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Originally published In Press as doi:10.1074/jbc.M604613200 on August 31, 2006

J. Biol. Chem., Vol. 281, Issue 45, 34333-34340, November 10, 2006
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Structure of the Calmodulin {alpha}II-Spectrin Complex Provides Insight into the Regulation of Cell Plasticity*

Miljan Simonovic{ddagger}1, Zhushan Zhang§, Carol D. Cianci§, Thomas A. Steitz{ddagger}, and Jon S. Morrow§2

From the {ddagger}Howard Hughes Medical Institute, Yale University, New Haven, Connecticut 06520 and the §Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06520

Received for publication, May 12, 2006 , and in revised form, August 21, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
{alpha}II-spectrin is a major cortical cytoskeletal protein contributing to membrane organization and integrity. The Ca2+-activated binding of calmodulin to an unstructured insert in the 11th repeat unit of {alpha}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 {alpha}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 calmodulin-binding domain of human {alpha}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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
 REFERENCES
 
Spectrin {alpha}beta heterodimers are organized into filamentous structures composed of two related subunits. The {alpha} 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 spectrin-based 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 out-growth (19, 20). It has been observed that during the early stages of apoptosis, a characteristic 150-kDa fragment of {alpha}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 enter-opathogenic bacteria, catalyzes spectrin fragmentation and the consequent collapse of the cell membrane (22-24). In each case, the cellular response ensuing from such proteolysis is determined by cleavage at a specific site in the {alpha}II spectrin molecule.

The 11th repeat of {alpha}II-spectrin contains an unusual, protease-hypersensitive insert in helix C. This insert contains recognition sites for µ-calpain, caspases 2, 3, and 7, and the serine proteinase Pet toxin (13, 23, 25, 26). The N-terminal side of the 11th repeat (herein termed {alpha}IIspec) is flanked by an SH3 domain, whereas the C-terminal portion encompasses a calmodulin-binding domain that is proposed to be unstructured in solution (27). The Ca2+-dependent calmodulin (CaM)3 binding to human {alpha}II-spectrin regulates the proteolytic susceptibility of spectrin, enhancing its cleavage by µ-calpain, while blocking caspase 2, 3, and 7 cleavage (28-30). Cleavage by calpain is also controlled by phosphorylation of residue Tyr1176 by c-Src, a kinase that binds to the flanking SH3 domain in the {alpha}10 repeat unit (31). When phosphorylated, {alpha}II-spectrin becomes resistant to calpain proteolytic activity (31, 32). Thus, an insert in the {alpha}11 repeat of the human {alpha}II-spectrin represents the convergence point of two crucial cellular signaling cascades.

The mechanism(s) of both the CaM-dependent regulation of spectrin proteolysis and the cross-talk between the two signaling networks at the site of {alpha}IIspec is not well understood. To address these questions, we have determined the three-dimensional structure of the complex between bovine calmodulin and the proposed calmodulin-binding domain of human {alpha}II-spectrin (CaM-{alpha}IIspec) (25). The structure of this complex revealed that the CaM-{alpha}IIspec-binding interface is composed entirely of hydrophobic residues, in striking contrast to previously determined CaM-peptide structures. CaM binding also induced a 23-residue stretch of {alpha}IIspec to fold into an amphiphilic {alpha}-helix with hydrophobic side chains facing the CaM hydrophobic groove. These numerous hydrophobic interactions are reminiscent of those seen within a protein core, suggesting that complex formation is driven by an increase in solvent entropy. A detailed comparison of our structure with those of other CaM-peptide complex structures also reveals that {alpha}IIspec is positioned uniquely in the CaM groove relative to previously known ligands (33-36). The crystal structure of the CaM-{alpha}IIspec complex thus provides a compelling structural basis for understanding how calmodulin regulates the susceptibility of {alpha}II-spectrin to proteolysis and thereby other downstream cytoskeletal signals.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
 REFERENCES
 
Complex Preparation and Crystallization—Bovine calmodulin was purchased from Sigma. The human {alpha}II-spectrin calmodulin-binding domain containing cleavage sites for calpain, caspases, and Pet toxin was cloned into pGEX4T vector and expressed and purified as a GST fusion protein from Escherichia coli BL21(DE3) using the following procedure. An over-night culture of the CaM-binding domain of spectrin was diluted 1/10 into fresh Luria-Bertani medium containing 100 µg/ml ampicillin, grown with shaking at 37 °C for 1 h when it was induced with 1 mM isopropyl-1-thio-beta-D-galactopyranoside. The growth was continued for an additional 4 h at 37°C. The cells were spun down at 5,000 x g/10 min. The cell pellet was resuspended in 50 mM Tris-HCl, pH 8.0, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 0.2 mg/ml lysozyme, and 1% Triton X-100 and incubated for 30 min at 4 °C with gentle rocking. The culture was then diluted with an additional 10 ml of the resuspension buffer, and while on ice, sonicated with three 10-s pulses. The lysate was then centrifuged at 18,000 rpm for 20 min. at 4 °C. The soluble fraction was loaded onto a glutathione agarose matrix and washed with Tris-buffered saline buffer, and GST-{alpha}IIspec was eluted in 4 ml of 50 mM Tris-HCl, pH 8.0, 1 mM EDTA, 1 mM dithiothreitol, and 5 mM reduced glutathione. The protein was then further purified on a calmodulin-Sepharose 4B column (Amersham Biosciences). The column was equilibrated with 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM beta-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, 1 mM CaCl2. Prior to loading the column, CaCl2 was added to the sample to a final concentration of 10 mM. The column was then washed with the above buffer until the baseline absorbance was restored. The GST-{alpha}IIspec was eluted with 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 5 mM beta-mercaptoethanol, 0.1 mM phenylmethylsulfonyl fluoride, and 5 mM EGTA. GST was subsequently removed by thrombin cleavage and removed by absorption to glutathione-Sepharose. The {alpha}II-spec-CaM complex was formed from an equimolar solution of calmodulin and peptide. Crystals were obtained by the sitting drop vapor diffusion method, mixing equal volumes of the protein complex and the well solution (50 mM Tris-HCl, pH 8.0, 30% polyethylene glycol 8000, 0.1 M (NH4)2SO4) in a total volume of 40 µl. The crystals belong to space group P212121 (a = 44.29, b = 57.73, c = 69.75 Å) with one molecule per asymmetric unit and 49% solvent content.

Data Collection, Structure Determination, and Refinement— Diffraction data were collected at room temperature on a Rigaku RAXIS II detector using a rotating copper anode as an x-ray source. The data were indexed in Denzo and scaled and reduced in Scalepack (37). The initial phases were determined by molecular replacement using the structure of calmodulin (PDB accession code: 1CDM [PDB] ). The search model was broken into two domains (residues 4-70 and 79-144), omitting the linker loop containing residues 71-80. Molecular replacement calculations were performed with Phaser 1.3 (38, 39), searching for the two domains of calmodulin sequentially. The top solution had a Z score of 12 and good packing with no steric clashes. Upon a cycle of simulated annealing in CNS (40), the Fo - Fc map contoured at 2{sigma} revealed a three-turn helix of the spectrin calmodulin-binding domain. Further refinement was done in CNS (40), whereas electron density map inspection and manual model rebuilding were done in Quanta. Ten percent of the data was randomly assigned to an Rfree test set for cross-validation (41). The final stages of refinement, including individual B factor refinement, were performed against all the data. The final model yielded good geometry with 89.6% of residues in the most favored region and 10.4% in additional allowed orientations according to Procheck (42). It had an R factor of 24.5% and Rfree of 26.9%. The crystallographic data and final model statistics are presented in Table 1.


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  		TABLE 1
Data collection and refinement statistics

 
CD Spectroscopy—Circular dichroism studies of the purified {alpha}II-spectrin peptide alone, of CaM alone, and of the CaM-{alpha}II-spec complex, either with 0.1 mM CaCl2 or in the present of 0.1 mM EDTA, were carried out in 50 mM Tris-HCl, 20 mM NaCl, pH 8.0 at room temperature. The molar circular dichroism was measured from 190 to 260 nM, and the percentages of protein secondary structure were estimated using the program K2d available at the K2d home page (43).


   RESULTS AND DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
REFERENCES
 
Overall Structure—The crystal structure of the calmodulin-binding domain of {alpha}II-spectrin in complex with calmodulin was solved to 2.45 Å resolution and refined to a final R factor of 24.5% (Rfree = 26.9%) (Table 1). The final model contains 157 protein residues, 62 water molecules, and four calcium ions. The CaM-{alpha}IIspec complex is compact and globular, like other CaM-peptide structures solved previously (see Fig. 2A) (33-36, 44), with two calcium-binding lobes of CaM, designated as the N- and C-lobes, wrapped around the {alpha}IIspec peptide. All four calcium-binding sites are similar when compared with other CaM-ligand complex structures.

To crystallize the complex of CaM with {alpha}IIspec, a 42-residue peptide of the proposed calmodulin-binding domain of {alpha}II-spectrin was prepared (Fig. 1A). This peptide contains cleavage sites for µ-calpain, caspases 2, 3, and 7, and the recently identified Pet toxin proteinases (23, 25, 28, 30). A 23-residue-long stretch (residues Ala1189-Arg1211) of the peptide was ordered and adopted an {alpha}-helical conformation (Fig. 2). Five N-terminal residues of CaM, loop 70 (residues 74-80) and residue 116, were disordered and consequently could not be modeled into the CaM-{alpha}IIspec structure. Likewise, the 18 N-terminal residues of {alpha}IIspec, encompassing the calpain and caspase cleavage sites, and 3 extreme C-terminal residues, were missing from the map.

CaM-{alpha}IIspec Binding Is Mediated Entirely by Hydrophobic Interactions—Analysis of the CD spectra of the calmodulin-binding domain of {alpha}II-spectrin as well as earlier modeling studies (27) show that it is disordered in solution (Fig. 3). The structure of the CaM-{alpha}IIspec complex revealed that upon calmodulin binding, {alpha}IIspec folds into an amphiphilic helix beginning with a proline residue at position 1191 (based on the human {alpha}II-spectrin numbering). The combined buried surface area at the interface between the two molecules is 2,590 Å2, which is a half of the {alpha}IIspec surface area. A more detailed analysis of {alpha}IIspec binding revealed a striking difference between the CaM-spectrin complex and other previously solved CaM-peptide structures.


Figure 1
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  		FIGURE 1.
Sequence and functional sites in {alpha}IIspec. A, schematic diagram of a portion of the human {alpha}II-spectrin domain structure depicting {alpha}9/10 (orange) and {alpha}11 (blue and green) repeats. The SH3 domain responsible for c-Src kinase binding is within the {alpha}9/10 repeat (light orange). A complete sequence of helix C from the {alpha}11 repeat containing an unusual insert that binds CaM is shown. The insert is in red letters, whereas the proposed CaM-binding domain is in bold red italicized letters. A peptide ({alpha}IIspec) spanning residues Glu1174-Glu1210 was used in this study. The helix symbol designates the visible part of the {alpha}IIspec peptide in the CaM-{alpha}IIspec structure presented here, showing that the proposed CaM-binding sequence fits perfectly with the structured region of the peptide. Hydrophobic residues Trp1192, Met1198, and Phe1205, the landmarks of the 1-8-14 subclass of CaM-binding proteins, are labeled with an asterisk. The proteinase recognition sites are labeled accordingly, and the calpain cleavage site (Tyr1176) is also a target residue for c-Src kinase. B, sequence alignment of {alpha}IIspec with other canonical CaM ligands. Typically, there is minimal sequence similarity; the major hydrophobic residues responsible for binding CaM are shown in green, and other hydrophobic anchor points are depicted in red. Sequence alignment was done using the program Multalin (59).

 
It is well established that upon binding of Ca2+, two calmodulin lobes, designated as the N- and C-lobe, undergo a structural rearrangement to expose hydrophobic pockets and allow various ligands to bind. Flexibility in helix D (hD) that connects the two CaM lobes allows an additional conformational change upon ligand binding. In such a complex, two CaM lobes wrap around the target ligand, stabilizing the closed conformation of the CaM-peptide complex. The typical CaM ligand is a helix of about 20 residues containing two hydrophobic side chains separated by 8, 10, 14, or 16 residues that provide anchor points for the ligand (46, 47). The proposed CaM-binding sequence of the {alpha}IIspec contains 3 hydrophobic residues, whereas the rest of the sequence does not bear any similarity with other known CaM ligands (Fig. 1B). These hydrophobic residues put the {alpha}II-spectrin CaM-binding domain into the 1-14 class of CaM-binding peptides, or more precisely, into its 1-8-14 subclass (47). The class/subclass name designates the spacing between the critical anchor residues in the CaM ligand. In all previously described CaM-peptide complexes, the majority of interactions with the ligand are mediated through extensive hydrogen bonding networks that are supported with limited hydrophobic interactions.

However, unlike the other CaM-binding sequences that have been characterized, the interacting surface of the CaM-{alpha}IIspec complex is composed almost entirely of hydrophobic residues clustered into three hydrophobic pockets (Fig. 4). The first CaM-binding pocket is composed of the C-lobe residues from helices F (hF) and H (hH), the extreme C terminus of CaM, and the loop connecting hF and helix G (hG). Two methionine residues, Met124 and Met144, form the first methionine cleft that binds the side-chain Trp1192 from {alpha}IIspec. At the bottom of the pocket are Phe92, Leu105, Met109, Phe141, and Met145 from CaM. The side chains of Phe92 and Met109 interact with Ala1195 from {alpha}IIspec, a residue positioned on the same face of the helix as Trp1192. The Van der Waals contacts between Trp1192 and the first CaM methionine cleft anchor the N-terminal end of {alpha}IIspec into the C-lobe of CaM (Fig. 4, A and B). Hydrophobic interactions between a tryptophan side chain at the N terminus of a ligand and the first hydrophobic pocket of CaM have been described previously (34, 35). In those structures, the tryptophan side chain forms a hydrogen bond with the carbonyl oxygen of Met124 of CaM. Conversely, in the CaM-{alpha}IIspec complex, the Trp1192 side chain adopts a different rotamer conformation and interacts with the first methionine pocket via exclusively hydrophobic interactions (Fig. 4B).


Figure 2
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  		FIGURE 2.
Overall structure of the CaM-{alpha}IIspec complex. A, stereo view ribbon diagram of the complex between CaM and the calmodulin-binding domain of {alpha}II-spectrin ({alpha}IIspec). The N-lobe of CaM is shown in green, the C-lobe is shown in blue, {alpha}IIspec is shown in red, and calcium ions are shown as gold spheres. The secondary structure elements of CaM are labeled as under "Results," whereas the disordered regions are represented with a dashed line (images were produced in Molscript (60) and rendered in Raster 3D (61)). C-term., C terminus; N-term., N terminus. B, stereo view of the simulated annealing omit |2Fo - Fc|{alpha}C electron density map of the N-terminal end of {alpha}IIspec (residues Trp1192-His1200) contoured at a level of 2{sigma}.

 
The second binding pocket is composed of residues from the N-lobe helix A (hA), C-lobe helix E (hE), and the loop connecting hF and hG. The {alpha}IIspec residue Leu1197 interacts with Ala15 and Leu18, whereas Val1199 interacts with Ile85, Ala88, and Met145. The anchor residue in the second pocket is clearly Met1198, which makes contacts with Val35, Leu39, and Ala112 (Fig. 4C). The pocket expands further with Met36 from CaM, which interacts with side chains of Thr1201 and Val1202 from {alpha}IIspec. This pocket represents the region where the hydrophobic surfaces of the two CaM lobes fuse into one continuous region.

The third pocket is composed of residues from helices B, C, and D of the N-lobe of CaM. CaM side chains Phe16, Phe19, Phe61, Ile63, Phe65, and Phe68 form the bottom of the pocket, whereas the wall is lined up with Leu32, Val55, Met71, and Met72. Finally, the pocket roof is composed of Met36 and Met51. Three methionine residues Met36, Met51, and Met71 establish the second methionine cleft that interacts primarily with {alpha}IIspec residue Phe1205 (Fig. 4B). Other {alpha}IIspec residues, such as Thr1201, Val1202, and Ile1204, contribute with additional Van der Waals contacts. It is clear that the hydrophobic side chains of the 2 {alpha}IIspec residues Trp1192 and Phe1205, located at the opposite ends of the helix, interact with two methionine clefts located within different CaM lobes (Fig. 4, A and B). These residues, along with Met1198, serve as anchor points for the {alpha}IIspec helix, playing a crucial role in CaM-{alpha}IIspec recognition.


Figure 3
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  		FIGURE 3.
CD measurements of the {alpha}IIspec at pH 8.0 reveal {alpha}IIspec to be random coil in solution. The blue line is {alpha}IIspec, the red line is CaM, and the green line is CaM-{alpha}IIspec. The helical content is calculated to be 5% for {alpha}IIspec and 30% both for CaM and for CaM-{alpha}IIspec using the K2d algorithm (43).

 
The only two ionic interactions observed in the entire complex are the following: Lys1193 of {alpha}IIspec makes a hydrogen bond with Glu14 (N-lobe; hA), whereas Asn1206 forms hydrogen bonds with both the Gln41 (N-lobe; hB) and the Glu84 (C-lobe; hE) side chains. Interestingly, these ionic interactions are contiguous to the major anchor pockets both sequentially and spatially. These small hydrogen bond networks further stabilize the orientation of the {alpha}IIspec helix. Finally, {alpha}IIspec residues from Asn1206 to the C terminus and other polar side chains do not contribute to the binding with CaM.

The Calmodulin-binding Domain of Spectrin Binds Differently in the CaM-binding Groove—We have compared our structure of the calmodulin-binding domain of spectrin with the following CaM-peptide complexes: CaM-CaMKII (36), CaM-MARCKS (44), CaM-NOSIII (33), CaM-smMLCK (35), and CaM-CaMKI (34). The ligands used in the comparison belong to different classes of calmodulin-binding proteins based on the motifs they contain; CaM-CaMKII is from the 1-10 class, CaM-MARCKS is from the basic class, whereas the others are from the 1-14 class of calmodulin-binding proteins (47). Further, CaM-smMLCK belongs to the basic 1-8-14, CaM-CaMKI is in the 1-14 and CaM-NOSIII is in the 1-5-8-14 subclass of the more general 1-14 class (47). The superimposition of C{alpha} atoms of either the N-lobe (6-72) or the C-lobe residues (83-147) of CaM in our structure with those of other CaM-peptide complexes gave the following values: N-lobe r.m.s.d. = 0.77-1.31 Å (67 C{alpha} atoms superimposed) and C-lobe r.m.s.d. = 0.68-1.19 Å (65 C{alpha} atoms superimposed). These results suggest that the backbone conformation of the lobes in different CaM-peptide complexes is very similar.


Figure 4
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  		FIGURE 4.
The CaM-{alpha}IIspec-binding surface is composed entirely of hydrophobic residues. A, molecular surface representations of the complex of CaM bound to {alpha}IIspec peptide. Left, the hydrophobic residues of CaM-binding groove are in yellow, whereas the rest of the molecule is in white. The {alpha}IIspec helix is shown as sticks with hydrophobic residues shown in green and polar, charged and uncharged, residues shown in blue. Right, surface charge distribution representation of CaM-{alpha}IIspec complex. The arrows point into the two CaM methionine clefts, labeled Met cleft 1 and Met cleft 2. The N- and C-terminal residues of {alpha}IIspec are labeled as under "Results" (produced in Grasp (62)). The view is rotated 90° clockwise in the plane relative to Fig. 2A. B, stereo view of the major side-chain contacts between the N- (green) and C-lobes (blue) of CaM with {alpha}IIspec (red). The residues, shown as balls-and-stick, are labeled as under "Results," whereas both methionine clefts are marked with an asterisk (produced in Molscript (60) and rendered in Raster 3D (61)). C, the second hydrophobic pocket positioned between two methionine clefts is composed of residues from both CaM lobes. The anchoring residues from {alpha}IIspec, Met1198 and Val1199, interact with hydrophobic residues from both lobes of CaM. This view is rotated 90° clockwise around the horizontal axis relative to Fig. 4B.

 
Further, our data suggest that the relative orientation of the lobes differ in various complexes due to adjustments in the CaM molecule upon ligand binding. The calculated r.m.s.d. values were much higher, 2.02-2.94 Å, for the CaM-smMLCK and CaM-MARCKS complexes, respectively, when 132 C{alpha} atoms (residues 6-72 and 83-147) were used for superimposition. Interestingly, calmodulin adopted a more open conformation upon binding to the {alpha}IIspec when compared with that seen in the previously solved complexes. The N-terminal hA and the C-lobe hG are pushed apart, and a shift is visible both in the second calcium-binding site (loop 60) and in the N-lobe hD (Fig. 5A). The flexibility of loop 60 is reflected in higher B factor values (~70 Å-2) when compared with the rest of the molecule (~46.4 Å-2) (Fig. 5D). It is possible that CaM had to open up more to accommodate the much longer peptide used in this study. Small spatial rearrangements of both the N-lobes and the C-lobes caused the exposure of the continuous hydrophobic binding pocket. This reflects the adaptability of CaM to different ligands. Further, because of the length of the ligand, the structure of the complex between CaM and the {alpha}IIspec that was derived here may be a better representation of the physiological CaM-ligand complex. The superposition of CaM molecules derived from different CaM-ligand complexes allowed examination of the differences in the ligand positions within the CaM-binding site.

When the CaM-{alpha}IIspec complex is viewed in the orientation shown in Fig. 2A, it becomes evident that {alpha}IIspec binds differently in the CaM groove than do other CaM ligands (Fig. 5, B and C). The {alpha}IIspec is tilted upwards toward the second methionine pocket, where the side chain of Asn1206 provides an additional hydrogen bond network stabilizing the ligand position. This tilt of the helix is present even when compared with the CaM-smMLCK complex that bears the greatest structural similarity with CaM-{alpha}IIspec. In all previously described complexes, both the second and the third hydrophobic pockets are either empty (CaM-NOSIII, CaM-CaMKII, and CaM-MARCKS) or occupied by a small hydrophobic residue making a limited number of Van der Waals contacts with CaM (CaM-smMLCK). Conversely, in the CaM-{alpha}II-spec complex, there are numerous hydrophobic interactions between the ligand and the last two hydrophobic binding pockets of calmodulin. These hydrophobic interactions are reminiscent of those within the hydrophobic core of proteins, suggesting that complex formation with {alpha}IIspec is extremely stable and driven by increases in solvent entropy. The dissociation of CaM from {alpha}II-spectrin thus is likely to require dramatic changes in calmodulin structure, presumably the unfolding of calmodulin due to a fall in cellular Ca+2 concentration.


Figure 5
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  		FIGURE 5.
The spectrin {alpha}IIspec binds differently to a more open CaM when compared with ligands of previously solved CaM-peptide complexes. A, the superimposition of the CaM lobes from different CaM-peptide complex structures revealed that CaM adopted a more open conformation once bound to {alpha}II-spectrin. The CaM-{alpha}IIspec is blue, whereas others are in beige (CaM-MARCKS, CaM-CaMKII, CaM-NOSIII). Only the backbones of the CaM molecules are shown. Similar results have been obtained with other CaM-peptide complexes (CaM-smMLCK and CaM-CaMKI; not shown for clarity). The superimposition using 132 C{alpha} atoms of CaM (residues 6-72 and 83-147) was calculated using the CCP4 module Polypose (63). B, the {alpha}IIspec calmodulin-binding domain is tilted upward toward the second methionine cleft when compared with other CaM ligands. CaM is shown in an all-atom representation, {alpha}IIspec is in red, the MARCKS peptide is in green (PDB ID: 1IWQ), the CaMKII peptide is in blue (PDB ID: 1CDM), and the NOSIII peptide is in gold (PDB ID: 1NIW). The CaM molecule is in the same orientation as in A. C, the {alpha}IIspec helix direction is different from that of other CaM ligands. The {alpha}IIspec is tilted up and twisted toward the C-terminal end of CaM. This view is rotated ~90° clockwise around the vertical axis relative to B (produced in Molscript (60) and rendered in Raster 3D (61)). The peptide ligand positions are generated upon superimposing C{alpha} atoms of the N- and C-lobes of CaM using the CCP4 module Polypose (63). D, B factor (average main chain) versus residue plots for CaM-{alpha}IIspec complex reveals flexible regions in CaM. The average B factor value for CaM is 46.4 Å-2 and for the {alpha}IIspec peptide is 36.2 Å-2. The most flexible regions in CaM are indicated on the plot and are labeled as under "Results." The calculation was performed using the CCP4 module BAVERAGE (45).

 
Proteolysis Inhibition by Steric Hindrance and Signal Transduction by Domain Folding—The {alpha}11 repeat contains several proteinase recognition sites (Fig. 1A). Caspases cleave at Asp1185, µ-calpain cleaves at Tyr1176, and Pet toxin cleaves at Met1198 (13, 23, 25). Each of these cleavages activates distinct cellular responses such as cell shape change, neuronal growth elongation, and apoptosis. The structure of the complex between CaM and the {alpha}IIspec provides a structural basis for understanding many of these phenomena. It is apparent that calmodulin inhibits caspase- and Pet toxin-catalyzed proteolysis of {alpha}II-spectrin by steric hindrance, whereas the stimulation of calpain proteolytic activity is probably accomplished through direct effects on the conformation of both the substrate and the target proteinase. The caspase cleavage site, 1182DEDT1185, is only 3 residues removed from Ala1189, suggesting that calmodulin binding inhibits the caspase-catalyzed proteolysis of {alpha}II-spectrin by steric hindrance. Once the calmodulin molecule binds to {alpha}II-spectrin, caspase cannot approach the recognition site. It is important to note that caspase cleaves the DETD sequence efficiently, suggesting that the cleavage site assumes a substrate-like conformation when {alpha}IIspec is not in complex with CaM. The CaM-dependent inhibition of caspase activity abolishes the degradation of {alpha}beta-spectrin heterodimer and retards the completion of the apoptotic cascade. In a fashion similar to caspase inhibition, calmodulin also inhibits the Pet toxin-catalyzed proteolysis of spectrin by steric hindrance. The target residue for Pet toxin is Met1198. In the CaM-{alpha}IIspec complex, Met1198 lies buried in the second hydrophobic pocket interacting with CaM residues Val35, Leu39, and Ala112 (Fig. 4B).

The recognition site for the cysteine proteinase milli- or µ-calpain is located adjacent to a putative PEST domain (48) at residue Tyr1176, juxtaposed to helix C of the {alpha}11 repeat of {alpha}II-spectrin (Fig. 1A). Calmodulin binding to {alpha}II-spectrin accelerates the rate of µ-calpain cleavage of {alpha}II-spectrin, renders an adjacent betaII-spectrin subunit susceptible to µ-calpain cleavage at Gln1441-Ser1442, and modulates the quaternary association state of the post-cleaved spectrin heterodimer/heterotetramer (25, 29, 49). The consequences of calpain proteolysis of {alpha}II-spectrin are thus quite distinct from the action of the caspases, inducing the reorganization of membrane subdomains and cell shape transformation rather than cell membrane fragmentation and blebbing (13, 15-17, 50). Calpain cleaves spectrin significantly only in the presence of calmodulin in vitro (30, 49). Based on our structure, it is likely that these events are directly coupled to the CaM-induced folding of the {alpha}II-spectrin insertion containing the calpain cleavage site. The folding of the insert would constrain the conformation of the loop that contains the calpain cleavage site, forcing it to adopt a substrate-like conformation in which the scissile P1-P1' bond (51) would assume the optimal orientation for calpain binding and proteolysis. These conclusions fit well with findings that the primary sequence of the natural calpain targets evolved to be suboptimal (52). The constraints in the spectrin insert presumably might also reorient the packing of helix C of the 11th spectrin repeat unit, triggering the conformational change that unmasks the adjacent beta-spectrin subunit to attack by calpain.

In addition to the effects on the substrate conformation, calmodulin might affect the attacking calpain molecule through a direct interaction. The classic calpains are heterodimers consisting of large (L) and small (S) chains. The L-chain is the proteinase domain, whereas the S-chain serves as a chaperone, and the two interact through their calmodulin-like domains (53). An interesting regulatory mechanism involving dissociation of the chaperone-like S-chain has been suggested (54, 55), along with the proposal that calpain activity might be regulated either by binding to endogenous activators (56) or by interaction with phospholipids (57, 58) in vivo. It is plausible, therefore, that CaM interacts directly with the calmodulin-like domain of the L-chain of calpain stimulating the dissociation of the regulatory S-chain. Thus, we postulate that calmodulin might stimulate calpain activity in two ways: (i) by presenting the substrate to the proteinase in the optimal conformation and (ii) by tethering and stabilizing the tertiary structure of the attacking proteinase.

As noted above, calpain cleavage is also controlled by phosphorylation of the calpain target residue Tyr1176 (31, 32). Thus, the CaM-{alpha}IIspec complex represents a point of convergence of at least two cellular signaling cascades. The structure of the CaM-{alpha}IIspec complex presented here is the first attempt to understand the calmodulin-dependent regulation of {alpha}II-spectrin proteolysis and the downstream cytoskeletal signal transduction from a structural perspective. Further structural and biochemical studies employing progressively larger multisubunit complexes will be necessary to decipher the precise mechanisms of the cross-talk between different signaling cascades that converge at the {alpha} subunit of spectrin.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 2FOT) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported in part by National Institutes of Health Grants R01-HL28560 and P01-NS35476 (Ment PI) (to J. S. M.), National Institutes of Health Grant GM22778 (to T. A. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 A recipient of the Jane Coffin Childs Postdoctoral Fellowship. Back

2 To whom correspondence was addressed: Dept. of Pathology, Yale University School of Medicine, 310 Cedar St. BML 140, New Haven, CT 06520. Tel.: 203-785-3624; Fax: 203-785-7037; E-mail: jon.morrow{at}yale.edu.

3 The abbreviations used are: CaM, calmodulin; CamK, calmodulin-dependent protein kinase; PDB, Protein Data Bank; GST, glutathione S-transferase; MARCKS, myristoylated alanine-rich C-kinase substrate; NosIII, nitric oxide synthase 3; smMLCK, smooth muscle myosin light chain kinases; r.m.s.d., root mean square deviation. Back


   ACKNOWLEDGMENTS
 
We thank C. Axel Innis and Peter G. W. Gettins for reading the manuscript and for comments and suggestions.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS AND DISCUSSION
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