mu-1, ... (8 entities in total)
• Functional Keywords: adaptor protein complex, ap-3, lysosomal transport, endosomal transport, protein trafficking, transport protein
• Biological source: Homo sapiens (human); More
• Total number of polymer chains: 14
• Total formula weight: 517301.52

Authors

Begley, M.C.,Baker, R.W. (deposition date: 2024-06-06, release date: 2024-12-18, Last modification date: 2025-03-05)

Primary citation

Begley, M.,Aragon, M.,Baker, R.W.
A structure-based mechanism for initiation of AP-3 coated vesicle formation.
Proc.Natl.Acad.Sci.USA, 121:e2411974121-e2411974121, 2024

PubMed Abstract: Adaptor protein complex-3 (AP-3) mediates cargo sorting from endosomes to lysosomes and lysosome-related organelles. Recently, it was shown that AP-3 adopts a constitutively open conformation compared to the related AP-1 and AP-2 coat complexes, which are inactive until undergoing large conformational changes upon membrane recruitment. How AP-3 is regulated is therefore an open question. To understand the mechanism of AP-3 membrane recruitment and activation, we reconstituted human AP-3 and determined multiple structures in the soluble and membrane-bound states using electron cryo-microscopy. Similar to yeast AP-3, human AP-3 is in a constitutively open conformation. To reconstitute AP-3 activation by adenosine di-phosphate (ADP)-ribosylation factor 1 (Arf1), a small guanosine tri-phosphate (GTP)ase, we used lipid nanodiscs to build Arf1-AP-3 complexes on membranes and determined three structures showing the stepwise conformational changes required for formation of AP-3 coated vesicles. First, membrane recruitment is driven by one of two predicted Arf1 binding sites, which flexibly tethers AP-3 to the membrane. Second, cargo binding causes AP-3 to adopt a fixed position and rigidifies the complex, which stabilizes binding for a second Arf1 molecule. Finally, binding of the second Arf1 molecule provides the template for AP-3 dimerization, providing a glimpse into the first step of coat polymerization. We propose coat polymerization only occurs after cargo engagement, thereby linking cargo sorting with assembly of higher-order coat structures. Additionally, we provide evidence for two amphipathic helices in AP-3, suggesting that AP-3 contributes to membrane deformation during coat assembly. In total, these data provide evidence for the first stages of AP-3-mediated vesicle coat assembly.

PubMed: 39705307
DOI: 10.1073/pnas.2411974121

Experimental method

ELECTRON MICROSCOPY (4.3 Å)