Fig. 1. AP2 can bind clathrin only when it is attached to a PtdIns(4,5)P2-
and cargo-containing membrane. (A) AP2 schematic, color-coded by subunit: a, blue; b2, green; N-m2, dark magenta; C-m2, pale magenta; s, cyan. The
unstructured a and b2 hinge and appendage subdomains, together with the
core of the complex, are indicated. Parts shown in gray are not present in FLb.
AP2. Schematics of constructs are shown in fig. S1. (B) FLb.AP2 showing the
positions of the clathrin box and purification tags. (C) Coomassie-stained SDS–
polyacrylamide gel electrophoresis (SDS-PAGE) of glutathione-sepharose pull-
downs using GST-FLb.AP2 or GST-b2-h+app. Supernatant (s) and pellet (p). The
band marked with an asterisk results from proteolysis of b2. MW, molecular
weight. (D) Coomassie-stained SDS-PAGE of clathrin cage assembly assays
using 2.5 mM clathrin and 1.5 mM adaptors (as indicated), overnight at 21°C,
centrifuged to separate clathrin cages (p) from supernatants (s). (E) Coomassie-
stained SDS-PAGE of liposome pulldown assays. Liposomes were sequentially
incubated with adaptors and clathrin, then centrifuged to separate unbound
material (s) from the liposome pellet (p).
Fig. 2. The AP2 b2 subunit LLNLD
clathrin binding motif is buried in
the center of the core. Overall (A)
and closeup (B) views of the structure of bhingeHis6.AP2. The residues
of the hinge resolved in the structure
are shown in green as a stick representation. The AP2 core is depicted in
a surface representation, colored as
in Fig. 1. The residues of the buried
hinge are indicated in (B), with electron density shown as mesh (2mFo – DFc
map, contoured at 0.34 e Å−3). Q, Gln.
Also shown are the positions of the
selenium (Se) sites found in the bowl
for each of the methionine (M) mutants
indicated, showing good agreement
with the positions of the corresponding
wild-type residues that were mutated.
Individual log-likelihood gradient maps
are shown in fig. S3. (C) Ligplot+ (25)
diagram showing interactions between
buried hinge residues (in pale green)
with residues of a (blue), m2 (magenta),
and b2 (dark green). Red fans indicate
hydrophobic interactions; dashed green
lines indicate hydrogen bonds. The residues of the clathrin-binding motif are
boxed. See also fig. S4. A, Ala; C, Cys; E,
Glu; G, Gly; H, His; I, Ile; K, Lys; P, Pro; S,
Ser; and V, Val. (D) Clathrin cage assembly
assays. (D) is identical to Fig. 1D (2.5 mM
clathrin, 1.5 mM adaptors) with the addition
of the FLb.AP2.DCm2 lane. (E) Assays
performed as in (D) but at 28°C and with
2 m M clathrin and 4 m M adaptors as shown.
