THE MEMBRANE-BINDING DOMAIN OF ANKYRIN CONTAINS 4 INDEPENDENTLY FOLDED SUBDOMAINS, EACH COMPRISED OF 6 ANKYRIN REPEATS

Ankyrin repeats are a 33-amino acid motif present in a number of prote ins of diverse functions including transcription factors, cell differe ntiation molecules, and structural proteins. This motif has been shown to mediate protein interactions in the case of ankyrin as well as sev eral other repeat-bearing proteins. In ankyrin, 24 tandemly arrayed re peats are arranged to form a globular, membrane-binding domain. This r eport provides evidence that the repeats in this domain fold into four independently folded subdomains of six repeats each. Limited proteoly tic digestions of defined regions of the membrane-binding domain ident ified protease-sensitive sites, which divided this domain into subdoma ins of approximately six repeats each. Hydro-dynamic measurements and circular dichroism spectroscopy of expressed subdomains confirmed that these six-repeat regions exist as folded, globular structures. The re quirement of a complete set of six repeats for proper folding was dete rmined using a series of protein constructs, which sequentially delete d repeats from the last subdomain. Deletion of even one repeat resulte d in a 40% loss of alpha-helicity. Deletions removing three or more re peats abolished the helical signal completely. The spherical shapes of the intact domain and of the subdomains (inferred from hydrodynamic v alues) suggest that the four subdomains are organized in either a tetr ahedral or square planar configuration. Two six-repeat subdomains were found to be required for high affinity association with the anion exc hanger, suggesting that at least some of the protein interactions medi ated by ankyrin repeats involve multiple subdomains.