Human medium chain μ2 adaptin subunit (AP50) of adaptor protein complex 2

Jo Bùi '22 and Isabelle Freeman '23

Contents:

I. Introduction

The human medium chain μ2 adaptin subunit is a component of the clathrin-associated adaptor protein complex 2 (AP-2)*, a heterotetramer of 4 subunits: , , , and .

The complex functions in endocytosis, specifically cargo selection and vesicle formation, and is activated by the phospholipid component . Its function consists of 3 main sequential activities: recruitment to the plasma membrane, binding specific signals from cargo transmembrane proteins, and anchoring the polymerization of clathrin into a polyhedral vesicle coat.

The μ2 subunit is mainly involved in the second activity, recognizing tyrosine-based (x is any amino acid and φ a bulky hydrophobic amino acid that can be Leu, Ile, Met, Phe, or Val) . However, it is only accessible for signal binding when the complex is in the unlocked(or activated) conformation, where the binding sites are not occluded by other subunits.

*There have been no successful attempts to crystallize the phosphorylated AP50 subunit in the unlocked conformation all displayed open complexes are merely theoretical models.

II. General Structure

AP50, when complexed with CTLA-4, is an asymmetrical heterodimer of 34.21 kDa, composed of 229 residues making up two unique polypeptide chains.

The is comprised of a N-terminal domain that is tightly bound into the core (α and β2) and a C-terminal domain (CTD) that swings out from the core upon activation of the complex . The two domains are connected by a linker that also changes conformation upon activation.

μ2 CTD, the more active domain, is a nearly all β-sheet sandwich structure that consists of 2 subdomains, and .

III. μ2 Active Sites

The linker connecting N- and C- terminal domains contains (although it is referenced in other papers as Thr156), which is a site of phosphorylation by AAK1 (α-appendage binding kinase) or cyclin-G associated kinase (GAK. Thr375 phosphorylation promotes cargo interactions with AP-2 in vitro and in vivo.

Subdomain A of μ2 CTD contains the for Yxxφ signals that is occluded by part of β2 subunit in the locked conformation

Subdomain B contains a PI(4,5)P2 binding site that is solvent exposed in both the locked and unlocked conformations. It does not play a role in the PI(4,5)P2 binding-mediated recruitment of AP-2 to the membrane, however it is important in AP-2 activation.

There are on subdomain μ2, close to the Yxxφ motif binding site. Though these site are accessible in the locked state, they have a minor effect on AP-2 activity .

Figure 1

IV. AP-2 Unlocking and Stabilization

Video 1
N-terminal domain C-terminal domain

The first step in AP-2 activation is its recruitment onto the plasma membrane, primarily by binding of α or β2 to PI(4,5)P2. Once attached, the and subsequent binding of μ2 CTD to the high local concentration of PI(4,5)P2 causes AP-2 to adopt the open conformation.

The , disordered in the locked conformation, can now fold into an α-helix in the slot formed between N-μ2 and β2, again stabilizing the open form. Further stabilization may be achieved through phosphorylation of μ2 Thr375 by AAK1 or GAK.

The resulting electropositive environment leads to tighter binding of AP-2 to cargo-containing membranes.

V. Binding Yxxφ signals

Binding of Yxxφ signals, the most important function of the μ2 subunit, is high-affinity and high-specificity and happens only when AP-2 is in the unlocked conformation. Yxxφ signals bind to μ2 CTD by β-augmentation to one edge of a sheet on subdomain A.

‘‘β-augmentation’’ refers to a peptide or protein binding mode involving the incorporation of the bound ligand as one or more additional strands of the β-sheet. Specificity is conferred by two hydrophobic pockets that bind the Y and φ residues. The aromatic side-chain of Y stacks with those of surrounding Trp, Tyr, and Arg residues , and its hydroxyl is hydrogen-bonded with a conserved Asp residue .

The φ residue is bound in a hydrophobic pocket formed at the juncture of the β-sheet sandwich .


VI. References

Canagarajah, B. J., Ren, X., Bonifacino, J. S., & Hurley, J. H. (2013). The clathrin adaptor complexes as a paradigm for membrane-associated allostery. Protein Science, 22(5), 517-529.

Follows, E. R., McPheat, J. C., Minshull, C., Moore, N. C., Pauptit, R. A., Rowsell, S., ... & Abbott, W. M. (2001). Study of the interaction of the medium chain μ2 subunit of the clathrin-associated adapter protein complex 2 with cytotoxic T-lymphocyte antigen 4 and CD28. Biochemical Journal, 359(2), 427-434.

(Figure 1)(Video 1) Jackson, L. P., Kelly, B. T., McCoy, A. J., Gaffry, T., James, L. C., Collins, B. M., ... & Owen, D. J. (2010). A large-scale conformational change couples membrane recruitment to cargo binding in the AP2 clathrin adaptor complex. Cell, 141(7), 1220-1229.

Olusanya,O.,Andrews, P. D., Swedlow, J. R., & Smythe, E. (2001). Phosphorylation of threonine 156 of the μ2 subunit of the AP2 complex is essential for endocytosis in vitro and in vivo. Current biology : CB, 11(11), 896–900. https://doi.org/10.1016/s0960-9822(01)00240-8

Owen, D. J., & Evans, P. R. (1998). A structural explanation for the recognition of tyrosine-based endocytotic signals. Science, 282(5392), 1327-1332.

Partlow, E. A., Baker, R. W., Beacham, G. M., Chappie, J. S., Leschziner, A. E., & Hollopeter, G. (2019). A structural mechanism for phosphorylation-dependent inactivation of the AP2 complex. Elife, 8, e50003.

Back to Top