Spectrin and ankyrin participate in membrane organization, stability, signal transduction, and protein targeting; their interaction is critical for erythrocyte stability. Repeats 14 and 15 of ␤I-spectrin are crucial for ankyrin recognition, yet the way spectrin binds ankyrin while preserving its repeat structure is unknown. We have solved the crystal structure of the ␤I-spectrin 14,15 di-repeat unit to 2.1 Å resolution and found 14 residues critical for ankyrin binding that map to the end of the helix C of repeat 14, the linker region, and the B-C loop of repeat 15. The tilt (64°) across the 14,15 linker is greater than in any published di-repeat structure, suggesting that the relative positioning of the two repeats is important for ankyrin binding. We propose that a lack of structural constraints on linker and inter-helix loops allows proteins containing spectrin-like di-repeats to evolve diverse but specific ligand-recognition sites without compromising the structure of the repeat unit. The linker regions between repeats are thus critical determinants of both spectrin's flexibility and polyfunctionality. The putative coupling of flexibility and ligand binding suggests a mechanism by which spectrin might participate in mechanosensory regulation. (Blood. 2009;113:5377-5384) IntroductionFirst discovered in the human erythrocyte and closely associated with a variety of familial hemolytic anemias, the spectrin-ankyrin cytoskeleton has emerged as the classical paradigm of a polyfunctional organizing membrane scaffold. Ubiquitous in higher eukaryotes, the spectrin-ankyrin skeleton contributes to membrane stability, the organization of membrane proteins and lipids, the recruitment to membranes of cytosolic proteins and signaling complexes, the tethering of organized protein mosaics to filamentous actin or to the motors effecting microtubule-directed transport, and the facilitated transport of membrane proteins through the secretory and endocytic pathways. 1 Reflecting these diverse but fundamental roles, hereditary or experimental disruption of spectrin or ankyrin leads to many pathologies, including hemolytic disease, 2 embryonic lethality, cancer and developmental defects, 3 pump and channel failures and endoplasmic reticulum (ER) retention disorders, 4 neuromuscular syndromes and sudden cardiac death. 5,6 The participation of spectrin and ankyrin in so many cellular processes reflects their ability to organize multiple membrane and cytosolic proteins and lipids into membrane microdomains, linking them to the filamentous skeleton. Polyfunctionality is a critical attribute of both proteins. Ankyrins derive this capacity largely by the juxtaposition of multiple 33-residue repeat units, each composed of 2 helices linked by a ␤-turn. 7 Selectivity is achieved by minor sequence variation within the ␤-turn of each ankyrin-repeat and by the juxtaposition of repeats with differing sequence. Less is known about how spectrin binds its ligands with high affinity and specificity. In humans, there are 7 spectrin genes encoding 5...