PDBsum entry 1hg2

Endocytosis

PDB id: 1hg2

Contents

Protein chain

263 a.a. *

Ligands

IP2

Waters ×128

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Simultaneous binding of ptdins(4,5)p2 and clathrin by ap180 in the nucleation of clathrin lattices on membranes.

Authors

M.G.Ford, B.M.Pearse, M.K.Higgins, Y.Vallis, D.J.Owen, A.Gibson, C.R.Hopkins, P.R.Evans, H.T.Mcmahon.

Ref.

Science, 2001, 291, 1051-1055. [DOI no: 10.1126/science.291.5506.1051]

PubMed id

11161218

Abstract

Adaptor protein 180 (AP180) and its homolog, clathrin assembly lymphoid myeloid leukemia protein (CALM), are closely related proteins that play important roles in clathrin-mediated endocytosis. Here, we present the structure of the NH2-terminal domain of CALM bound to phosphatidylinositol-4,5- bisphosphate via a lysine-rich motif. This motif is found in other proteins predicted to have domains of similar structure (for example, Huntingtin interacting protein 1). The structure is in part similar to the epsin NH2-terminal (ENTH) domain, but epsin lacks the PtdIns(4,5)P2-binding site. Because AP180 could bind to PtdIns(4,5)P2 and clathrin simultaneously, it may serve to tether clathrin to the membrane. This was shown by using purified components and a budding assay on preformed lipid monolayers. In the presence of AP180, clathrin lattices formed on the monolayer. When AP2 was also present, coated pits were formed.

Figure 2.
Fig. 2. The structure of CALM-N bound to PtdIns(4,5)P[2]. (A) Ribbon diagram of CALM-N, colored from green at the NH[2]-terminus to gold at the COOH-terminus. (B) The ENTH domain of epsin in the same orientation [PDB code 1edu (23)]. (C) The surface of CALM-N colored by electrostatic potential, red +10 kT e^ 1, blue -10 kT e^ 1. This is a slightly different view from that in (A), to show the strong positive patch that binds PtdIns(4,5)P[2]. (D) Close-up of PtdIns(4,5)P[2]-binding site, showing a difference electron density map omitting the ligand, contoured at 2 . There was strong density only for the 4- and 5-phosphates, weak density for the inositol ring and the 1-phosphate, and none for the lipid chains. (E) Ins(4,5)P[2] also shows most density for the phosphates: it was modeled as a 50:50 mixture of two binding modes interchanging the 4- and 5-phosphates. (F) InsP[6] was probably bound in multiple orientations, and the orientation of the inositol ring was different from that of the bisphosphates. (G) Sequence alignments of the very similar CALM-N and AP180-N (81% identical, unshaded, further conserved residues shaded mauve), and the structurally similar epsin ENTH domain (16% sequence identity, shaded orange). Helices are shown as cylinders, colored as in A and B. PtdIns(4,5)P[2]-binding residues are marked with arrows. Also shown is the PtdIns(4,5)P[2]-binding region of -adaptin, with the conserved PtdIns(4,5)P[2]-binding motif and predicted helices. (H) The 1 to 2 loop regions for three families of proteins: AP180/CALM family with the PtdIns(4,5)P[2]-binding motif (blue); some other proteins with the PtdIns(4,5)P[2]-binding motif (blue); epsin family with the (D/E)PW motif (orange). Other conserved residues are colored purple. Yeast-SLA2 is Yeast-SLA2p.

Figure 3.
Fig. 3. AP180-N binds, and has specificity for, PtdIns(4,5)P[2]. (A) AP180-N and CALM-N are sedimented by lipid tubules containing 10% PtdIns(4,5)P[2]. The measurements on the abscissa refer to the amount of PtdIns(4,5)P[2] in the experiment; the amount of tubules is therefore 10 times this value. Each assay contained 0.05 mg/ml protein. (B) Liposomes containing 10% cholesterol, 40% phosphatidylethanolamine, 40% phosphatidylcholine, and 10% of a test lipid were prepared and used to evaluate the lipid specificity of AP180-N. Each experiment contained 0.05 mg/ml protein and 50 µM of the lipid under investigation; each experiment, therefore, contained a total lipid concentration of 500 µM. (C) AP180 recruits clathrin to PtdIns(4,5)P[2] containing liposomes (27). Pellets (P) and supernatants (S) were separated by centrifugation. In AP180mut, lysines 38 and 40 were changed to glutamic acids. Although AP2 alone sedimented approximately as efficiently as AP180, it was not capable of sedimenting together with clathrin, possibly because of a requirement for cargo and/or oligomerization. Averages of at least three experiments are shown in the bar graph. Experiments contained 0.05 mg/ml AP180, 0.05 mg/ml AP2, 0.025 mg/ml clathrin in a final volume of 100 µl. All gels were stained with Coomassie Blue.

The above figures are reprinted by permission from the AAAs: Science (2001, 291, 1051-1055) copyright 2001.