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Protein Science (2005), 14:1214-1221. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 The Protein Society
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The crystal structure of calcium- and integrin-binding protein 1: Insights into redox regulated functions

Chad J. Blamey1,2, Christopher Ceccarelli1, Ulhas P. Naik1,2 and Brian J. Bahnson1

1 Department of Chemistry and Biochemistry and 2 Department of Biological Sciences, University of Delaware, Newark, Delaware 19716, USA

Reprint requests to: Ulhas P. Naik, Department of Biological Sciences, University of Delaware, Newark, DE 19716, USA; e-mail: unaik{at}udel.edu; fax: (302) 831-2281; or Brian J. Bahnson, Department of Chemistry and Biochemistry, University of Delaware, Newark, DE 19716, USA; e-mail: bahnson{at}udel.edu; fax: (302) 831-6335.

(RECEIVED December 6, 2004; FINAL REVISION January 28, 2005; ACCEPTED February 1, 2005)


   Abstract
TOP
 Abstract
Introduction
 Results and Discussion
 Materials and methods
 References
 
Calcium- and integrin-binding protein 1 (CIB1) is involved in the process of platelet aggregation by binding the cytoplasmic tail of the {alpha}IIb subunit of the platelet-specific integrin {alpha}Iib{beta}3. Although poorly understood, it is widely believed that CIB1 acts as a global signaling regulator because it is expressed in many tissues that do not express integrin {alpha}Iib{beta}3. We report the structure of human CIB1 to a resolution of 2.3 Å, crystallized as a dimer. The dimer interface includes an extensive hydrophobic patch in a crystal form with 80% solvent content. Although the dimer form of CIB1 may not be physiologically relevant, this intersub-unit surface is likely to be linked to {alpha}IIb binding and to the binding of other signaling partner proteins. The C-terminal domain of CIB1 is structurally similar to other EF-hand proteins such as calmodulin and calcineurin B. Despite structural homology to the C-terminal domain, the N-terminal domain of CIB1 lacks calcium-binding sites. The structure of CIB1 revealed a complex with a molecule of glutathione in the reduced state bond to the N-terminal domain of one of the two subunits poised to interact with the free thiol of C35. Glutathione bound in this fashion suggests CIB1 may be redox regulated. Next to the bound GSH, the orientation of residues C35, H31, and S48 is suggestive of a cysteine-type protein phosphatase active site. The potential enzymatic activity of CIB1 is discussed and suggests a mechanism by which it regulates a wide variety of proteins in cells in addition to platelets.

Keywords: platelets; calmyrin; CIB; CIB1; integrin {alpha}Iib{beta}3; GPIIb/IIIa; glutathione; cysteine phosphatase

Abbreviations: CaM, calmodulin • CIB1, calcium- and integrin-binding protein 1 • CnB, calcineurin B • GSH, glutathione • MAD, multiwavelength anomalous diffraction • NCS, non-crystallographic symmetry • PP1, protein phosphatase-1 • PDB, Protein Data Bank • RMSD, root mean square deviation

Article and publication are at http://www.proteinscience.org/cgi/doi/10.1110/ps.041270805.


   Introduction
TOP
 Abstract
 Introduction
Results and Discussion
 Materials and methods
 References
 
Platelets are anucleated cells that play an important role in hemostasis and thrombosis. In response to vascular injury, circulating platelets adhere to exposed subendothelial matrix proteins, such as collagen and von Willebrand factor, at the site of injury. Adherent platelets become activated and recruit more platelets to form a hemostatic plug. In this plug, platelets are cross-linked through fibrinogen binding to integrin {alpha}Iib{beta}3 (also known as GPIIb/IIIa), the platelet fibrinogen receptor (Weiss et al. 1991). This integrin is in a low affinity state on circulating platelets where it is exclusively expressed. Upon activation by an agonist through inside-out signaling {alpha}Iib{beta}3 is rapidly converted to a high affinity state capable of binding soluble fibrinogen (Naik and Parise 1997; Shattil et al. 1998). This binding triggers an outside-in signaling cascade through integrin {alpha}Iib{beta}3 that initiates a cascade of signaling leading to platelet aggregation (Aplin et al. 1998). While a number of proteins have been shown to interact with the {alpha}Iib{beta}3 cytoplasmic tails (Rojiani et al. 1991; Shattil et al. 1995; Hannigan et al. 1996; Law et al. 1996; Tadokoro et al. 2003), how they regulate bidirectional signaling through the integrin is not well understood.

We previously identified calcium- and integrin-binding protein 1 (CIB1) and showed that it binds to the cytoplasmic tail of the integrin {alpha}IIb subunit in a calcium-dependent manner (Naik et al. 1997; Shock et al. 1999). Although CIB1 has been shown to activate integrin {alpha}Iib{beta}3 (Tsuboi 2002), recent reports indicate that CIB1 may be involved in outside-in signaling (Vallar et al. 1999; Naik and Naik 2003a,b). When platelets are activated by agonists, such as thrombin or collagen, CIB1 translocates to the cytoskeleton in an aggregation-dependent manner (Shock et al. 1999). Once the activated {alpha}Iib{beta}3 binds to the ligand, CIB1 in turn binds to the integrin’s cytoplasmic tail forming a complex (Shock et al. 1999). This interaction between CIB1 and {alpha}Iib{beta}3 is required for platelet spreading on immobilized fibrinogen (Naik and Naik 2003a,b). CIB1, which shares significant homology with calcineurin B (CnB) and calmodulin (CaM), contains four calcium binding EF-hand motifs. Only the two most C-terminal are predicted to be functional, EF-hands 3 and 4 (Naik et al. 1997; Stabler et al. 1999). Like CaM, experimental observations have suggested that CIB1 also changes its conformation considerably with calcium binding (Yamniuk et al. 2004).

Studies have also described CIB1 in multiple tissues that do not express {alpha}Iib{beta}3, suggesting that CIB1 may play a wider regulatory role (Shock et al. 1999; Stabler et al. 1999). Since our original report describing the discovery of CIB1 and its interaction with {alpha}Iib{beta}3 (Naik et al. 1997), it has been shown that CIB1 also binds to a number of other proteins (Wu and Lieber 1997; Kauselmann et al. 1999; Stabler et al. 1999; Haataja et al. 2002; Whitehouse et al. 2002; Ma et al. 2003). Notably among these proteins are focal adhesion kinase, presenilin 2, polo-like kinase 2 and 3, Rac3, and DNA-dependent protein kinase (Wu and Lieber 1997; Kauselmann et al. 1999; Stabler et al. 1999; Haataja et al. 2002; Ma et al. 2003; Naik and Naik 2003a). It is not clear how CIB1 associates, and possibly regulates, proteins with such diverse functions. To gain insight into how CIB1 regulates these signaling proteins, other laboratories have modeled CIB1 based on its known homology to the CaM family of calcium-binding proteins (Hwang and Vogel 2000; Barry et al. 2002). In order to better understand the role of CIB1, we felt it was necessary to understand the molecular structure of this protein. We therefore initiated studies to elucidate the X-ray structure of CIB1.

Here we report the crystal structure of a calcium bound form of human CIB1 to a resolution of 2.3 Å. As expected the structure of CIB1 is homologous to CnB and CaM. The greatest structural similarity is in the C-terminal domain, which contains two EF-hand motifs, like those found in CaM. In addition to presenting the structure of CIB1, we predict the interactions of CIB1 responsible for binding to the {alpha}IIb subunit cytoplasmic tail. The N-terminal domain of our structure also revealed a coordinated molecule of reduced glutathione (GSH) and a putative cysteine-type phosphatase active site. The GSH-binding site resides just outside the putative active site and suggests a redox regulated component to CIB1’s function.


   Results and Discussion
TOP
 Abstract
 Introduction
 Results and Discussion
Materials and methods
 References
 
Overall structure of CIB1
The final model of human {Delta}-1–8–CIB1 is a homodimer solved to a resolution of 2.3 Å (Fig. 1Go). Each subunit contains 183 amino acid residues (residues 9–191 of full-length CIB1) and is made up of mostly {alpha}-helices with a small two stranded {beta}-sheet in the N-terminal domain. A summary of the X-ray data collection and refinement of CIB1 is presented in Tables 1Go and 2Go. An additional surprise of the crystal structure was the presence of a bound molecule of GSH. The C-terminal domain of CIB1 contains two functional EF-hand motifs (EF-hand 3 and 4) bound to calcium as previously predicted (Naik et al. 1997; Stabler et al. 1999). The N-terminal domain EF-hands 1 and 2 vary considerably from functional EF-hands rendering them nonfunctional. Both are missing key aspartic acid residues at 38, 40, and 42 along with 78, 80, and 87 for EF-hand 1 and 2, respectively, plus EF-hand 1 contains an additional loop, residues 43–50.




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  		Figure 1. The structure of Ca2+-CIB1. (A) CIB1 crystallized as a homodimer in the asymmetric unit with chain A (yellow) and chain B (blue) complexed to two Ca2+ atoms (gray) in the EF-hands 3 and 4. The N and C terminus of chain A are labeled in red. Chain B has a bound GSH (green) in the reduced form. The subunits are oriented in a head-to-tail fashion with the N-terminal domain of one chain interacting with the C-terminal domain of the neighboring chain. (B) The subunit interface of chain A is rotated toward the viewer displaying in purple ball and stick a hydrophobic patch of the C-terminal domain on the right (I106, Y110, I114, F115, F117, A184, F187) and the N-terminal domain on the left (F21, F69, I73, V76, F77, V97, F98, P104). Also shown in the N-terminal domain is a patch of negative charged residues in red ball and stick (E89, E90, E93). (C) The electrostatic potential of the subunit interface surface of chain A is displayed in the identical view to B using the program GRASP (Honig and Nicholls 1995). The surface is colored according to convention with red indicating negative charge, blue positive charge, and white net neutral charge. The electrostatic potential surface further highlights the hydrophobic patch of the N- and C-terminal domains, as well as the same negative patch indicated in B of the N-terminal domain surface.

 

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  		Table 1. Crystal and X-ray data of CIB1
 

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  		Table 2. Refinement and model statistics of Ca2+-CIB1
 
Within a monomer, the N- and C-terminal domains are distinct from each other. The space between the domains is occupied by water, making CIB1, at first glance, resemble CaM and CnB. A structurally corrected sequence alignment of the entire CIB1 molecule showed no significant homology to other structures. However, alignments of the C-terminal domain alone did share a perceptible homology with parts of both CaM and CnB shown in Fig. 2AGo. Although, the N-terminal domain shares the protein fold similarity with its C-terminal end with an aligned RMSD of 1.67 Å (Fig. 2BGo), it does not contain calcium-binding sites so its structural similarity with other calcium binding proteins was low.



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  		Figure 2. Structurally corrected sequence alignments for the C-terminal domain of CIB1. Residue numbering on top of the alignment is based on the C-terminal domain of CIB1. Structurally equivalent residues are shown within the boxed regions. Secondary structures are shown beneath the alignments. {alpha}-Helices are depicted in magenta; {beta}-strands in green. Calcium-binding residues are shown in yellow. The sequence identities within structurally equivalent regions are shown at the ends of the alignments. (A) Alignment of C-terminal domain of CIB1 vs. C-terminal domain of CnB (PDB 1AUI [PDB] ) and the N-terminal domain of CaM (PDB 1CLL [PDB] ). The RMSD values for pairwise fits to CIB1 are 1.34 Å for 61 C{alpha} atoms of CnB, and 1.99 Å for 63 C{alpha} atoms of CaM. (B) Alignment of the C-terminal (CIB1-C) vs. the N-terminal (CIB1-N) domains of CIB1. The numbering below the alignment refers to the N-terminal domain. The RMSD for a pairwise fit of the two domains is 1.67 Å for 46 C{alpha} atoms in the alignment.

 
CIB1 crystallizes as a dimer
The oligomeric state of CIB1 under physiological conditions is still an open question. Despite an extremely high solvent content of 80%, which is often linked to limited crystal contacts, chain A and B of CIB1 share an extensive contact surface (Fig. 1Go). Typically such an extensive interface points to a physiologically relevant interacting surface of a protein, thus suggesting a possible dimerization during the physiological lifetime of CIB1. The dimer is formed in a head-to-tail fashion with pseudosymmetric interactions from a hydrophobic patch of both the N and C terminus of CIB1. These hydrophobic surfaces are displayed as ball and stick (Fig. 1BGo) and as an electrostatic potential surface (Fig. 1CGo). CIB1 may form a dimer to prevent aggregation via this extensive hydrophobic surface (Fig. 1BGo) in the absence of a physiological binding partner. Alternatively, the dimerization found in the crystal may be an anomaly linked to the high protein concentration of crystallization trials (1 mM) and the crystal contact forces that exist between molecules. It is expected that interaction of CIB1 with physiological partners, such as the cytoplasmic tail of integrin {alpha}IIb, would be exclusively mediated as a monomer since there is no evidence so far as we are aware that CIB1 acts as a dimer. The connection between the N- and C-terminal domains of one monomer spans the domains in a flexible fashion near the dimer interface. Therefore it is reasonable to consider that one CIB1 subunit could fold back on itself with the hydrophobic surfaces from both the N- and C-terminal domains contributing to a functionally relevant interaction. This conformational rearrangement would be analogous to the current model for CaM binding to its signaling partners. It is noteworthy that at lower concentrations (0.15 mM) and in the absence of a physiological binding partner, CIB1 was shown to have a hydrodynamic radius similar to monomeric proteins of a comparable size (Weljie et al. 2003). Regardless of the functional significance of a CIB1 dimer, the surface of the protein highlighted by this extensive subunit–subunit interface is likely to be critical for CIB1 binding its functionally relevant partners, such as the cytoplasmic tail of {alpha}IIb subunit.

CIB1–{alpha}IIb cytoplasmic tail binding surface
CIB1 has been shown to functionally bind to the cytoplasmic tail of the integrin {alpha}IIb from residue 983–997, which corresponds to the sequence LVLAMWKVGFFKRNR. These {alpha}IIb tail residues are a mix of hydrophobic and positively charged residues. This leads one to look for a complementary surface on CIB1 that is directly involved in {alpha}IIb tail binding. The hydrophobic patch on the C terminus of CIB1(I106, Y110, I114, F115, F117, A184, F187) contains a few of the residues (I114, F115, F117) that were previously shown to bind the {alpha}IIb tail by modeling and mutagenesis studies (Barry et al. 2002). However, the majority of these residues (L131, V132, L135, L152, I153, F173, I177) are in the hydrophobic core of CIB1’s C-terminal domain and are not likely to be directly involved in {alpha}IIb tail recognition. An additional region revealed to be critical for {alpha}IIb tail binding from in vivo experiments with a peptide corresponds to the C-terminal region of CIB1 from residue 179–188 (Tsuboi 2002). Although this region only contains two residues on the hydrophobic surface of CIB1 (A184 and F187, Fig. 1BGo), the C terminus is solvent exposed, adjacent to the hydrophobic region and therefore likely to have important interactions with the {alpha}IIb tail. The hydrophobic surface of CIB1 shown in Figure 1, B and CGo, extends to the N-terminal domain, and therefore may also participate in {alpha}IIb tail binding. In addition to the extension of the hydrophobic patch, the N-terminal domain contains a patch of negative charge (Fig. 1BGo, red residues) on this surface that may associate with positively charged residues of the {alpha}IIb tail.

Although it is difficult to predict precisely how and in what orientation CIB1 binds the {alpha}IIb tail on platelets, the surface of CIB1 discussed above is consistent with the expected functional binding area for {alpha}IIb (Barry et al. 2002). CIB1 has been shown to be N-terminally myristoylated, and therefore membrane anchored (Stabler et al. 1999; Barry et al. 2002). Although our structure of {Delta}-1–8–CIB1 lacks the first eight amino acids, which includes the putative myristoylation site, it does not appear from the position of the N terminus (Fig. 1A,BGo) that this region would interfere with the surface discussed above to interact with the {alpha}IIb tail. We performed intrinsic tryptophan fluorescence studies to show that deletion of these amino acids did not affect the binding of {Delta}-1–8–CIB1 to the {alpha}IIb tail (data not shown). Although our results show that myristoylation and N-terminal residues are not required for {alpha}IIb-peptide binding, it is possible that wild-type protein with a membrane anchor may be needed for CIB1 to bind key residues of {alpha}IIb, which are thought to be buried in the membrane (Armulik et al. 1999; Barry et al. 2002). The demonstrated binding of CIB1 to this putative membrane buried region (Barry et al. 2002) is significant, as it is tied to a recent model of integrin signaling, which has been proposed to be mediated by a conformational tilting of the transmembrane domains of the integrin, thereby leading to the intracellular exposure of what were previously membrane buried residues (Stefansson et al. 2004). Although speculative at this point, CIB1 binding to these residues on the {alpha}IIb tail may be critical in integrin signaling.

The EF-hand 4 in the C-terminal domain of CIB1 may be setup to control target protein binding in a manner analogous to CaM. It has been reported that the greatest structural change occurs when EF-hand 4 becomes occupied, thereby stabilizing significant secondary and tertiary structure (Yamniuk et al. 2004). The residue responsible for calcium specificity at EF-hand 4, E172 (Yamniuk et al. 2004), is adjacent to the hydrophobic core residue F173; this could be the determining signal to setup the hydrophobic-binding surface for its functional interaction. These predictions await the structural determination of a CIB1–target protein complex. It should be noted that crystal screening with a peptide corresponding to the recognition sequence of the {alpha}IIb tail has only yielded small crystals of poor quality, perhaps due to the limited solubility of the CIB1–{alpha}IIb complex.

GSH-binding site and possible CIB1 activities
After placing the complete model of CIB1 protein atoms, a large area of positive electron density on chain B remained. Reviewing the CIB1 purification conditions led us to hypothesize that a molecule of GSH was bound at this site. The free thiol group of GSH faces the free thiol group of C35 and the sulfur atoms are 6 Å apart (Fig. 3AGo). Further scrutiny of the difference electron density map revealed positive electron density between the C35 side chain and ultimately joining the imidazole ring of H31. Initially, this was modeled as cysteine sulfenic acid. In a recent report (Barrett et al. 1999a), a redox regulatory mechanism is described in which an active site cysteine is oxidized to cysteine sulfenic acid and then reduced to the free sulfhydryl active form by GSH. We originally predicted the cysteine sulfenic acid intermediate was trapped, stabilized by the neighboring H31 imidazole. Although cysteine sulfenic acid fits, a cysteine plus a water molecule that interacted with both the cysteine thiol and the H31 imidazole fits better (Fig. 3AGo). The crystals were grown in the presence of 0.25 mM DTT so they are expected to have reduced thiol at C35. However, it should be emphasized that there remains positive and negative Fo-Fc difference in density in and around the GSH-binding site, which suggests there may be an ensemble of minor states contributing to the diffraction pattern. It cannot be ruled out that among this ensemble of minor states there may be different oxidation states of cysteine.



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  		Figure 3. The GSH-binding site of CIB1. (A) The GSH-binding site on the N-terminal domain of chain B is displayed relative to H31, C35, a bound water that sits between these residues, and the thiol of GSH. A hydrogen bond of 2.8 Å bridges the cysteine amide of GSH and the backbone carbonyl of L49 of CIB1. A difference electron density map (2Fo-Fc) is displayed around the bound water and GSH. (B) The alignment of the N-terminal domains of chain A (yellow) and the GSH bound chain B (blue) of CIB1. The binding of GSH causes the loop (residues 39–51) to lose helical character and adopt an alternate conformation. The residue S48, which is adjacent to H31 and C35 in the GSH-free structure (chain A), moves by more than 7 Å with GSH binding (chain B). Note, the view in B is rotated slightly from A.

 
A structural overlay of chains A and B near the GSH-binding site showed a large displacement of residues 39–51 by as much as 16 Å (Fig. 3BGo). The N-terminal helix of the protein is shifted away from the center of the GSH site by 2.3 Å. Here, the most intriguing structural change is a switch from {alpha}-helical character for a four-residue region (V45 to S48) present in chain A, to random coil in chain B upon binding GSH. A noteworthy residue from this loop, S48, is adjacent to residues H31 and C35 in the GSH-free chain A (Fig. 3BGo). Upon GSH binding the loop swings S48 away by 7 Å, suggesting this residue may be functionally important.

On chain A this site resembles several classes of phosphatases that utilize a cysteine nucleophile like the dual specific CDC25 or protein tyrosine phosphatase 1B (PTP 1B) (Wang et al. 2003). CIB1 shares no consensus sequence or protein fold homology with these or any other phosphoryltransfer enzyme structures reported to date. On the basis of these phosphatases, which operate with a cysteine nucleophile, we speculate that functional residues of the CIB1 active site would be made up of at least H31, C35, and S48. Additionally, R32 is adjacent to this putative active site and could be mechanistically useful. Furthermore, several cysteine nucleophile phosphatases have been demonstrated to be redox regulated by GSH; for example, the active site cysteine of PTP 1B is glutathiolated (Barrett et al. 1999b). The structure of CIB1 presented here with bound GSH, is suspiciously setup to serve a redox role, but we cannot rule out its presence as an artifact of purification. However, an initial phosphatase assay using p-nitrophenylphosphate as a substrate did not show evidence of activity, both in the presence and absence of {alpha}IIb tail peptide (data not shown). This could be because p-nitrophenylphosphate is not a physiological substrate. It is plausible that a CIB1 phosphatase activity will only be activated upon binding to its physiological protein substrate. It should be noted that in addition to a seemingly redox sensitivity, CIB1 is already known to be highly regulated by multiple ions (Yamniuk et al. 2004) and binds multiple protein kinases (Wu and Lieber 1997; Kauselmann et al. 1999; Ma et al. 2003; Naik and Naik 2003a).

Of interest is the known involvement of the {alpha}Iib{beta}3 integrin in the regulation of protein-tyrosine phophatases (Ezumi et al. 1995). Recently it was shown that CIB1 and protein phosphatase-1 (PP1) have overlapping binding sites on the {alpha}IIb cytoplasmic tail (Vijayan et al. 2004). Interestingly, PP1 is bound to this region in an unactivated state; in contrast, CIB1 binds only after activation of {alpha}Iib{beta}3 (Vallar et al. 1999; Tsuboi 2002; Naik and Naik 2003b; Vijayan et al. 2004). Therefore, it is an attractive hypothesis that {alpha}IIb acts in part as a location for phosphatases to regulate platelet aggregation. Phosphatases, such as PP1 and calcineurin, require association with a regulatory subunit to form distinct holoenzymes (Vijayan et al. 2004). The {alpha}IIb tail may act in this manner for both CIB1 and PP1. Interestingly, it has been shown that dephosphorylation of T753 of the cytoplasmic tail of integrin {beta}3 is a necessary step during platelet activation (Lerea et al. 1999; Kirk et al. 2000). It is interesting to speculate whether CIB1 serves as a phosphatase of this T753 residue upon its association with the {alpha}Iib{beta}3 integrin.

The presence of bound GSH outside the C35 and H31 site points to an alternate possible enzymatic role of CIB1. The C35 site of CIB1 may function as a single cysteine peroxiredoxin, where the cysteine would sense redox poise through a sulfenic intermediate (Poole et al. 2004). The structure of CIB1 bound GSH has a site that may be regulated by the formation and removal of a possible sulfenic intermediate form of C35. However, this possibility needs further exploration.

Concluding remarks
Since its discovery, CIB1 has been shown to associate with a myriad of proteins in platelets and other cells. Based on its homology to the CaM family of proteins it was believed that CIB1’s role in signaling was limited to regulation through binding its partners. The crystal structure presented here suggests a putative enzymatic function for CIB1. Thus CIB1 may function either as a protein phosphatase or a single cysteine peroxiredoxin in order to regulate diverse functional proteins in a wide variety of cells. Furthermore, the bound GSH structure adds a novel redox regulated dimension to the physiological function of CIB1 so far understood. Study is underway to conclusively determine both the enzymatic function as well as the redox regulation of CIB1.


   Materials and methods
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
References
 
Expression and purification of CIB1
Cloning and expression of full-length CIB1 (NCBI gi: 1848271) has previously been reported (Naik et al. 1997). The CIB1 protein was expressed as glutathione S-transferase fusion protein using BL-21 plus RIL cells (Stratagene). Cells were resuspended in 20 mM Tris at pH 8.0, 500 mM NaCl, 1 mM phenylmethanesulfonyl fluoride, 10 µg/mL aprotinin, 10 µg/mL leupeptin, and 5 mM DTT. The CIB1 protein was purified using GSH-affinity chromatography (Amersham-Pharmacia) followed by column thrombin cleavage of the fusion protein with 25 units/mL of thrombin protease (Enzyme Research Laboratories) at 4°C. The proteolysis was carried out under reducing condition (5 mM DTT), thereby leading to the release of reduced GSH from the GSH–Sepharose resin, which is manufactured as oxidized glutathione. The thrombin protease was removed immediately using a benzamidine TRAP column (Amersham-Pharmacia). Dialysis was performed for 12 h in 5 mM HEPES at pH 7.5, 125 mM NaCl, 5 mM CaCl2, and 0.25 mM DTT. Protein was concentrated to 20 mg/mL, aliquoted, flash-frozen, and stored at –80°C until further use. The protein was purified in reduced conditions in the presence of DTT and degassed. We have observed, and it has also been suggested by other investigators, that a reducing environment is a key requirement for maintaining a native-like conformation of CIB1 (Yamniuk et al. 2004).

Crystallization of CIB1 and {Delta}-1–8–CIB1
Crystallization trials were performed using the hanging-drop vapor diffusion method with the crystal screens 1 and 2 (Hampton Research). Our crystals with full-length CIB1 were found to be unreliable in terms of crystal growth and of poor diffraction quality. Secondary structure prediction using the program JPRED (www.compbio.dundee.ac.uk/~www-jpred/) indicated a lack of secondary structure for the first 8 residues of the N terminus. We therefore truncated the N terminus of CIB1 so that it begins at residue 9. The resulting truncated form of the protein ({Delta}-1–8–CIB1) was purified as described above and used exclusively in the work reported. The best quality crystals of {Delta}-1–8–CIB1 (200 x 100 x 100 µm) were obtained with 20 mg/mL of {Delta}-1–8–CIB1 in 3.0 M formate at pH 7.0, 50 mM HEPES at pH 7.0, 0.3 M NaCl, and 1% dimethylsulfoxide at 4°C. Of note, the presence of 0.25 mM DTT from the final dialysis step in the protein solution seemed to be critical for optimal crystallization. Addition of fresh DTT just before the crystallization trials or in excess of 1 mM during dialysis inhibited nucleation and crystal growth.

Cryo-conditions and introducing holmium for phasing
Crystals were frozen using liquid nitrogen in a cryo-solution made from the above crystallization solution by the addition of 15% glycerol. Holmium is known to replace calcium in EF-hands (Veenstra et al. 1995) and has been successfully used to solve structures by mutiwavelength anomalous diffraction (MAD) phasing (Weis et al. 1991). We soaked crystals in Ca2+-free cryo-solution containing 5 mM HoCl3 for 24 h at 4°C.

MAD phasing crystal structure of CIB1
The crystallization conditions listed above yielded two different crystal forms (tetragonal and hexagonal). Crystal data were collected with a Rigaku RUH3R-Raxis IV system, at Brookhaven NSLS (beamlines x12C, x26, and x25) and at APS beamline BIO-CARS 14-IDB. The data presented here are that of hexagonal crystals and were collected at APS from a single Ca2+–CIB1 crystal for a native data set extending to 2.3 Å resolution and a single Ho3+–CIB1 crystal for derivative data sets extending to 3.1 Å resolution. Data sets to 3.1 Å resolution were collected at the holmium L-absorption edge, at the f'' peak value, and at a high-energy remote wavelength. The program SOLVE (Terwilliger and Berendzen 1999) was used to identify heavy atom sites and to phase the reflections to 3.1 Å. Four holmium sites and two subunits (chains A and B) of CIB1 were found in the asymmetric unit. Density modification starting from phases from the program SOLVE and including noncrystallographic symmetry (NCS) was performed using the program RESOLVE. The results from the program SCALEIT (Howell and Smith 1992; Collaborative Computational Project 1994) suggested that the derivative and native crystal structures were isomorphous. Hence, the Ca2+–CIB1 native amplitudes were combined with the Ho3+–CIB1 phases to 3.1 Å. Density modification in RESOLVE was begun at this resolution, and subsequently extended to the native data diffraction limit of 2.3 Å. RESOLVE was used to build a partial model of Ca2+–CIB1 (Terwilliger 2002) with about 85% of the residues modeled in.

The structure was refined in the program CNS by rigid body refinement, followed by simulated annealing using torsion angle dynamics (Brunger et al. 1998). NCS restraints were initially applied. Programs O (Jones et al. 1991) and XtalView (McRee 1999) were used for model building. EF-hands 3 and 4 were fit with Ca2+ and side chains were adjusted. Alternate steps of model building and refinement led to the complete placement of all protein atoms. During the water placement an unusually large region of positive difference electron density was observed near C35 of chain B and significant deviations from NCS in the N-terminal domain were noted. Residues 9–15 and 39–51 were problematic on the chain B because of the displacement caused by this region of electron density. It was apparent to us during the refinement process that this was most likely GSH considering the materials used for protein purification and crystallization. The density was modeled as GSH, which was supported by refinement. There is no corresponding GSH site near chain A, therefore we reasoned that this caused the large NCS deviations. As a result of these differences, NCS restraints during refinement were turned off. The CNS topology and parameter files for GSH were obtained from XPLO2D (Kleywegt and Jones 1997).

Water molecules were built in during successive cycles of model building, adjustment, and refinement. A final 2Fo-Fc annealed composite omit map for the Ca2+–CIB1 model was used to confirm the validity of the entire final model. The final Rworking and Rfree values were 0.236 and 0.251, respectively. Figures depicting protein structures were rendered using MOLSCRIPT (Kraulis 1991), POVSCRIPT (Fenn et al. 2003), and POVRAY (www.povray.org). Structure stereochemistry and data quality were validated using the programs PROCHECK and SFCHECK, respectively (Collaborative Computational Project 1994). The atomic coordinates and structure factors for the structure of human CIB1 have been deposited in the Protein Data Bank (PDB; http://www.pdb.org/) as entry 1Y1A.


   Acknowledgments
 
This work was supported in part by NIH Grant HL057630 to U.P.N. and NIH COBRE Grant P20RR015583 to U.P.N. and B.J.B. We thank Bryan Berger, Bramie Lenhoff, and Colin Thorpe for their helpful discussions. We acknowledge Howard Robinson (mail-in program) as well as Michael Becker (x25 beamline) for initial data collection at the National Synchrotron Light Source with financial support from the Offices of Biological and Environmental Research and of Basic Energy Sciences of the U.S. Department of Energy, and from the NCRR of the NIH. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, under Contract No. W-31-109-Eng-38. The NIH and NCRR supported use of the BioCARS Sector 14, under grant number RR07707.


   References
TOP
 Abstract
 Introduction
 Results and Discussion
 Materials and methods
 References
 
Aplin, A.E., Howe, A., Alahari, S.K., and Juliano, R.L. 1998. Signal transduction and signal modulation by cell adhesion receptors: The role of integrins, cadherins, immunoglobulin-cell adhesion molecules, and selectins. Pharmacol. Rev. 50: 197–263.[Abstract/Free Full Text]

Armulik, A., Nilsson, I., von Heijne, G., and Johansson, S. 1999. Determination of the border between the transmembrane and cytoplasmic domains of human integrin subunits. J. Biol. Chem. 274: 37030–37034.[Abstract/Free Full Text]

Barrett, W.C., DeGnore, J.P., Keng, Y.F., Zhang, Z.Y., Yim, M.B., and Chock, P.B. 1999a. Roles of superoxide radical anion in signal transduction mediated by reversible regulation of protein-tyrosine phosphatase 1B. J. Biol. Chem. 274: 34543–34546.[Abstract/Free Full Text]

Barrett, W.C., DeGnore, J.P., Konig, S., Fales, H.M., Keng, Y.F., Zhang, Z.Y., Yim, M.B., and Chock, P.B. 1999b. Regulation of PTP1B via glutathionylation of the active site cysteine 215. Biochemistry 38: 6699–6705.[CrossRef][Medline]

Barry, W.T., Boudignon-Proudhon, C., Shock, D.D., McFadden, A., Weiss, J.M., Sondek, J., and Parise, L.V. 2002. Molecular basis of CIB binding to the integrin {alpha}IIb cytoplasmic domain. J. Biol. Chem. 277: 28877–28883.[Abstract/Free Full Text]

Brunger, A.T. 1992. The free R value: A novel statistical quantity for assessing the accuracy of crystal structures. Nature 355: 472–474.[CrossRef][Medline]

Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. 1998. Crystallography & NMR system: A new software suite for macro-molecular structure determination. Acta Crystallogr. D Biol. Crystallogr, 54: 905–921.[CrossRef][Medline]

Collaborative Computational Project, Number 4. 1994. The CCP4 suite: Programs for protein crystallography. Acta Crystallogr. D Biol. Crystallogr. 50: 760–763.[CrossRef][Medline]

Ezumi, Y., Takayama, H., and Okuma, M. 1995. Differential regulation of protein-tyrosine phosphatases by integrin {alpha} IIb {beta} 3 through cytoskeletal reorganization and tyrosine phosphorylation in human platelets. J. Biol. Chem. 270: 11927–11934.[Abstract/Free Full Text]

Fenn, T.D., Ringe, D., and Petsko, G.A. 2003. POVScript+: A program for model and data visualization using persistence of vision ray-tracing. J. Appl. Crystallogr. 36: 944–947.[CrossRef]

Haataja, L., Kaartinen, V., Groffen, J., and Heisterkamp, N. 2002. The small GTPase Rac3 interacts with the integrin-binding protein CIB and promotes integrin {alpha}(IIb){beta} (3)-mediated adhesion and spreading. J. Biol. Chem. 277: 8321–8328.[Abstract/Free Full Text]

Hannigan, G.E., Leung-Hagesteijn, C., Fitz-Gibbon, L., Coppolino, M.G., Radeva, G., Filmus, J., Bell, J.C., and Dedhar, S. 1996. Regulation of cell adhesion and anchorage-dependent growth by a new {beta} 1-integrin-linked protein kinase. Nature 379: 91–96.[CrossRef][Medline]

Honig, B. and Nicholls, A. 1995. Classical electrostatics in biology and chemistry. Science 268: 1144–1149.[Abstract/Free Full Text]

Howell, P.L. and Smith, G.D. 1992. Identification of heavy-atom derivatives by normal probability methods. J. Appl. Cryst. 25: 81–86.

Hwang, P.M. and Vogel, H.J. 2000. Structures of the platelet calcium- and integrin-binding protein and the {alpha}IIb-integrin cytoplasmic domain suggest a mechanism for calcium-regulated recognition; homology modelling and NMR studies. J. Mol. Recognit. 13: 83–92.[CrossRef][Medline]

Jones, T.A., Zou, J.Y., Cowan, S.W., and Kjeldgaard, M. 1991. Improved methods for binding protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47: 110–119.

Kauselmann, G., Weiler, M., Wulff, P., Jessberger, S., Konietzko, U., Scafidi, J., Staubli, U., Bereiter-Hahn, J., Strebhardt, K., and Kuhl, D. 1999. The polo-like protein kinases Fnk and Snk associate with a Ca(2+)- and integrin-binding protein and are regulated dynamically with synaptic plasticity. EMBO J. 18: 5528–5539.[CrossRef][Medline]

Kirk, R.I., Sanderson, M.R., and Lerea, K.M. 2000. Threonine phosphorylation of the {beta} 3 integrin cytoplasmic tail, at a site recognized by PDK1 and Akt/PKB in vitro, regulates Shc binding. J. Biol. Chem. 275: 30901–30906.[Abstract/Free Full Text]

Kleywegt, G.J. and Jones, T.A. 1997. Model building and refinement practice. Methods Enzymol. 277: 208–230.[Medline]

Kraulis, P.J. 1991. MOLSCRIPT: A program to produce both detailed and schematic plots of protein structures. J. Appl. Cryst. 24: 946–950.[CrossRef]

Law, D.A., Nannizzi-Alaimo, L., and Phillips, D.R. 1996. Outside-in integrin signal transduction. {alpha} IIb {beta} 3-(GP IIb IIIa) tyrosine phosphorylation induced by platelet aggregation. J. Biol. Chem. 271: 10811–10815.[Abstract/Free Full Text]

Lerea, K.M., Cordero, K.P., Sakariassen, K.S., Kirk, R.I., and Fried, V.A. 1999. Phosphorylation sites in the integrin {beta}3 cytoplasmic domain in intact platelets. J. Biol. Chem. 274: 1914–1919.[Abstract/Free Full Text]

Ma, S., Liu, M.A., Yuan, Y.L., and Erikson, R.L. 2003. The serum-inducible protein kinase Snk is a G1 phase polo-like kinase that is inhibited by the calcium- and integrin-binding protein CIB. Mol. Cancer Res. 1: 376–384.[Abstract/Free Full Text]

McRee, D.E. 1999. XtalView/Xfit—A versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125: 156–165.[CrossRef][Medline]

Naik, M.U. and Naik, U.P. 2003a. Calcium- and integrin-binding protein regulates focal adhesion kinase activity during platelet spreading on immobilized fibrinogen. Blood 102: 3629–3636.[Abstract/Free Full Text]

Naik, U.P. and Naik, M.U. 2003b. Association of CIB with GPIIb/IIIa during outside-in signaling is required for platelet spreading on fibrinogen. Blood 102: 1355–1362.[Abstract/Free Full Text]

Naik, U.P. and Parise, L.V. 1997. Structure and function of platelet {alpha} IIb {beta} 3. Curr. Opin. Hematol. 4: 317–322.[Medline]

Naik, U.P., Patel, P.M., and Parise, L.V. 1997. Identification of a novel calcium-binding protein that interacts with the integrin {alpha}IIb cytoplasmic domain. J. Biol. Chem. 272: 4651–4654.[Abstract/Free Full Text]

Poole, L.B., Karplus, P.A., and Claiborne, A. 2004. Protein sulfenic acids in redox signaling. Annu. Rev. Pharmacol. Toxicol. 44: 325–347.[CrossRef][Medline]

Rojiani, M.V., Finlay, B.B., Gray, V., and Dedhar, S. 1991. In vitro interaction of a polypeptide homologous to human Ro/SS-A antigen (calreticulin) with a highly conserved amino acid sequence in the cytoplasmic domain of integrin {alpha} subunits. Biochemistry 30: 9859–9866.[CrossRef][Medline]

Shattil, S.J., O’Toole, T., Eigenthaler, M., Thon, V., Williams, M., Babior, B.M., and Ginsberg, M.H. 1995. Beta 3-endonexin, a novel polypeptide that interacts specifically with the cytoplasmic tail of the integrin {beta} 3 subunit. J. Cell Biol. 131: 807–816.[Abstract/Free Full Text]

Shattil, S.J., Kashiwagi, H., and Pampori, N. 1998. Integrin signaling: The platelet paradigm. Blood 91: 2645–2657.[Free Full Text]

Shock, D.D., Naik, U.P., Brittain, J.E., Alahari, S.K., Sondek, J., and Parise, L.V. 1999. Calcium-dependent properties of CIB binding to the integrin {alpha}IIb cytoplasmic domain and translocation to the platelet cytoskeleton. Biochem. J. 342 Pt 3: 729–735.

Stabler, S.M., Ostrowski, L.L., Janicki, S.M., and Monteiro, M.J. 1999. A myristoylated calcium-binding protein that preferentially interacts with the Alzheimer’s disease presenilin 2 protein. J. Cell Biol. 145: 1277–1292.[Abstract/Free Full Text]

Stefansson, A., Armulik, A., Nilsson, I., von Heijne, G., and Johansson, S. 2004. Determination of N- and C-terminal borders of the transmembrane domain of integrin subunits. J. Biol. Chem. 279: 21200–21205.[Abstract/Free Full Text]

Tadokoro, S., Shattil, S.J., Eto, K., Tai, V., Liddington, R.C., de Pereda, J.M., Ginsberg, M.H., and Calderwood, D.A. 2003. Talin binding to integrin {beta} tails: A final common step in integrin activation. Science 302: 103–106.[Abstract/Free Full Text]

Terwilliger, T.C. 2002. Automated main-chain model-building by template-matching and iterative fragment extension. Acta Crystallogr. D Biol. Crystallogr. 59: 34–44.

Terwilliger, T.C. and Berendzen, J. 1999. Automated MAD and MIR structure solution. Acta Crystallogr. D Biol. Crystallogr. 55 (Pt. 4): 849–861.[CrossRef][Medline]

Tsuboi, S. 2002. Calcium integrin-binding protein activates platelet integrin {alpha} IIbbeta 3. J. Biol. Chem. 277: 1919–1923.[Abstract/Free Full Text]

Vallar, L., Melchior, C., Plancon, S., Drobecq, H., Lippens, G., Regnault, V., and Kieffer, N. 1999. Divalent cations differentially regulate integrin {alpha}IIb cytoplasmic tail binding to {beta}3 and to calcium- and integrin-binding protein. J. Biol. Chem. 274: 17257–17266.[Abstract/Free Full Text]

Veenstra, T.D., Gross, M.D., Hunziker, W., and Kumar, R. 1995. Identification of metal-binding sites in rat brain calcium-binding protein. J. Biol. Chem. 270: 30353–30358.[Abstract/Free Full Text]

Vijayan, K.V., Liu, Y., Li, T.T., and Bray, P.F. 2004. Protein phosphatase 1 associates with the integrin {alpha}IIb subunit and regulates signaling. J. Biol. Chem. 279: 33039–33042.[Abstract/Free Full Text]

Wang, W.Q., Sun, J.P., and Zhang, Z.Y. 2003. An overview of the protein tyrosine phosphatase superfamily. Curr. Top. Med. Chem. 3: 739–748.[CrossRef][Medline]

Weis, W.I., Kahn, R., Fourme, R., Drickamer, K., and Hendrickson, W.A. 1991. Structure of the calcium-dependent lectin domain from a rat mannose-binding protein determined by MAD phasing. Science 254: 1608–1615.[Abstract/Free Full Text]

Weiss, H.J., Turitto, V.T., and Baumgartner, H.R. 1991. Further evidence that glycoprotein IIb-IIIa mediates platelet spreading on subendothelium. Thromb. Haemost. 65: 202–205.[Medline]

Weljie, A.M., Yamniuk, A.P., Yoshino, H., Izumi, Y., and Vogel, H.J. 2003. Protein conformational changes studied by diffusion NMR spectroscopy: Application to helix-loop-helix calcium binding proteins. Protein Sci. 12: 228–236.[Abstract/Free Full Text]

Whitehouse, C., Chambers, J., Howe, K., Cobourne, M., Sharpe, P., and Solomon, E. 2002. NBR1 interacts with fasciculation and elongation protein {zeta}-1 (FEZ1) and calcium and integrin binding protein (CIB) and shows developmentally restricted expression in the neural tube. Eur. J. Biochem. 269: 538–545.[Medline]

Wu, X. and Lieber, M.R. 1997. Interaction between DNA-dependent protein kinase and a novel protein, KIP. Mutat. Res. 385: 13–20.[Medline]

Yamniuk, A.P., Nguyen, L.T., Hoang, T.T., and Vogel, H.J. 2004. Metal ion binding properties and conformational states of calcium- and integrin-binding protein. Biochemistry 43: 2558–2568.[CrossRef][Medline]


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