JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M603963200 on July 6, 2006

J. Biol. Chem., Vol. 281, Issue 36, 26455-26464, September 8, 2006
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Supplemental Data
Right arrow All Versions of this Article:
281/36/26455    most recent
M603963200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yamniuk, A. P.
Right arrow Articles by Vogel, H. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yamniuk, A. P.
Right arrow Articles by Vogel, H. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

The Interaction between Calcium- and Integrin-binding Protein 1 and the {alpha}IIb Integrin Cytoplasmic Domain Involves a Novel C-terminal Displacement Mechanism*Formula

Aaron P. Yamniuk1, Hiroaki Ishida2, and Hans J. Vogel, Holds a senior scientist award from the Alberta Heritage Foundation for Medical Research3

From the Structural Biology Research Group, Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada

Received for publication, April 25, 2006 , and in revised form, June 21, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Calcium- and integrin-binding protein 1 (CIB1) regulates platelet aggregation in hemostasis through a specific interaction with the {alpha}IIb cytoplasmic domain of platelet integrin {alpha}IIbbeta3.In this work we report the structural characteristics of CIB1 in solution and the mechanistic details of its interaction with a synthetic peptide derived from the {alpha}IIb cytoplasmic domain. NMR spectroscopy experiments using perdeuterated CIB1 together with heteronuclear nuclear Overhauser effect experiments have revealed a well folded {alpha}-helical structure for both the ligand-free and {alpha}IIb-bound forms of the protein. Residual dipolar coupling experiments have shown that the N and C domains of CIB1 are positioned side by side, and chemical shift perturbation mapping has identified the {alpha}IIb-binding site as a hydrophobic channel spanning the entire C domain and part of the N domain. Data obtained with a truncated version of CIB1 suggest that the extreme C-terminal end of the protein weakly interacts with this channel in the absence of a biological target, but it is displaced by the {alpha}IIb cytoplasmic domain, suggesting a novel mechanism to increase binding specificity.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The platelet-specific heterodimeric transmembrane integrin receptor {alpha}IIbbeta3 plays a central role in hemostasis and thrombosis (1). At the site of vascular injury, platelet agonists such as thrombin trigger "inside-out" signaling events that activate {alpha}IIbbeta3, resulting in ligand binding by the integrin extracellular domains, integrin cross-linking, and ultimately, platelet aggregation. Ligand occupancy also generates "outside-in" signals that lead to granular secretion of ADP, cytoskeletal reorganization, and platelet spreading (2). The small EF-hand calcium-binding protein CIB1,4 (calcium- and integrin-binding protein 1, also known as CIB, calmyrin, KIP) binds specifically to the {alpha}IIb cytoplasmic domain (3), and the interaction has been implicated in both inside-out and outside-in signaling events (4-6). The binding of CIB1 to synthetic {alpha}IIb peptides can occur in vitro with a dissociation constant in the high nanomolar range (7, 8); however, the mechanistic details of the interaction are not well understood. Because inappropriate platelet activation is a major contributor to cardiovascular disease (9), understanding the interaction between CIB1 and {alpha}IIb could be an important step toward the development of novel anti-platelet therapeutics.

CIB1 shares significant sequence homology with calcineurin B (CnB), calcineurin homologous protein-1, and the neuronal calcium sensor (NCS) family of EF-hand proteins. Like these proteins, CIB1 is myristoylated on its N-terminal glycine residue and is membrane-associated in vivo (10, 11). However, myristoylation is not required for {alpha}IIb binding (7, 8, 10). Recent x-ray crystal structures (Protein Data Bank codes 1XO5 [PDB] and 1Y1A) have shown that like its homologs, calcium-bound CIB1 (Ca2+-CIB1) folds into N- and C-terminal globular domains, each composed of two EF-hand motifs, with extended N- and C-terminal regions (12, 13). Ca2+ is bound to the canonical C domain EF-hands (EF-III and EF-IV) but not to the divergent N domain EF-hands (EF-I and EF-II), consistent with biochemical studies (14). Although the core dual helix-loop-helix EF-hand structure of Ca2+-CIB1 is very similar in both 1XO5 and 1Y1A, there are distinct differences in the protein's oligomeric state, the orientation of the two domains, and the conformation of the N- and C-terminal extensions (supplemental Fig. 1). In 1XO5, Ca2+-CIB1 was crystallized as a monomer with similar domain orientation to CnB, calcineurin homologous protein-1, and the NCS proteins (13). CIB1 is also monomeric in solution as revealed by diffusion NMR spectroscopy (15), sedimentation equilibrium, and gel filtration studies (13, 16). However, in 1Y1A the protein was crystallized as a head-to-tail dimer, with a different domain orientation and structurally distinct N- and C-terminal extensions (12). The structure of the N-terminal extension is important since it would affect the placement of the myristoyl group and orientation of CIB1 with respect to the cytoplasmic membrane. The structure of the C-terminal extension is interesting because in each crystal structure it shields the C domain hydrophobic channel that had previously been proposed to be the {alpha}IIb-binding site based on mutagenesis and molecular modeling data (10, 17). In this study we have utilized solution NMR spectroscopy, optical spectroscopy and microcalorimetry of CIB1, and truncated CIB1 proteins to investigate the solution structure of Ca2+-CIB1 and characterize its interaction with an acetylated synthetic peptide ({alpha}IIb-L) encompassing the entire {alpha}IIb cytoplasmic domain and part of the transmembrane domain (Ac-LVLAMWKVGFFKRNRPPLEEDDEEGQ-OH). Our data reveal the structural characteristics of peptide-free and peptide-bound Ca2+-CIB1 in solution and reveals a novel binding mode for the {alpha}IIb cytoplasmic domain, in which the C-terminal extension of CIB1 plays an important role in controlling binding specificity.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Protein Expression and Purification—Wild type histidine10-tagged-CIB1 (herein referred to as CIB1) was expressed in Escherichia coli strain ER2566 (New England Biolabs) from a pET19b vector (Novagen) as previously described (14). The total protein construct is 213 residues long (24 kDa) including the 23 residue N-terminal tag. 15N- and 13C,15N-labeled CIB1 were expressed from the same bacterial strain in M9 minimal medium containing 15NH4Cl and either 3 g/liter unlabeled glucose or 3 g/liter [13C]glucose, respectively. Perdeuteration required that the bacterial strain be first acclimatized to M9 minimal media with increasing percentages of D2O (0, 30, 60, 99.9%) (18, 19). 2H,13C,15N-Labeled CIB1 was ultimately expressed in 99.9% D2O medium containing [13C]glucose and 15NH4Cl. All proteins were purified by Ni2+-Sepharose affinity chromatography (14), and concentrations were determined using the predicted molar extinction coefficient ({epsilon}276 = 2900). Mass spectrometry revealed that the level of perdeuteration was ~87%.

Generation of Mutant CIB1 Proteins—The CIB1{Delta}H10 construct was generated by introducing a stop codon (TGA) in place of the Ser-180 codon (AGC) using the QuikChange site-directed mutagenesis kit (Stratagene) and the forward and reverse primers 5'-CAGCATGTGATCAGCCGCTGACCAGATTTTGCGAGC-3' (FWD) and 5'-GCTCGCAAAATCTGGTCAGCGGCTGATCACATGCTG-3' (REV). The coding region for residues 96-191 of CIB1 was amplified from the CIB1-pET19b template using PCR and the primers 5'-AGATTTTCTGGATCATATGAGCGTGTTTAGCG-3' (FWD) and 5'-TTAGCAGCCGGATCCTCACAGCAC-3' (REV). The purified PCR product and empty pET19b expression vector were each digested with NdeI and BamHI (New England Biolabs) and then ligated using T4 DNA ligase (Invitrogen) to generate the CIB1-C-pET19b construct. The integrity of both constructs was confirmed by DNA sequencing, and each protein was purified to homogeneity identically to wild type CIB1. Concentrations were determined using the predicted molar extinction coefficient for CIB1{Delta}H10 ({epsilon}276 = 2900), and CIB1-C ({epsilon}276 = 1450).

Peptides—All peptides were synthesized commercially and determined to be more than 95% pure by matrix-assisted laser desorption/ionization mass spectroscopy and high pressure liquid chromatography. Peptide {alpha}IIb-L (Ac-LVLAMWKVGFFKRNRPPLEEDDEEGQ-OH) corresponds to amino acids 983-1008 of the platelet integrin {alpha}IIb subunit, with Gln-1008 as the C-terminal residue (20). Peptide H10p (Ac-SPDFASSFKIVL-OH) corresponds to residues 180-191 of CIB1. Peptide smooth muscle myosin light chain kinase smMLCKp (Ac-ARRKWQKTGHAVRAIGRLSS-NH2) is the calmodulin (CaM)-binding site of chicken smooth muscle myosin light chain kinase, encompassing amino acids 36-55. The concentration of each peptide was determined using their predicted molar extinction coefficients: {alpha}IIb-L, {epsilon}280 = 5690; H10p, {epsilon}258 = 390; smMLCKp, {epsilon}280 = 5690.

NMR Spectroscopy—All NMR spectra were acquired on Bruker AVANCE 500 MHz or 700 MHz NMR spectrometers equipped with either a triple resonance inverse cryoprobe with single axis z gradient or triple-axis gradient triple broad band inverse detection (TBI) probe. Samples used for resonance assignments contained 650 µM 2H,13C,15N-labeld CIB1 in 20 mM HEPES, 100 mM KCl, 10 mM d10-dithiothreitol (d10-DTT), 3 mM CaCl2, 10% D2O, ~0.5 mM NaN3, pH 7.5 ± 0.1, with and without 720 µM (1.1 molar equivalent) {alpha}IIb-L. Sequence-specific assignments of the 1HN, 13C{alpha}, 13Cbeta, 13C, and 15N resonances for Ca2+-CIB1 and Ca2+-CIB1 in complex with {alpha}IIb-L were obtained manually using 1H,15N heteronuclear single quantum coherence (HSQC) spectra and a combination of transverse relaxation optimized spectroscopy (TROSY)-based triple-resonance experiments including three-dimensional HNCACB, HN(CA)CO, HN(CO)CACB, and HNCO experiments (21-23). All spectra were acquired at 310 K. Proton chemical shifts were referenced to the internal standard, 2,2-dimethyl-2-silapentane-5-sulfonate, and both 13C and 15N were referenced indirectly (24). Spectral analysis was performed using NMRPipe/NMRDraw (25) and NMRView (26) software, and chemical shift values were corrected for shifts induced by TROSY and perdeuteration (27).

The secondary structure of Ca2+-CIB1 alone and in complex with {alpha}IIb-L was determined using the weighted average secondary shift method, where the chemical shift deviation from random coil for the backbone C{alpha}, C, and N nuclei are normalized to the random coil range for each nucleus, and then averaged (Equation 1).

Formula 1(Eq. 1)

Chemical shift changes between Ca2+-CIB1 and the Ca2+-CIB1-{alpha}IIb-L complex were analyzed using the chemical shift perturbation (CSP) method described by Wingfield and co-workers (28), but for illustrative purposes the contributions of the HN and N nuclei are displayed separately from the contribution of the C{alpha} and Cbeta nuclei (Equation 2).

Formula 2(Eq. 2)


Figure 1
View larger version (29K):
[in this window]
[in a new window]
  		FIGURE 1.
Secondary structure and backbone flexibility of Ca2+-CIB1. A, schematic representation of the secondary structure for Ca2+-CIB1 determined by x-ray crystallography (Protein Data Bank codes 1XO5 and 1Y1A), with {alpha}-helices and beta-strands represented by boxes and arrows, respectively. Labeling of {alpha}-helices H1-H10 was taken from Gentry et al. Ref. 13. H0 is an additional {alpha}-helix that is observed in the NMR data and 1Y1A but not in 1XO5 and is defined herein. B, weighted average secondary shifts (WASS) for the Ca2+-CIB1 N, C{alpha}, and C nuclei reveal the secondary structure of the protein in solution. Positive weighted average secondary shift values are characteristic of {alpha}-helices, whereas negative values are characteristic of beta-strands and extended conformations. Residues for which no backbone amide was observed are indicated by solid circles in the lower part of the panel, and proline residues are indicated by open circles. C,{1H}-15N NOE data for Ca2+-CIB1. Values greater than ~0.6 are typical for folded globular domains, whereas smaller values suggest increased flexibility in the protein backbone.

 
{1H}-15N NOE experiments were acquired at 310 K and 700 MHz with and without 1H saturation using a recycle delay of 4.5 s (29). Samples consisted of 600 µM 15N-labeled CIB1 in 20 mM HEPES, 100 mM KCl, 10 mM d10-DTT, 3 mM CaCl2, 10% D2O, ~0.5 mM NaN3,pH7.5 ± 0.1, with and without 660 µM {alpha}IIb-L. {1H}-15N NOE data were processed and analyzed using NMRPipe/NMRDraw and NMRView software, with a noise floor of 7 and 10% for Ca2+-CIB1 and Ca2+-CIB1-{alpha}IIb-L, respectively.

1H,15N backbone dipolar couplings were measured at 310 K and 700 MHz using a sensitivity-enhanced IPAP-type 1H,15N HSQC experiment (30). Isotropic samples contained 650 µM 15N-labeled CIB1 in 20 mM HEPES, 100 mM KCl, 10 mM d10-DTT, 3 mM CaCl2, 10% D2O, ~0.5 mM NaN3,pH7.5 ± 0.1, and aligned samples contained 500 µM 15N-labeled CIB1 in the same buffer plus 12 mg/ml Pf1 phage (Asla Biotech). Data processing and analysis were performed using NMRPipe/NMRDraw, NMR-View, and PALES software (31). In the PALES analysis the correlation between residual dipolar couplings (RDC) data and a protein structure is given by the correlation coefficient (R) and the quality factor (Q), which each range from 0 to 1, with a better correlation represented by higher R values and lower Q values.

Isothermal Titration Calorimetry—All isothermal titration calorimetry (ITC) experiments were performed on a MicroCal VP-ITC microcalorimeter. Protein preparation included an overnight incubation in 10 mM DTT to reduce any disulfide bonds and subsequent DTT removal by gel filtration as previously described (14). Titrations consisted of sequential injections of 300-500 µM CIB1, CIB1{Delta}H10, or CIB1-C in 20 mM HEPES, 100 mM KCl, 2 mM CaCl2, pH 7.5, into a sample cell containing 10-20 µM {alpha}IIb-L or H10p in the same buffer at temperatures ranging from 20 to 37 °C. The non-linear heat of dilution exhibited by CIB1{Delta}H10 required the subtraction of a complete reference experiment for each peptide titration at all temperatures. Titrations of 400 µM smMLCKp into 20 µM CIB1 or CIB1{Delta}H10 were also performed under similar conditions. All data were fit to a one-site binding model using MicroCal Origin software to obtain values for the stoichiometry (N), association constant (Ka), and enthalpy change ({Delta}H), whereas values for the entropy change (T{Delta}S) and change in heat capacity ({Delta}Cp) were calculated using standard thermodynamic equations.

Fluorescence Spectroscopy—Steady state 8-anilino-1-naphalenesulfonate (ANS) fluorescence spectra were recorded at 37 °C on a Varian Cary Eclipse spectrofluorimeter using samples of 40 µM ANS in 20 mM HEPES, 100 mM KCl, 2 mM CaCl2, 1 mM DTT, pH 7.5 ± 0.1, with and without 10 µM CIB1 or CIB1{Delta}H10. Samples were excited at 370 nm using 5 nm excitation slits, and steady state fluorescence emission spectra were recorded from 400 to 600 nm at a scan rate of 600 nm/min using 10-nm emission slits.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
NMR Characterization of Ca2+-CIB1—Using TROSY-based triple resonance NMR spectroscopy experiments, we were able to obtain nearly complete assignments of the HSQC spectrum of Ca2+-CIB1, representing 88% of the expected backbone signals for the native polypeptide sequence (supplemental Fig. 2, A and C). Most of the amino acid residues that could not be detected were from solvent-exposed loops or at the N terminus, which is predicted to be solvent-exposed and flexible due to the high glycine and serine content in this region (13). Therefore, the signals for these residues are probably broadened by conformational exchange and/or the rapid amide hydrogen exchange that occurs with solvent H2O under these conditions (pH 7.5, 37 °C). We note that lowering the experimental pH or temperature reduced protein solubility and spectral quality, and no additional signals could be detected at higher temperatures (data not shown).


Figure 2
View larger version (27K):
[in this window]
[in a new window]
  		FIGURE 2.
Domain orientation of Ca2+-CIB1 revealed by residual dipolar coupling NMR data. Correlation between the observed dipolar couplings and those calculated from 1XO5 (A) or 1Y1A (B). The absolute difference between the observed and expected dipolar couplings {Delta}D is plotted as a function of amino acid residue for 1XO5 (C) and 1Y1A (D). Note that only the N terminus shows a poor correlation with 1XO5, whereas the entire protein backbone shows a poor correlation with 1Y1A.

 
A strong correlation between backbone chemical shift values and protein secondary structure is well established (32, 33). Therefore, we used the chemical shift data to predict the secondary structure of Ca2+-CIB1 in solution. As shown in Fig. 1, A and B, the NMR data confirmed that Ca2+-CIB1 is highly {alpha}-helical and composed of four EF-hand motifs. Both of the canonical Ca2+ binding EF-hand motifs in the C domain (EF-III and EF-IV) show the expected helix-loop-helix structure with a short beta-strand in the center of each Ca2+ binding loop. EF-II within the N domain also displays the typical helix-loop-helix structure, whereas the more divergent EF-I consists of a long {alpha}-helix (H1) and then a mixture of short helical segments (H2, H3a, and H3b) and connecting loops. Importantly, an N-terminal {alpha}-helix (H0) from K10-D18 is clearly observed in the NMR data, similar to crystal structure 1Y1A. The C-terminal extension adopts a short {alpha}-helical segment from Asp-182—Ser-185 followed by an extended conformation from Ser-186 —Leu-191, which is also similar to the secondary structure observed in 1Y1A. However, the signals for most of the extreme C terminus (Phe-187—Leu-191) displayed greater than average line-broadening, suggesting that these residues might be partially solvent-exposed or in chemical exchange between multiple conformations (supplemental Fig. 2A).

The backbone flexibility of Ca2+-CIB1 was further examined using {1H}-15N NOE experiments. This analysis revealed that almost the entire protein backbone has flexibility consistent with typical well folded globular domains, having average {1H}-15N NOE values near ~0.8 (Fig. 1C). This includes the residues within the short central linker between the N and C domains, suggesting that the two domains might adopt a fixed orientation in solution. The most flexible region of the protein is the loop connecting EF-III and EF-IV, which also has very high temperature factors in the crystallographic data (12, 13). The {1H}-15N NOE values also decrease toward the C terminus, suggesting increased mobility, but unfortunately the signals for the last three residues (I189-L191) could not be analyzed in the {1H}-15N NOE data due to chemical exchange broadening.

Domain Orientation of Ca2+-CIB1 Revealed by Residual Dipolar Couplings—To determine the N and C domain orientation of Ca2+-CIB1 in solution, we measured one-bond backbone 1H,15N RDC and compared the results to the RDC predicted from crystal structures 1XO5 and 1Y1A (Fig. 2, Table 1). Analysis of the entire protein backbone revealed a much better correlation between the NMR data and the RDC predicted from crystal structure 1XO5 [PDB] in comparison to 1Y1A. However, a good correlation was observed when the N and C domains of either 1XO5 or 1Y1A were analyzed separately. This suggests that the poor overall correlation with 1Y1A is due to a difference in domain orientation rather than domain structure and that the domains of Ca2+-CIB1 are oriented side by side in solution, similar to the monomeric crystal form, 1XO5.


View this table:
[in this window]
[in a new window]
  		TABLE 1
Correlation between the residual dipolar couplings determined by solution NMR spectroscopy and those calculated from the x-ray crystal structures of Ca2+-CIB1, 1XO5, and 1Y1A A better agreement is represented by a higher correlation coefficient (R) and lower quality factor (Q).

 


Figure 3
View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 3.
Secondary structure and backbone flexibility of Ca2+-CIB1 bound to {alpha}IIb-L. A, weighted average secondary shifts (WASS) for the Ca2+-CIB1 N, C{alpha}, and C nuclei reveal the secondary structure of the protein when bound to {alpha}IIb-L. Residues for which no backbone amide was observed are indicated by solid circles in the lower part of the panel, and proline residues are indicated by open circles. B, comparison of the {1H}-15N NOE data for Ca2+-CIB1 alone (•) and Ca2+-CIB1 in complex with {alpha}IIb-L ({circ}). Note the high flexibility (small {1H}-15N NOE values) for the C-terminal extension of Ca2+-CIB1 in complex with the {alpha}IIb-L peptide.

 
Omitting the N-terminal extension from the analysis significantly improves the correlation between the NMR data and 1XO5 but not 1Y1A (Table 1). This confirms that the N-terminal extension adopts an {alpha}-helical structure (H0) and orientation similar to 1Y1A rather than the extended conformation observed in 1XO5, which is also consistent with the secondary structure analysis. Omitting the C-terminal extension from the analysis of the C domain improves the correlation with each crystal structure only marginally. However, only three residues were available for RDC analysis in this region (Ser-180, Ser-186, Val-190) due to overlap and low signal intensity, making it difficult to exclude the conformation observed in either of the crystal structures. Considering the aforementioned chemical exchange in this region, it is also possible that the C-terminal extension simply exhibits some conformational flexibility.

NMR Characterization of the Ca2+-CIB1-{alpha}IIb-L Complex— The complex of Ca2+-CIB1 with the {alpha}IIb-L peptide produced NMR spectra of similar quality to the peptide-free protein and, consequently, also required perdeuteration and TROSY to obtain resonance assignments (supplemental Fig. 2B). Most of the residues that did not give rise to HSQC signals in peptide-free Ca2+-CIB1 were also not observed in the presence of the {alpha}IIb-L peptide, indicating that peptide binding does not alter the exchange in these regions of the protein (supplemental Fig. 2C). The chemical shift analysis also revealed that the secondary structure of Ca2+-CIB1 remains intact in complex with {alpha}IIb-L (Fig. 3A), which is typical for EF-hand proteins in complex with their targets (34). Moreover, the {1H}-15N NOE data showed similar backbone flexibility (or lack thereof) for {alpha}-helices H0-H9, their connecting loops, and all four short beta-strands (Fig. 3B). The one distinct difference that is observed upon peptide binding is that the NMR signals for much of the C-terminal extension (Asp-182, Phe-183, Ser-185—Lys-188) become broadened beyond detection, suggesting that this region undergoes conformation exchange on the intermediate NMR timescale. Moreover, the previously exchange-broadened signals for Ile-189, Val-190, and Leu-191 at the extreme C terminus dramatically increase in intensity and display very small or negative {1H}-15N NOE values (-0.3-0.1) characteristic of a high degree of conformational flexibility (Fig. 3). Therefore, these data indicate that {alpha}IIb-L binding significantly increases the flexibility of the C-terminal extension of Ca2+-CIB1.

Identification of the {alpha}IIb-binding Site on Ca2+-CIB1—To further characterize the regions of Ca2+-CIB1 that are affected by {alpha}IIb-L binding, CSP analysis was performed. This analysis demonstrated that {alpha}IIb-L binding has the largest effect on non-polar residues of Ca2+-CIB1, consistent with the predominantly hydrophobic interaction predicted from previous experiments (7, 8, 10). The largest CSP values were generally to residues from the C domain, especially residues on the hydrophobic face of H6, H7, and H8, as well as most of H9 and the C-terminal extension (Fig. 4). However, significant changes were also observed in regions of the N domain, suggesting that both domains interact with the peptide. In the N domain the largest CSP values were within the loop between H0 and H1, Arg-33, and H3a/H3b as well as the C-terminal end of H5.

Mapping the CSP data onto the structure of Ca2+-CIB1 localizes the {alpha}IIb-binding site to the face of each domain that is opposite to the Ca2+ binding loops (functional and non-functional) (Fig. 4, B and C). H5 and residues from H3a/H3b form a small hydrophobic pocket on the surface of the N domain that is solvent-exposed in the absence of peptide. The linker between H0 and H1 interacts with the C-terminal region of H5 in both 1XO5 and 1Y1A and is likely perturbed by the movement of this helix in the complex. The side chain of Arg-33 projects toward this pocket (Fig. 4, B and C) and could be involved in a salt-bridge with the acidic C terminus of {alpha}IIb, as was initially proposed based on homology modeling studies (17). The large upfield C{alpha} and Cbeta shifts for Arg-33 are also consistent with salt bridge formation (Fig. 4A). However, we note that this electrostatic interaction has little effect on the binding affinity since neither the R32A/R33A double mutation nor removal of the acidic {alpha}IIb-L C terminus has a significant effect on the interaction (8, 10).


Figure 4
View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 4.
Identification of the {alpha}IIb-L-binding site on Ca2+-CIB1. A, CSP induced upon {alpha}IIb-L binding to Ca2+-CIB1 plotted as a function of amino acid residue. The CSP for HN and N are shown together as open bars, whereas the CSP for C{alpha} and Cbeta are shown together as solid bars. The secondary structure of Ca2+-CIB1 is shown in the top part of the panel with {alpha}-helices and beta-strands represented by boxes and arrows, respectively, and residues that were unavailable for analysis are represented by solid circles in the lower part of the panel. B, residues with CSP >0.1 are mapped in red onto the backbone structure of Ca2+-CIB1 (1XO5). Ca2+ ions are represented by black spheres, and {alpha}-helices H0-H10 are labeled. C, space-fill representation of B including side chains. For clarity, the C-terminal extension (Ser-180 —Leu-191) of Ca2+-CIB1 is shown in green line representations in both B and C, and Arg-33 is indicated in panels A, B, and C. D, Ribbon representation of the x-ray crystal structure of Ca2+-CnB bound to residues 347-372 of CnA (Protein Data Bank code 1AUI). Ca2+-CnB, CnA, and the four bound Ca2+ ions are shown in ivory, magenta, and black, respectively. The orientation of Ca2+-CnB in panel D is similar to that of Ca2+-CIB1 in panels B and C.

 
The most perturbed regions within the C domain are a hydrophobic cleft formed by EF-III and EF-IV and the C-terminal extension that shields this cleft in the absence of {alpha}IIb-L. Together with the N domain, this cleft forms a continuous hydrophobic channel that is similar to the CnA-binding site of CnB (Fig. 4D). Several of the residues within the C domain portion of this channel have also been shown to be essential for {alpha}IIb binding by mutagenesis (10). In fact, previous studies have suggested that the C domain of Ca2+-CIB1 may be sufficient for {alpha}IIb binding, likely interacting with the hydrophobic N-terminal region of the {alpha}IIb cytoplasmic/transmembrane domain (5, 10, 17). To test this hypothesis, we generated a C domain construct (CIB1-C) consisting of residues 96-191 of CIB1 and an N-terminal His10-tag. Like full-length Ca2+-CIB1 (14), circular dichroism spectra showed that CIB1-C is folded and {alpha}-helical in the presence of Ca2+ (data not shown). However, ITC experiments revealed no significant interaction between Ca2+-CIB1-C and {alpha}IIb-L (Fig. 5A). Subsequent NMR spectra showed that despite maintaining a folded {alpha}-helical structure, the tertiary structure of the isolated C domain is not identical to the C domain structure in the full-length Ca2+-CIB1 protein (Fig. 5B). In addition to confirming the importance of the N domain in {alpha}IIb-binding, this suggests that an interdomain interaction is necessary to maintain the C domain in the correct native conformation.

Defining the Function of the C-terminal Extension—The change in flexibility and large CSP values for the C-terminal extension of Ca2+-CIB1 suggests that it must undergo a large conformational change upon binding of the {alpha}IIb-L peptide. To determine whether this region is directly involved in {alpha}IIb-L binding or is simply displaced from the binding site by the peptide, we generated a deletion mutant of CIB1 having the entire C-terminal extension (Ser-180 —Leu-191) removed (CIB1{Delta}H10). Steady state fluorescence spectroscopy experiments using the hydrophobic probe 8-anilino-1-naphalenesulfonate show that Ca2+-CIB1{Delta}H10 has a significantly larger solvent-exposed hydrophobic surface than full-length Ca2+-CIB1 (Fig. 6A). This is consistent with the hypothesis that removal of the C-terminal extension exposes the hydrophobic channel of the C domain. ITC experiments clearly demonstrate that Ca2+-CIB1{Delta}H10 retains the ability to bind to the {alpha}IIb-L peptide with similar affinity to the wild type protein (Ka ~ 106) (Fig. 6B, Table 2) (8). Therefore, the C terminus of Ca2+-CIB1 must not be directly involved in binding to the peptide but instead must be displaced from the binding site. The more negative change in heat capacity associated with {alpha}IIb-L binding to Ca2+-CIB1{Delta}H10 (-1.6 kJ/mol K) in comparison to full-length Ca2+-CIB1 (-1.1 kJ/mol K) (8) indicates that a larger hydrophobic surface becomes buried in the complex with the truncated protein, which is also consistent with the C terminus displacement model. Additional ITC experiments revealed no interaction between Ca2+-CIB1{Delta}H10 and an acetylated synthetic peptide (H10p) encompassing the truncated portion of the C-terminal extension (Ac-180SPDFASSFKIVL191) of CIB1 (Fig. 6B). This indicates that the C-terminal extension itself has a low intrinsic affinity for the C domain. Together with our NMR data, these results suggest that the C-terminal extension interacts weakly with the C domain and is easily displaced by {alpha}IIb-L.


View this table:
[in this window]
[in a new window]
  		TABLE 2
Thermodynamics of Ca2+-CIB1{Delta}H10 binding to {alpha}IIb-L or smMLCKp, as determined by ITC The values and errors for the 37 °C experiments with {alpha}IIb-L are the average and S.D. of three independent titrations. All other errors are calculated from the curve fitting. Note that there is no error associated with T{Delta}S because it is a calculated value.

 


Figure 5
View larger version (26K):
[in this window]
[in a new window]
  		FIGURE 5.
Structural and {alpha}IIb-L binding properties of CIB1-C. A, top panel, base-line corrected raw ITC titrations of Ca2+-CIB1-C or Ca2+-CIB1 into {alpha}IIb-L at 37 °C. Lower panel, derived binding isotherms for the Ca2+-CIB1-C (•) or Ca2+-CIB1 ({blacktriangleup}) titrations after subtraction of the heat of dilution control experiments. The data for Ca2+-CIB1 were taken Yamniuk and Vogel (8). B, overlay of the 1H,15N HSQC NMR spectra for Ca2+-15N-labeled CIB1 (black) and Ca2+-15N-CIB1-C (red) suggest that the isolated C domain is not completely folded correctly (spectra recorded at 37 °C and 500 MHz). Some C domain peaks for Ca2+-CIB1 are labeled.

 


Figure 6
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 6.
Surface properties and peptide binding to Ca2+-CIB1{Delta}H10. A, steady state fluorescence emission spectra of 8-anilino-1-naphalenesulfonate alone (solid line) in the presence of full-length Ca2+-CIB1 (dashed line) or in the presence of Ca2+-CIB1{Delta}H10 (dotted line) show an increase in hydrophobic surface for the C-terminal-truncated protein. a.u., arbitrary units. B, top panel, base-line-corrected raw ITC data for the titration of Ca2+-CIB1{Delta}H10 into buffer (heat of dilution), peptide H10p (H10p), or peptide {alpha}IIb-L ({alpha}IIb-L) performed at 37 °C. Bottom panel, derived binding isotherms for the H10p (•) and {alpha}IIb-L ({blacksquare}) titrations after subtraction of the HOD experiment. C, top panel, base-line-corrected raw ITC data and (bottom panel) derived binding isotherm for titrations of smMLCKp into Ca2+-CIB1 ({blacktriangleup}) or Ca2+-CIB1{Delta}H10 ({diamondsuit}) performed at 37 °C. The binding to Ca2+-CIB1 was too weak to fit the data to any binding model, whereas the titration data with Ca2+-CIB1{Delta}H10 could be fit to a one-site model with a Ka of ~4 x 104 M-1. Note that there was essentially no heat of dilution for smMLCKp itself (not shown).

 
In our ITC experiments we noted a distinct heat of dissociation for Ca2+-CIB1{Delta}H10 (Fig. 6B, top panel) that was not observed with wild type Ca2+-CIB1. This suggested that removal of the C terminus causes weak self-association of Ca2+-CIB1{Delta}H10 at high concentrations in the ITC syringe but dissociation upon dilution into the ITC cell, which is not surprising considering the increase in hydrophobic surface area for this truncated protein (Fig. 6A). However, this behavior also implied that the function of the C terminus in vivo could be to reduce nonspecific hydrophobic interactions. This could be important since the target sequences of other EF-hand proteins typically have similar properties to the CIB1-binding region of {alpha}IIb, including the propensity to form amphipathic {alpha}-helices with basic and hydrophobic faces. We, therefore, tested the binding of Ca2+-CIB1 and Ca2+-CIB1{Delta}H10 to a synthetic peptide derived from smMLCK (smMLCKp), a well known target sequence of CaM (35). Although full-length Ca2+-CIB1 showed a very weak interaction with smMLCKp with affinity outside the range of the ITC (Ka ~ 103 M-1), Ca2+-CIB1{Delta}H10 yielded a much larger release of heat that could be fit to a one site binding isotherm to yield a Ka of 4 x 104 M-1 (Fig. 6C, Table 2). These results are consistent with the notion that the C-terminal extension of CIB1 is important for increasing target-protein specificity.


Figure 7
View larger version (18K):
[in this window]
[in a new window]
  		FIGURE 7.
Model for the interaction between CIB1 and the {alpha}IIb subunit of platelet integrin {alpha}IIbbeta3. A, in resting platelets the CIB1 binding region of {alpha}IIb is sequestered by beta3 and is partially buried within the cytoplasmic membrane. B, agonist-induced disruption of interactions between the cytoplasmic and transmembrane domains of {alpha}IIb and beta3 exposes the CIB1-binding site of {alpha}IIb. The binding of CIB1 to {alpha}IIb involves the displacement of the C-terminal extension (green).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our NMR spectroscopy studies of Ca2+-CIB1 have shown that the core helix-loop-helix EF-hand structure of the two lobes is similar in solution to the structures observed in each of the recent crystal forms of the protein. However, the N domain and C domain are oriented side by side, similar to the monomeric crystal form 1XO5, consistent with the monomeric state of the protein in solution. The relatively high {1H}-15N NOE values for the central linker are consistent with a folded structure rather than a flexible loop, and these data together with the interdomain interactions suggested from studies with CIB1-C imply that the N and C domains of Ca2+-CIB1 tumble together in solution with a fixed orientation. This contrasts with CaM, which has a flexible central linker ({1H}-15N NOE values of 0.2-0.4) and N and C domains, which tumble independently in solution (36). An interdomain interaction would result in more rapid T2 relaxation for the domains of Ca2+-CIB1 in comparison to the independently tumbling domains of CaM and could explain why perdeuteration was necessary to obtain NMR spectra of suitable quality for resonance assignment. Interdomain interactions are also important to the structure and function of CnB (37) and many NCS proteins, and in fact a single mutation in the domain interface of some NCS proteins can severely alter their activity (38). The close association of the N and C domains is one feature that distinguishes this class of EF-hand proteins from CaM, troponin C, and related proteins, which have independent domains that retain similar structures and ligand binding properties in isolation (39, 40).

Another characteristic that distinguishes CIB1 from CaM is the mechanism of target protein binding. Like CnB and the NCS proteins, the side-by-side domain orientation of the Ca2+-CIB1 domains generates a single binding surface. This differs from the N and C lobes of CaM, which adopt numerous distinct orientations to accommodate different target sequences (41). CaM also lacks N- and C-terminal extensions, and its hydrophobic target protein binding surfaces are exposed to the solvent in the absence of a binding partner, whereas the binding site of CIB1 is partially shielded by the C-terminal extension. The interaction between the C-terminal extension and the C domain of CIB1 is conceivable since downstream of Asp-182 it has hydrophobic/basic character and a similar amino acid content to the CIB1 binding region of {alpha}IIb. Our interpretation is that this shielding mechanism is necessary to prevent self-association and reduce nonspecific binding of CIB1 to the hydrophobic sequences of non-biological targets. However, our data suggest that the C terminus is only weakly coordinated in this hydrophobic channel and can be easily displaced by {alpha}IIb, thereby providing a simple mechanism to increase target specificity. Sequestering the target binding site of CIB1 could be important since it is mostly composed of hydrophobic Leu, Ile, Val, and Phe residues, in contrast to the binding patches of CaM, which are very rich in more polar Met residues (42-44). The hydrophobic face of the EF-hands in many CIB1 homologs also have a similar amino acid content to CIB1 and are either partially or completely covered by their C-terminal extensions (45). However, the primary target binding site of recoverin has been mapped to the N domain (46), and truncation of the C-terminal extension from the NCS protein KChIP1 completely abolishes binding to its biological target (47). Therefore, the C-terminal extensions of different members within this protein family appear to have evolved other unique functions as well.

Our NMR data have shown that the N-terminal extension of Ca2+-CIB1 adopts a structure similar to that observed in 1Y1A, including an {alpha}-helix (H0) from Lys-10 —Asp-18. Therefore, the extended structure of this region in 1XO5 must be an artifact of crystal formation, as was initially hypothesized by Parise and co-workers (13). The position of this H0 helix implies a role in extruding the myristoyl group into solution away from the protein for interaction with the cytoplasmic membrane. The glycine-rich extreme N terminus of Ca2+-CIB1 probably acts as a flexible tether between the myristoyl group and H0, allowing the membrane-anchored protein to adjust its orientation in solution to optimally interact with the {alpha}IIb cytoplasmic/transmembrane domain. The N-terminal region of many CIB1 homologs also have similarly placed {alpha}-helices and flexible N termini that perform a similar function (47, 48). Like CnB (49) and some NCS proteins (50), the association of CIB1 with biological membranes is Ca2+-independent, indicating that the protein does not utilize a Ca2+-myristoyl switch mechanism (51). This suggests the potential for constitutive membrane tethering of CIB1, which would increase the probability of binding to the membrane-anchored {alpha}IIb cytoplasmic/transmembrane sequence. The binding of CIB1 to {alpha}IIb is also Ca2+-independent, since the binding is of similar affinity in vitro in the presence of Ca2+ or physiological Mg2+ concentrations (8). Therefore, regulation of the CIB1-{alpha}IIb interaction is likely achieved in vivo through a dynamic interplay between conformational changes in the {alpha}IIb and beta3 subunits and the competitive binding of other regulatory proteins to the cytoplasmic domains. For example, Johansson and co-workers (52) have suggested that the primary CIB1 binding region of {alpha}IIb may alternate between cytoplasmic and membrane-buried environments depending on the activation state of {alpha}IIbbeta3 (Fig. 7). The binding of CIB1 might also require disrupting interactions between the {alpha}IIb and beta3 cytoplasmic or transmembrane domains themselves (53, 54). Once the binding site becomes accessible, regulation could be fine-tuned through competition between CIB1 and other proteins that have overlapping binding sites (55) or through phosphorylation of the integrin domains or CIB1 itself. We note with interest that the C-terminal extension of CIB1, which becomes exposed upon binding to {alpha}IIb, contains a consensus protein kinase C (PKC) phosphorylation site (3), and PKC has been linked to {alpha}IIbbeta3 signaling (56). The interplay between these different regulatory mechanisms would allow for strict control of the activation state of {alpha}IIbbeta3 and the hemostatic processes that it regulates.


   FOOTNOTES
 
* This research was supported by the Canadian Institutes of Health Research. 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

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2. Back

1 Funded by studentships from Alberta Heritage Foundation for Medical Research and the Natural Sciences and Engineering Research Council of Canada. Back

2 Holds a postdoctoral fellowship from Alberta Heritage Foundation for Medical Research. Back

3 To whom correspondence should be addressed: Structural Biology Research Group, Dept. of Biological Sciences, University of Calgary, 2500 University Dr., N. W. Calgary, AB T2N 1N4, Canada. Tel.: 403-220-6006; Fax: 403-289-9311; E-mail: vogel{at}ucalgary.ca.

4 The abbreviations used are: CIB1, calcium- and integrin-binding protein 1; CnB, calcineurin B; NCS, neuronal calcium sensor; DTT, dithiothreitol; HSQC, heteronuclear single quantum coherence; TROSY, transverse relaxation optimized spectroscopy; CSP, chemical shift perturbation; ITC, isothermal titration calorimetry; RDC, residual dipolar couplings; CaM, calmodulin; smMLCKp, smooth muscle myosin light chain kinase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Deane McIntyre for tireless maintenance and upkeep of the NMR spectrometers and Dr. Karla Krewulak for helpful advice on performing mutagenesis experiments. The NMR and biophysical equipment was purchased with funds provided by Canada Foundation for Innovation and the Alberta Science Research Authority. The Bio-NMR center at the University of Calgary is maintained through funds provided by the Canadian Institutes of Health Research and the University of Calgary.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ni, H., and Freedman, J. (2003) Transfus. Apher. Sci. 28, 257-264
  2. Ginsberg, M. H., Partridge, A., and Shattil, S. J. (2005) Curr. Opin. Cell Biol. 17, 509-516[CrossRef][Medline] [Order article via Infotrieve]
  3. Naik, U. P., Patel, P. M., and Parise, L. V. (1997) J. Biol. Chem. 272, 4651-4654[Abstract/Free Full Text]
  4. Naik, U. P., and Naik, M. U. (2003) Blood 102, 1355-1362[Abstract/Free Full Text]
  5. Tsuboi, S. (2002) J. Biol. Chem. 277, 1919-1923[Abstract/Free Full Text]
  6. Yuan, W., Leisner, T. M., McFadden, A. W., Wang, Z., Larson, M. K., Clark, S., Boudignon-Proudhon, C., Lam, S. C., and Parise, L. V. (2006) J. Cell Biol. 172, 169-175[Abstract/Free Full Text]
  7. Shock, D. D., Naik, U. P., Brittain, J. E., Alahari, S. K., Sondek, J., and Parise, L. V. (1999) Biochem. J. 342, 729-735
  8. Yamniuk, A. P., and Vogel, H. J. (2005) Protein Sci. 14, 1429-1437[Abstract/Free Full Text]
  9. Behan, M. W., and Storey, R. F. (2004) Postgrad. Med. J. 80, 155-164[Abstract/Free Full Text]
  10. Barry, W. T., Boudignon-Proudhon, C., Shock, D. D., McFadden, A., Weiss, J. M., Sondek, J., and Parise, L. V. (2002) J. Biol. Chem. 277, 28877-28883[Abstract/Free Full Text]
  11. Stabler, S. M., Ostrowski, L. L., Janicki, S. M., and Monteiro, M. J. (1999) J. Cell Biol. 145, 1277-1292[Abstract/Free Full Text]
  12. Blamey, C. J., Ceccarelli, C., Naik, U. P., and Bahnson, B. J. (2005) Protein Sci. 14, 1214-1221[Abstract/Free Full Text]
  13. Gentry, H. R., Singer, A. U., Betts, L., Yang, C., Ferrara, J. D., Sondek, J., and Parise, L. V. (2005) J. Biol. Chem. 280, 8407-8415[Abstract/Free Full Text]
  14. Yamniuk, A. P., Nguyen, L. T., Hoang, T. T., and Vogel, H. J. (2004) Biochemistry 43, 2558-2568[CrossRef][Medline] [Order article via Infotrieve]
  15. Weljie, A. M., Yamniuk, A. P., Yoshino, H., Izumi, Y., and Vogel, H. J. (2003) Protein Sci. 12, 228-236[Abstract/Free Full Text]
  16. Sobczak, A., Blazejczyk, M., Piszczek, G., Zhao, G., Kuznicki, J., and Wojda, U. (2005) Acta Biochim. Pol. 52, 469-476[Medline] [Order article via Infotrieve]
  17. Hwang, P. M., and Vogel, H. J. (2000) J. Mol. Recognit. 13, 83-92[CrossRef][Medline] [Order article via Infotrieve]
  18. Gardner, K. H., and Kay, L. E. (1998) Annu. Rev. Biophys. Biomol. Struct. 27, 357-406[CrossRef][Medline] [Order article via Infotrieve]
  19. Shan, X., Gardner, K. H., Muhandiram, D., Rao, N., Arrowsmith, C., and Kay, L. E. (1996) J. Am. Chem. Soc. 118, 6570-6579[CrossRef]
  20. Calvete, J. J., Schafer, W., Henschen, A., and Gonzalez-Rodriquez, J. (1990) FEBS Lett. 263, 43-46[CrossRef][Medline] [Order article via Infotrieve]
  21. Eletsky, A., Kienhofer, A., and Pervushin, K. (2001) J. Biomol. NMR 20, 177-180[CrossRef][Medline] [Order article via Infotrieve]
  22. Salzmann, M., Wider, G., Pervushin, K., Senn, H., and Wuthrich, K. (1999) J. Am. Chem. Soc. 121, 844-848[CrossRef]
  23. Salzmann, M., Pervushin, K., Wider, G., Senn, H., and Wuthrich, K. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13585-13590[Abstract/Free Full Text]
  24. Wishart, D. S., Bigam, C. G., Yao, J., Abildgaard, F., Dyson, H. J., Oldfield, E., Markley, J. L., and Sykes, B. D. (1995) J. Biomol. NMR 6, 135-140[Medline] [Order article via Infotrieve]
  25. Delaglio, F., Grzesiek, S., Vuister, G. W., Zhu, G., Pfeifer, J., and Bax, A. (1995) J. Biomol. NMR 6, 277-293[Medline] [Order article via Infotrieve]
  26. Johnson, B. A., and Blevins, R. A. (1994) J. Biomol. NMR 4, 603-614[CrossRef]
  27. Shan, X., Gardner, K. H., Muhandiram, D. R., Kay, L. E., and Arrowsmith, C. H. (1998) J. Biomol. NMR 11, 307-318[CrossRef][Medline] [Order article via Infotrieve]
  28. Grzesiek, S., Bax, A., Clore, G. M., Gronenborn, A. M., Hu, J. S., Kaufman, J., Palmer, I., Stahl, S. J., and Wingfield, P. T. (1996) Nat. Struct. Biol. 3, 340-345[CrossRef][Medline] [Order article via Infotrieve]
  29. Farrow, N. A., Muhandiram, R., Singer, A. U., Pascal, S. M., Kay, C. M., Gish, G., Shoelson, S. E., Pawson, T., Forman-Kay, J. D., and Kay, L. E. (1994) Biochemistry 33, 5984-6003[CrossRef][Medline] [Order article via Infotrieve]
  30. Cordier, F., Dingley, A. J., and Grzesiek, S. (1999) J. Biomol. NMR 13, 175-180[CrossRef][Medline] [Order article via Infotrieve]
  31. Zweckstetter, M., and Bax, A. (2000) J. Am. Chem. Soc. 122, 3791-3792[CrossRef]
  32. Wishart, D. S., Sykes, B. D., and Richards, F. M. (1992) Biochemistry 31, 1647-1651[CrossRef][Medline] [Order article via Infotrieve]
  33. Wishart, D. S., and Nip, A. M. (1998) Biochem. Cell Biol. 76, 153-163[CrossRef][Medline] [Order article via Infotrieve]
  34. Anglister, J., Ren, H., Klee, C. B., and Bax, A. (1995) FEBS Lett. 375, 108-112[CrossRef][Medline] [Order article via Infotrieve]
  35. Wintrode, P. L., and Privalov, P. L. (1997) J. Mol. Biol. 266, 1050-1062[CrossRef][Medline] [Order article via Infotrieve]
  36. Barbato, G., Ikura, M., Kay, L. E., Pastor, R. W., and Bax, A. (1992) Biochemistry 31, 5269-5278[CrossRef][Medline] [Order article via Infotrieve]
  37. Jiang, G., and Wei, Q. (2003) Biol. Chem. 384, 1299-1303[CrossRef][Medline] [Order article via Infotrieve]
  38. Dizhoor, A. M., Boikov, S. G., and Olshevskaya, E. V. (1998) J. Biol. Chem. 273, 17311-17314[Abstract/Free Full Text]
  39. Drakenberg, T., Forsen, S., Thulin, E., and Vogel, H. J. (1987) J. Biol. Chem. 262, 672-678[Abstract/Free Full Text]
  40. Thulin, E., Andersson, A., Drakenberg, T., Forsen, S., and Vogel, H. J. (1984) Biochemistry 23, 1862-1870[CrossRef][Medline] [Order article via Infotrieve]
  41. Yamniuk, A. P., and Vogel, H. J. (2004) Mol. Biotechnol. 27, 33-57[CrossRef][Medline] [Order article via Infotrieve]
  42. Gellman, S. H. (1991) Biochemistry 30, 6633-6636[CrossRef][Medline] [Order article via Infotrieve]
  43. Walsh, M., and Stevens, F. C. (1978) Biochemistry 17, 3924-3928[CrossRef][Medline] [Order article via Infotrieve]
  44. Yuan, T., Ouyang, H., and Vogel, H. J. (1999) J. Biol. Chem. 274, 8411-8420[Abstract/Free Full Text]
  45. Ikura, M., and Ames, J. B. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 1159-1164[Abstract/Free Full Text]
  46. Tachibanaki, S., Nanda, K., Sasaki, K., Ozaki, K., and Kawamura, S. (2000) J. Biol. Chem. 275, 3313-3319[Abstract/Free Full Text]
  47. Zhou, W., Qian, Y., Kunjilwar, K., Pfaffinger, P. J., and Choe, S. (2004) Neuron 41, 573-586[CrossRef][Medline] [Order article via Infotrieve]
  48. Naoe, Y., Arita, K., Hashimoto, H., Kanazawa, H., Sato, M., and Shimizu, T. (2005) J. Biol. Chem. 280, 32372-32378[Abstract/Free Full Text]
  49. Kennedy, M. T., Brockman, H., and Rusnak, F. (1996) J. Biol. Chem. 271, 26517-26521[Abstract/Free Full Text]
  50. McFerran, B. W., Weiss, J. L., and Burgoyne, R. D. (1999) J. Biol. Chem. 274, 30258-30265[Abstract/Free Full Text]
  51. Blazejczyk, M., Wojda, U., Sobczak, A., Spilker, C., Bernstein, H. G., Gundelfinger, E. D., Kreutz, M. R., and Kuznicki, J. (2005) Biochim. Biophys. Acta 1762, 66-72
  52. Stefansson, A., Armulik, A., Nilsson, I., von Heijne, G., and Johansson, S. (2004) J. Biol. Chem. 279, 21200-21205[Abstract/Free Full Text]
  53. Hughes, P. E., Diaz-Gonzalez, F., Leong, L., Wu, C., McDonald, J. A., Shattil, S. J., and Ginsberg, M. H. (1996) J. Biol. Chem. 271, 6571-6574[Abstract/Free Full Text]
  54. Vinogradova, O., Velyvis, A., Velyviene, A., Hu, B., Haas, T., Plow, E., and Qin, J. (2002) Cell 110, 587-597[CrossRef][Medline]