The C-terminal Domain of AAGAB Mediates Tetramerization and Stability

In clathrin‐mediated endocytosis (CME), the coat protein clathrin relies on cargo adaptors such as the heterotetrameric AP2 adaptor complex to recruit cargo proteins to endocytic sites. It was previously thought that multimeric cargo adaptors such as AP2 adaptor assemble spontaneously. However, we recently discovered that AP2 adaptor assembly is an ordered process controlled by alpha and gamma adaptin binding protein (AAGAB). Without the assistance of AAGAB, AP2 adaptor fails to form, leading to CME defects. Heterozygous AAGAB mutations cause human diseases such as puncate palmoplantar keratoderma type 1 (PPKP1), a skin disorder characterized by lesions on palms and soles. However, the biochemical property and structure of AAGAB remain to be established. AAGAB is a soluble protein with two conserved regions – an N‐terminal G protein‐like domain (G domain) and a C‐terminal domain not similar to any known protein domain. We found that the full‐length AAGAB exists entirely as a homotetramer in solution and the tetramerization is mediated by its C‐terminal domain. Using X‐ray crystallography, we solved the atomic structure of the C‐terminal tetramerization domain (TD). The structure unveiled that the TD tetramer is formed by dimerization of two dimers. Furthermore, we observed that the expression level of the TD‐deficient AAGAB mutant (AAGABdeltaTD) in cells was drastically reduced compared to full‐length AAGAB. Together, our findings demonstrated that the TD mediates AAGAB tetramerization and is required for the stability of free AAGAB proteins prior to AP2 binding. These data also provide the first molecular explanation for the disease‐causing nonsense mutations found on AAGAB lacking the C‐terminal tetramerization domain. Support or Funding Information Florida State University startup funds to Yin; NIH/NIDDK R56 DK095367 to Shen; NIH/NIAID R21 AI128443 to Shen and Li.