FIGURE 6.

Tyr¹⁸⁷⁴ of loop-15BC is required for ankyrin binding to repeats 14–16 of β₂-spectrin. β₂-Spectrin variants with alanine mutations at 23 surface-exposed residues from helix-15B, loop-15BC, and helix-15C were screened for the ability to bind to the spectrin-binding domain (SBD) of ankyrin-B and ankyrin-G by yeast two-hybrid analyses. Shown in A is growth of yeast strains expressing the indicated spectrin variants fused to the Gal4 activation domain and the ankyrin-B spectrin-binding domain fused to the Gal4 DNA-binding domain on plates deficient for adenosine, histidine, leucine, and tryptophan. Similar results were observed in yeast strains expressing the spectrin variants together with the ankyrin-G spectrin-binding domain (yeast growth not shown). The location of individual mutations relative to secondary structure elements is shown in B along-side relative growth ability as indicated by ++ for strong growth and - for no growth. The location of the proposed ankyrin-binding site is indicated on the B14–16 structure in C.