PDBsum entry 1qtp

Membrane protein

PDB id: 1qtp

Contents

Protein chain

247 a.a. *

Waters ×266

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Crystal structure of the alpha appendage of ap-2 reveals a recruitment platform for clathrin-Coat assembly.

Authors

L.M.Traub, M.A.Downs, J.L.Westrich, D.H.Fremont.

Ref.

Proc Natl Acad Sci U S A, 1999, 96, 8907-8912. [DOI no: 10.1073/pnas.96.16.8907]

PubMed id

10430869

Abstract

AP-2 adaptors regulate clathrin-bud formation at the cell surface by recruiting clathrin trimers to the plasma membrane and by selecting certain membrane proteins for inclusion within the developing clathrin-coat structure. These functions are performed by discrete subunits of the adaptor heterotetramer. The carboxyl-terminal appendage of the AP-2 alpha subunit appears to regulate the translocation of several endocytic accessory proteins to the bud site. We have determined the crystal structure of the alpha appendage at 1.4-A resolution by multiwavelength anomalous diffraction phasing. It is composed of two distinct structural modules, a beta-sandwich domain and a mixed alpha-beta platform domain. Structure-based mutagenesis shows that alterations to the molecular surface of a highly conserved region on the platform domain differentially affect associations of the appendage with amphiphysin, eps15, epsin, and AP180, revealing a common protein-binding interface.

Figure 1.
Fig. 1. (A) Schematic illustration of the subunit organization of the AP-2 adaptor heterotetramer. Regions of the complex with known protein-binding functions are indicated. For tyrosine-based internalization signals, X is any amino acid and Ø represents a bulky hydrophobic residue (1, 3). (B) Functional protein associations with the GST- [C]-appendage fusion protein. Purified GST- [C] appendage (0-100 µg) or GST (100 µg), immobilized on 25 µl packed glutathione Sepharose, were incubated in 7.5 mg/ml rat brain cytosol for 60 min at 4°C. The Sepharose beads were then recovered by centrifugation, and aliquots corresponding to 1/150 of the supernatant (S) and 1/10 of each pellet (P) were resolved by SDS/PAGE and either stained with Coomassie blue (Left) or transferred to nitrocellulose (Right). Portions of the blots were probed with anti-epsin, anti-eps15, anti-amphiphysin, anti-AP180, or anti-dynamin antibodies. The position of the markers (kDa) is indicated on the left and only the relevant portion of each blot is shown.

Figure 4.
Fig. 4. [C] appendage-partner recognition surface. (A) Glutathione Sepharose (25 µl packed beads) containing either wild-type GST- [C] appendage ( [C]) or GST- [C] appendage F837A, R905A, R905A-F837A, R916A, or R916A-F837A mutants were incubated with rat brain cytosol for 60 min at 4°C. The Sepharose beads were then recovered by centrifugation, and aliquots corresponding to 1/150 of the supernatant (S) and 1/10 of each washed pellet (P) were resolved by SDS/PAGE and either stained with Coomassie blue (Left) or transferred to nitrocellulose (Right). Portions of the blots were probed with anti-epsin, anti-eps15, anti-amphiphysin, anti-AP180, or anti-dynamin antibodies. Immunoblots from assays performed at low ( 25 µg, L) or high ( 100 µg, H) GST- [C] density are indicated on the left. In the experiment performed at high density shown, the GST- [C] F837A mutant was not tested, and 1/40 of each supernatant was analyzed. The position of the markers (kDa) is indicated on the left, and only the relevant portion of each blot is shown. (B) Ribbon diagram (32) of the platform domain viewed from the top. Conserved residues that make up the upper surface of the platform are colored with invariant residues shaded magenta and conserved residues, yellow. The extended hydrogen-bonding network is shown as small gray balls with oxygen atoms in red, nitrogen in blue. The three highly exposed residues that have major consequences on partner binding when mutated to Ala are highlighted (F837, R905, R916).