Structure of the Calmodulin αII-Spectrin Complex Provides Insight into the Regulation of Cell Plasticity

Abstract

␣II-spectrin is a major cortical cytoskeletal protein contributing to membrane organization and integrity. The Ca 2؉ -activated binding of calmodulin to an unstructured insert in the 11th repeat unit of ␣II-spectrin enhances the susceptibility of spectrin to calpain cleavage but abolishes its sensitivity to several caspases and to at least one bacterially derived pathologic protease. Other regulatory inputs including phosphorylation by c-Src also modulate the proteolytic susceptibility of ␣II-spectrin. These pathways, acting through spectrin, appear to control membrane plasticity and integrity in several cell types. To provide a structural basis for understanding these crucial biological events, we have solved the crystal structure of a complex between bovine calmodulin and the calmodulinbinding domain of human ␣II-spectrin (Protein Data Bank ID code 2FOT). The structure revealed that the entire calmodulin-spectrin-binding interface is hydrophobic in nature. The spectrin domain is also unique in folding into an amphiphilic helix once positioned within the calmodulin-binding groove. The structure of this complex provides insight into the mechanisms by which calmodulin, calpain, caspase, and tyrosine phosphorylation act on spectrin to regulate essential cellular processes.Spectrin ␣␤ heterodimers are organized into filamentous structures composed of two related subunits. The ␣ subunit contains 21 triple-helical homologous repeats that are further organized into five protease-sensitive domains (1). The 11th repeat is the longest and contains a unique insert that is implicated to play a critical role in spectrin integrity. The spectrinbased cytoskeleton represents a scaffold attached to both the plasma membrane and the intracellular organelles by interactions involving both integral and peripheral membrane proteins as well as by direct lipid interactions (2-4). Spectrin integrity is critical for establishing organized membrane subdomains and their maintenance, supporting integral membrane protein durability (5, 6). Deletions in spectrin or dominant negative blockers of spectrin disrupt membrane-protein distributions and may lead to the total loss of critical signaling or transport proteins from the cell surface (7-10). Controlled spectrin proteolysis has been detected during both apoptotic and necrotic cell death processes (11-13), upon cell shape change (14), during cell differentiation (15-17), during lens development (18), and coincident with synaptic remodeling and dendrite outgrowth (19,20). It has been observed that during the early stages of apoptosis, a characteristic 150-kDa fragment of ␣II-spectrin is generated by caspases regardless of the cell type and the apoptosis stimulus (21). In neurons, calpain-catalyzed proteolysis has been linked with N-methyl-D-aspartate receptor activation and is thought to be crucial for synaptic and neuronal plasticity. Finally, the Pet toxin, a serine proteinase from enteropathogenic bacteria, catalyzes spectrin fragmentation and the consequent collapse of the cell membrane ...

Head of Myosin IX Binds Calmodulin and Moves Processively toward the Plus-end of Actin Filaments

“…Mutation of the two residues Trp 717 and Phe 730 abolished the binding of calmodulin. This is in agreement with the notion that these two residues serve as anchors for the C-and N-terminal domains of calmodulin, respectively (35,36). The identified calmodulin-binding sequence is highly conserved among the class IX myosins and present in class IX myosins only.…”

Sequential Degradation of αII and βII Spectrin by Calpain in Glutamate or Maitotoxin-Stimulated Cells

“…In the presence of bound CaM, new sites in αII spectrin (G 1230 ) and in βII spectrin (Q 1440 , S 1447 , and L 1482 ) become susceptible to calpain, and the rate of calpain cleavage of βII spectrin is enhanced many fold. Since recombinant αII spectrin peptides are not readily cleaved at the G 1230 bond (also see 48), it follows that CaM must create a new site favorable for cleavage by altering the tertiary conformation of αII spectrin about G1230., a finding consistent with the significant reordering of the 3-D structure of spectrin's calmodulin binding domain by bound CaM (51). Conversely, isolated βII spectrin recombinant peptides are readily cleaved by calpain at the same site as in CaM-loaded αII/βII spectrin.…”

Lipid Raft-dependent Endocytosis of Close Homolog of Adhesion Molecule L1 (CHL1) Promotes Neuritogenesis

“…Because lipid rafts are enriched in voltage-dependent Ca 2ϩ channels (22), CHL1 ligand-induced redistribution of the CHL1-␤II spectrin complex to lipid rafts may facilitate Ca 2ϩ -dependent disassembly of the complex. Spectrin ␣II contains Ca 2ϩ -binding EF-hands (39) and can bind to calmodulin (40), thus being directly linked to the Ca 2ϩ signaling network. An increase in Ca 2ϩ may also influence the integrity of the spectrin meshwork by activating the Ca 2ϩ -dependent actin-severing protein gelsolin, which is involved in spectrin meshwork remodeling (41).…”