FC0077
AP180  -  Clathrin

Biological function
Clathrin-mediated vesicular trafficking begins on the membrane, when clathrin assembly/adaptor proteins such as AP2 and AP180 are cooperatively recruited via interactions with the phosphoinositide PIP₂ (phosphatidylinositol 4,5- bisphosphate) and with membrane-bound cargo molecules. The nascent complex in turn recruits cytosolic clathrin and nucleates the formation of a clathrin-coated pit. As the coated pit grows, additional accessory proteins that promote membrane scission are recruited, allowing clathrin-coated vesicles to detach from the membrane. The clathrin-coated vesicles are then rapidly uncoated via a chaperone-mediated reaction that returns the coat proteins to the cytosol, and the liberated transport vesicles then deliver their cargo molecules to an appropriate subcellular compartment by membrane fusion.

Domain organization/sequence features
AP180 is a monomeric clathrin assembly protein with an ∼58 kDa C-terminal domain (CTD) with a highly repetitive structure that is unusually acidic and rich in proline, serine, threonine, and alanine residues, which was shown to be involved in clathrin binding and assembly. CTD revealed it to contain 12 degenerate repeats, each approximately 23 aa in length, each of which contains a central DLL/DLF sequence, which was proposed to serve as the clathrin binding element. The CTD is a largely disordered protein interspersed with multiple, short elements pre-structured to bind the clathrin TD, similarly to a line fishing mechanism, using multiple hooks as a bait. Interaction of each of these ’hooks’ with clathrin is weak and characterized by extremely fast association and dissociation rates, but the concentration of multiple clathrin binding elements at an endocytic site nevertheless provides for efficient clathrin recruitment, while the weakness of the individual interactions allows the recruited clathrin to have the motional freedom required for the dynamic assembly of the clathrin lattice at the endocytic site.

Structural evidence
Mutational, NMR chemical shift, and analytical ultracentrifugation analyses of the interaction between clathrin and a large fragment of the CBD (Clathrin binding domain) of the clathrin assembly protein AP180 defined two clathrin binding sites within this fragment, each of which is found to bind weakly to the N-terminal domain of the clathrin heavy chain (TD). The AP180 fragment is similarly unstructured in its bound and free states, with localized β-turn-like structures at the two clathrin binding sites both when free and when bound to clathrin. Weak binding by multiple, pre-structured clathrin binding elements regularly dispersed throughout a largely unstructured CBD allows efficient recruitment of clathrin to endocytic sites and dynamic assembly of the clathrin lattice. AP180 M5 revealed that AP180 M5 is predominantly unstructured in solution, in both the free and the clathrin-TD-bound states. This conclusion is based on the following observations: (1) All of the peaks representing the backbone of AP180 M5 are located within the region corresponding to backbone amide ¹ H chemical shifts from 7.9 to 8.5 ppm in both free and bound states (2) Secondary chemical shift analysis did not reveal patterns associated with α helices or β sheets in either state (3) NOESY experiments did not reveal any ¹H–¹H NOEs that would indicate the presence of α helices or β sheets in AP180 M5 either when free in solution or when bound to TD. (4) ¹⁵N– ¹H NOEs throughout the polypeptide chain are negative in both free and bound states, further indicating a lack of extensive secondary structure in AP180 M5. HSQC spectra in both the free and the bound forms indicates that binding is not associated with extensive folding of this fragment. Frictional coefficients measured by AUC closer to that of a fully unfolded polypeptide.

Biochemical evidence
While AP180 M5 is predominantly disordered, the two clathrin binding sites contain limited but persistent β-turn-like structure in both the free and the bound states. Binding of clathrin TD to each site is relatively weak, with K_d values in the ∼2×10⁻⁴ M range. Each site in the two-site AP180 M5 molecule binds a different clathrin TD. AUC suggests that both sites interact simultaneously with a single clathrin TD to enhance the affinity (by 1.5 fold) of the two-site AP180 M5. Dissociation rate constants are in the range of 2 × 10³ to 4 × 10³ s⁻¹, and association rate constants are in the range of 1×10⁷ to 2×10⁷ M⁻¹ s⁻¹ These are among the fastest association rates that have ever been measured for a protein-protein interaction.

Structure/Mechanism
(1) Each AP180 contains up to 12 clathrin binding sites; thus, if a clathrin molecule releases one site, it may remain engaged via interactions with other sites or is very likely to encounter and bind another such site. (2) This effect is further amplified because multiple AP180s will be concentrated at the endocytic site. (3) In addition to AP180, this endocytic site will contain other proteins that are known to have intrinsically disordered domains, which also contain multiple binding sites for clathrin, further increasing the number of clathrin binding elements concen- trated at the endocytic site. (4) Finally, since each clathrin triskelion contains three clathrin TDs and each TD may be able to bind more than one clathrin binding element, the likelihood of a clathrin triskelion being retained at the endocytic site through multiple, weak interactions is multiplied even further.

Mechanism category
tethering

Significance
Fuzziness enables a weak binding mode when multiple, pre-structured clathrin binding elements regularly dispersed throughout a largely unstructured CBD allow efficient recruitment of clathrin to endocytic sites and dynamic assembly of the clathrin lattice. This multivalent binding concentrates clathrin at a specific site without inhibiting the motions that allow the clathrin triskelia to establish the interactions that determine the final stability and geometry of the clathrin coat.