Clathrin-adaptor appendage domains

Appendage/ear domains recruit accessory proteins (see previous page) and are structurally and functionally conserved not merely amongst heterotetrameric AP complexes but also in subunits of the COPI coat complex and in GGAs (Golgi-localized, gamma-ear-containing, Arf-binding family of protein). There are 2 subdomains, a platform and a beta-sandwich (see illustration). In the movies below the overall structural conservation of binding sites is illustrated. The appendages of GGAs and the gamma subunit of AP-1 only have the beta-sandwich subdomain and they also bind peptide ligands. This has now led to a re-evaluation of possible peptide binding to beta-sandwich domains of other appendages and thus of accessory protein recruitment. This work is still ongoing. These pages are written to accompany a review by Mills et al 2004.
AP2 adaptor protein complex domain structure
Alpha-adaptin appendage (AP2 complex subunit)
2 ligand binding sites: one on the platform subdomain and the other on the beta-sandwich subdomain (taken from Brett et al 2002 and Owen et al 1999). The platform site has a hydrophobic pocket made from a conserved FxxxW that is present in most appendages having the platform sub-domain. This site binds to FxDxF motifs like the one found in the long splice varient of amphiphysin (although the last F in this motif is not well coordinated in the structure). The site can also bind to DPF and DPW motifs found in multiple copies in proteins like Eps15 and epsin1. The beta-sandwich binding site was found to bind a DPW motif but this interaction site is weak. We subsequently found that another motif, WVxF, can interact with the beta-sandwich subdomain and these motifs have a much higher affinity (see movie taken from Praefcke et al 2004, see also NMR structure in Ritter et al 2004). The top and side site interactions are often both used by the same protein to generate a high affinity yet reversible interaction (see Praefcke et al 2004).
Beta2-adaptin appendage (AP2 complex subunit)
1 binding site on the platform subdomain (taken from Owen et al 2000) centred around a hydrophobic pocket FxxxW like in the alpha-adaptin appendage. This site is assumed to bind DxF motifs.

Gamma-adaptin appendage (AP1 complex subunit)
Only one subdomain with a beta-sandwich structure (taken from Kent et al 2002 and Nogi et al 2002). No peptides were bound but mutagenesis showed the site which bound Eps15, gamma-synergin and rabaptin5. In Mills et al 2003 the gamma appendage was shown to bind DFxDF motifs in gamma-synergin and epsinR in the low micromolar range. This location of this binding site is similar to the binding sites on GGA1 and GGA3 appendages.
Gamma-COP appendage (COPI coat subunit)
1 binding site on the platform subdomain (taken from Hoffman et al 2003 and Watson et al 2004). A central FxxxW is conserved in the platform binding site like in the alpha and beta-adaptin appendages. This site was shown to bind ArfGAP.
GGA1 appendage
Only one subdomain with a beta-sandwich structure bound to a DFxxF peptide from p56 (taken from Collins et al 2003).
GGA3 appendage
Only one subdomain with a beta-sandwich structure bound to a DFxxL peptide from rabaptin5 (taken from Miller et al 2003).

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