The structure of the ankyrin-binding site of β-spectrin reveals how tandem spectrin-repeats generate unique ligand-binding properties

Abstract

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...

Crystal structure and functional interpretation of the erythrocyte spectrin tetramerization domain complex

“…As has been observed with all other phased, multirepeat spectrin structures to date, adjacent repeats are connected with ␣-helical linkers. [32][33][34][35][36][37][38] As predicted by structurebased homology modeling and biophysical measurements, the crystal structure confirms a complex dominated by ␣-helices. Finally, although the aforementioned studies contributed significantly in modeling the interactions present at the tetramerization interface, it is only with the structure of this complex that the precise nature of the molecular interactions can be discerned (Figure 3).…”

Cell organization, growth, and neural and cardiac development require αII-spectrin

“…When spectrin is concentrated at the membrane, it is most often at sites of specialized receptor function such as pre-and post-synaptic densities, rather than at sites that one would anticipate are subjected to high mechanical stress. However, recent data suggesting that spectrin might function as a mechanochemical signal transducer (Stabach et al, 2009) raises a speculative but interesting possibility that could reconcile the apparent fragility of spectrin-deficient axons (as observed in C. elegans) with the emerging role of spectrin as a major receptor-sorting and organizing scaffold. If stretch or bending of axons is a tropic stimulus for neuronal extension and maintenance (Van Essen, 1997), then the absence of such stimulus might activate pathways to prune unwanted axons and dendrites (Luo and O'Leary, 2005).…”

“…Three decades of study have implicated the spectrin heterodimer formed between aII-spectrin and any of five b-spectrins in a bewildering array of cellular processes. These include a role in the formation and maintenance of specialized plasma membrane domains defining apicalbasolateral and planar polarity in epithelial cells, muscle and neurons (Bennett and Baines, 2001); in the structural support of the plasma membrane and the maintenance of cell shape (Gallagher and Jarolim, 2000;Kizhatil et al, 2007); as a scaffold upon which calcium-mediated and tyrosine kinase-phosphatase signal transduction pathways converge (Nicolas et al, 2002;Nedrelow et al, 2003); as a tumor-suppressor protein involved in TGF-b-SMAD regulation (Tang et al, 2003); as a cargo selection mechanism in the secretory and endocytic pathways (De Matteis and Morrow, 2000); as a regulator of macropinocytosis (Xu et al, 2000); as a tether linking trafficking vesicles to microtubule motors (Holleran et al, 2001;Muresan et al, 2001); as a nuclear scaffold organizer (McMahon et al, 1999;Tse et al, 2001); and most recently, as a potential mechano-sensing ligand-binding switch (Stabach et al, 2009). Deletion of aII-spectrin in Drosophila melanogaster and Caenorhabditis elegans leads to late embryonic-early larval stage lethality (Moorthy et al, 2000;Dubreuil, 2006;Hammarlund et al, 2007), and recent knockdown studies of aII-spectrin in cultured cells have demonstrated growth and adhesion defects (Metral et al, 2009).…”

Dynamic Regulation of CD45 Lateral Mobility by the Spectrin-Ankyrin Cytoskeleton of T Cells

“…The spectrin-ankyrin interaction sites have recently been defined (40,41), and although the structure of the CD45 cytoplasmic domain is known (26), no structures of a CD45 complex with spectrin or ankyrin have been reported. The spectrinbinding site of CD45 maps to the base of Domain 2, and although ankyrin is known to bind directly to CD45 (13), its binding site remains undefined.…”