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Originally published In Press as doi:10.1074/jbc.M313974200 on April 14, 2004

J. Biol. Chem., Vol. 279, Issue 26, 26932-26938, June 25, 2004
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Dimerization of CtIP, a BRCA1- and CtBP-interacting Protein, Is Mediated by an N-terminal Coiled-coil Motif*

Manu J. Dubin{ddagger}§, Philippa H. Stokes{ddagger}§, Eleanor Y. M. Sum||, R. Scott Williams**, Valentina A. Valova{ddagger}{ddagger}, Phillip J. Robinson{ddagger}{ddagger}, Geoffrey J. Lindeman||§§, J. N. Mark Glover**, Jane E. Visvader||§§, and Jacqueline M. Matthews{ddagger}¶¶

From the {ddagger}School of Molecular and Microbial Biosciences, University of Sydney, New South Wales 2006, Australia, the ||Walter and Eliza Hall Institute of Medical Research and the Bone Marrow Research Laboratories, Royal Melbourne Hospital, Parkville, Victoria 3050, Australia, the {ddagger}{ddagger}Children's Medical Research Institute, Westmead, New South Wales 2145, Australia, and the **Department of Biochemistry, University of Alberta, Edmonton, AB T6G 2H7, Canada

Received for publication, December 22, 2003 , and in revised form, April 13, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CtIP is a transcriptional co-regulator that binds a number of proteins involved in cell cycle control and cell development, such as CtBP (C terminus-binding protein), BRCA1 (breast cancer-associated protein-1), and LMO4 (LIM-only protein-4). The only recognizable structural motifs within CtIP are two putative coiled-coil domains located near the N and C termini of the protein. We now show that the N-terminal coiled coil (residues 45–160), but not the C-terminal coiled coil, mediates homodimerization of CtIP in mammalian 293T cells. The N-terminal coiled coil did not facilitate binding to LMO4 and BRCA1 proteins in these cells. A protease-resistant domain (residues 27–168) that minimally encompasses the putative N-terminal coiled coil was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. This region is predicted to contain two smaller coiled-coil regions. The CtIP-(45–160) dimerization domain is helical and dimeric, indicating that the domain does form a coiled coil. The two smaller domains, CtIP-(45–92) and CtIP-(93–160), also formed dimers of lower binding affinity, but with less helical content than the longer peptide. The hydrodynamic radius of CtIP-(45–160) is the same as those of CtIP-(45–92) and CtIP-(93–160), implying that CtIP-(45–160) does not form a single long coiled coil, but a more compact structure involving homodimerization of the two smaller coiled coils, which fold back as a four-helix bundle or other compact structure. These results suggest a specific model for CtIP homodimerization via its N terminus and contribute to an improved understanding of how this protein might assemble other factors required for its role as a transcriptional corepressor.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CtIP (CtBP-interacting protein) is a transcriptional co-regulator that was originally isolated by its ability to bind CtBP (C terminus-binding protein; a co-regulator that binds the C terminus of E1A retrovirus) (1), BRCA1 (breast cancer-associated protein-1) (2, 3), and Rb (retinoblastoma protein) (4). More recently, CtIP has been shown to bind LMO4 and LMO2 (5), LIM-only proteins involved in regulating cell development, and members of the Ikaros family encoding Krüppel-like zinc finger proteins that regulate lymphocyte proliferation and homeostasis (6).

CtIP is a 897-residue nuclear protein that is widely expressed at variable levels (3–5). CtIP interacts with the BRCA1 C terminus (BRCT) repeats of BRCA1 and is likely to bind to the pocket domain of Rb (7) by interacting with Rb through an LECEE motif (4). BRCA1 C-terminus (BRCA1) and Rb are both tumor suppressor proteins, and CtIP is thought to play important roles in regulating their tumor-suppressive functions. BRCA1 is mutated in up to 20% of familial breast cancers (8), whereas Rb is a critical regulator of the cell cycle that is disrupted in up to 50% of all cancers (9). The cellular expression pattern of CtIP mirrors the cell cycle-dependent expression of BRCA1 (10). Mutations that abrogate binding of BRCA1 to CtIP have been shown to result in deregulation of the cell cycle, leading to oncogenesis (2, 3). CtIP can be phosphorylated by ATM kinase at residues 664 and 745 in response to ultraviolet or ionizing radiation (11). This leads to a radiation-induced cell cycle checkpoint between G2 and M phases by allowing the expression of DNA damage-response elements such as GADD45 (12). CtIP phosphorylation by ataxia telangiectasia mutant (ATM) kinase requires BRCA1 (13). Thus, it has been proposed that BRCA1 acts as a scaffold that links ATM to the phosphorylation of CtIP and other non-DNA-associated downstream substrates.

Ikaros family proteins bind to CtBP and repress transcription through recruitment of histone deacetylases (14). Initially, it was thought that the transcriptional regulatory activity of CtIP might result from its interaction with CtBP, which can modify chromatin structure through intrinsic histone deacetylase activity. More recently, Koipally and Georgopoulos (6) have demonstrated that CtIP cannot bind histone deacetylases directly and is thus a deacetylase-independent repressor. Rather, CtIP can interact directly with Ikaros and with the basal transcription protein transcription factor IIB. The ability to form the latter interaction is likely to be responsible for the histone deacetylase-independent corepressor properties of CtIP.

Although several protein-binding sites on CtIP have been identified, little is known about the structure of the protein. Apart from nuclear localization motifs and regions that mediate Rb and CtBP binding, the only recognizable domains are two putative coiled-coil motifs in the N- and C-terminal regions of the protein (see Fig. 1) (4). Coiled coils are the most commonly encountered oligomerization motif in proteins and comprise two to five right-handed amphipathic {alpha}-helices that coil around one another to form a left-handed supercoil (15). These usually stable motifs form the basis of fibrous proteins such as keratin and myosin, but are often found as dimerization domains in DNA-binding proteins. Numerous basic zipper and basic helix-loop-helix/zipper proteins such as GCN4 and c-Jun, respectively, must dimerize to bind DNA and may preferentially form homodimers or heterodimers. These putative coiled coils give CtIP the potential to homo- or heterodimerize or to form higher order oligomers. Here we report that only the N-terminal domain forms a coiled coil that mediates protein homodimerization.



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  		FIG. 1.
Schematic of CtIP. Residues 153–157 (LECEE) have been identified as binding Rb, residues 490–494 (PLDLS) as binding CtBP, residues 45–361 and 720–897 as binding LMO4 (5), and residues 133–379 as binding BRCA1 (residues 133–282 are essential, but not necessarily sufficient for binding (10)). The positions of putative nuclear localization sequences (stars) and coiled coils (CC; light gray boxes) are also shown. Expansions show the results from MULTICOIL sequence analysis. Residues with 0–1% (white boxes), 1–10% (dark gray boxes), and 10–95% (black boxes) probabilities of forming a coiled coil are indicated.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Protein Expression and Purification—Human CtIP constructs were subcloned into the pEF1{alpha}-puro vector carrying an N-terminal FLAG tag, pGEX-2T (Amersham Biosciences), or a modified pIH1119 plasmid (New England Biolabs Inc.). All proteins were expressed in Escherichia coli BL21(DE3) at 25 °C unless otherwise indicated. Maltose-binding protein (MBP)1 and MBP-CtIP-(1–369) were purified on amylose resin (New England Biolabs Inc.) according to the manufacturer's instructions. The proteins were further purified by anion exchange chromatography using a Mono QTM HR10/10 anion exchange column (Amersham Biosciences) in 20 mM Na2HPO4 (pH 8.0) containing 1 mM dithiothreitol at a flow rate of 2 ml/min with a gradient of 0–0.6 M NaCl over 5 column volumes. GST-CtIP proteins were purified on glutathione-Sepharose 4B (Amersham Biosciences) according to the manufacturer's instructions. CtIP peptides were released by treatment with thrombin at 25 °C for 15 h in 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1 mM CaCl2, and 0.05% (v/v) {beta}-mercaptoethanol. The eluted peptides were further purified by reversed-phase HPLC using a 10–90% acetonitrile and 0.1% trifluoroacetic acid gradient.

Immunoprecipitation Assays—Human embryonic kidney 293T cells were transiently transfected with 0.5 µg of each expression construct using FuGENE 6 (Roche Applied Science). Cell extracts were prepared, and proteins were immunoprecipitated and separated as described previously (5). After transfer to polyvinylidene difluoride membranes (Millipore Corp.), filters were blocked and incubated with mouse anti-FLAG monoclonal antibody (Sigma). Filters were then incubated with horseradish peroxidase-coupled secondary antibodies and developed by ECL (Amersham Biosciences).

Protein Concentrations—Protein concentrations were estimated using theoretical extinction coefficients at 280 nm where appropriate. Concentration estimates of CtIP-(45–160), CtIP-(45–92), and CtIP-(93–160), all of which lack tyrosine and tryptophan residues, were initially estimated by gas-phase amino acid analysis (Australian Proteome Analysis Facility, Macquarie University, Sydney, New South Wales, Australia) and Bradford assay (Bio-Rad, Regents Park, New South Wales).

Limited Proteolysis and Peptide Mapping—Trypsin or {alpha}-chymotrypsin protease was added to the target protein solution at a ratio of 1:500 protease:target protein. The reaction was carried out in ice for trypsin or at 37 °C for {alpha}-chymotrypsin. Samples (30 µl) were taken as indicated, quenched by adding SDS-PAGE loading buffer, and analyzed by Tris/Tricine-PAGE (16). In-gel trypsin digestion and analysis was performed as described previously (17).

Far-UV CD Spectropolarimetry—CD spectra were recorded on a Jasco J-720 spectropolarimeter equipped with a Neslab RTE-111 temperature controller. CD data were collected at 20 °C over a wavelength range of 195–260 nm in 0.1-, 1.0-, or 10-mm path length cells with a resolution of 0.5 nm, a bandwidth of 1 nm, and a response time of 1 s. Final spectra were the sum of three scans accumulated at a speed of 20 nm/min and were base line-corrected. Spectra were smoothed using five-point fast Fourier transform filtering (Origin Version 6.0, Microcal). The buffer used in all cases was 20 mM sodium phosphate (pH 7.0), except in the trifluoroethanol (TFE) experiments, for which 10 mM sodium phosphate (pH 7.0) was used.

Analytical Ultracentrifugation—Sedimentation equilibrium and velocity experiments were carried out using a Beckman XLA analytical ultracentrifuge equipped with an An-60ti rotor. Protein samples eluted from a SuperoseTM 12 HR10/30 gel filtration column (Amersham Biosciences) were diluted in gel filtration buffer to give three samples in a 12-mm path length with A230 nm = 0.6, 0.4, and 0.2, respectively, for equilibrium analysis, and one sample with A230 nm = 0.8 for velocity analysis. For equilibrium measurements, data sets were collected at 20 °C at several speeds over a range of 7500–42,000 rpm. Absorbance versus radius scans were acquired in 0.01-mm increments at 230 nm at 3-h intervals until equilibrium was reached. Data were corrected by the use of base lines acquired at 360 nm. Data analysis was carried out using NONLIN software (18) and Sednterp (19). For the sedimentation velocity experiment, data were collected at 10 °C at 58,000 rpm. Absorbance versus radius scans were acquired in 0.03-mm increments at intervals of 240 s. Data analysis was carried out using Sedfit software (20).

Light Scattering—Dynamic light scattering was performed using a DynaPro-MS/X dynamic light scattering/molecular sizing instrument (Protein Solutions). Lyophilized samples were resuspended in 20 mM Na2HPO4 (pH 7.5) and 1 mM dithiothreitol to final concentrations of 360 µM (CtIP-(45–92)), 390 µM (CtIP-(93–160)), and 110 µM (CtIP-(45–160)). rH values were taken from an average of 15 measurements at 25 °C. Data analysis was carried out using Dynamics graphical size analysis software (Protein Solutions).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
A Putative N-terminal Coiled-coil Domain of CtIP Mediates Homodimerization in Vivo—The coiled-coil prediction program MULTICOIL2 (21) was used to confirm the coiled coil-forming potential of residues in CtIP (Fig. 1) (4). When a default window size of 28 residues was used, obvious differences between the potentials for forming coiled coils between the two predicted coiled-coil regions were observed. Although the N-terminal region contains many residues with >90% probability of forming a coiled coil, the maximum probability of forming a coiled coil in the C-terminal leucine zipper was 5.4%. Furthermore, the coiled coil-forming propensities in the putative N-terminal coiled coil showed a significant decrease near residue 92. The first half (Fig. 1, CCA, residues 45–92) had moderate potential to form coiled coils (up to 10% probability), whereas the second half (Fig. 1, CCB, residues 93–160) had high potential to form coiled coils (up to 93% probability), and the halves were separated by a few residues (positions 89–94) with <1% probability of forming a coiled coil. In addition, residues 22–44 showed a very weak probability (<0.5%) of being included in a coiled coil.

To determine whether either of the putative coiled-coil domains in CtIP could dimerize in vivo, co-immunoprecipitation experiments were carried out using extracts from 293T cells transfected with constructs encoding hemagglutinin (HA)-tagged CtIP-(45–897) and a FLAG-tagged version of CtIP-(45–897), CtIP-(45–160), or CtIP-(665–800) (Fig. 2). CtIP-(45–897) is essentially full-length protein, but is expressed at much higher levels than CtIP-(1–897). CtIP-(45–160) comprises the putative N-terminal coiled-coil domain, and CtIP-(665–800) encompasses the putative C-terminal coiled-coil domain. Interactions with HA-tagged full-length CtIP protein were observed only with FLAG-tagged CtIP-(45–897) and CtIP-(45–160), but not with FLAG-tagged CtIP-(665–800). Thus, full-length CtIP can oligomerize in mammalian 293T cells, and oligomerization is mediated by the coiled-coil region in the N terminus of the protein (residues 45–160), but not by a second predicted coiled-coil domain near the C terminus (residues 665–800).



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  		FIG. 2.
CtIP homodimerization is mediated by an N-terminal region in the protein spanning residues 45–160. 293T cells were transfected with expression constructs encoding FLAG (F)-tagged derivatives of CtIP (lanes 1–3) together with that encoding HA-tagged CtIP (residues 45–897). Upper panels, lysates were prepared, and proteins were immunoprecipitated (IP) using anti-FLAG (F) or control isotype-matched (C) monoclonal antibody. After SDS-PAGE electrophoresis, Western blotting (W) was performed using anti-HA antibody. Lower panels, Western blot analysis confirmed appropriate expression levels of all the FLAG-CtIP and HA-CtIP proteins.

 
The interaction domains of CtIP for BRCA1 and LMO4 (2, 5) overlap with CtIP-(45–160). Co-immunoprecipitation experiments with extracts from transfected 293T cells coexpressing CtIP-(45–160) with BRCA1 or LMO4 did not revealed binding (data not shown). These results indicate that CtIP-(45–160) is not sufficient for association with BRCA1 or LMO4.

The N-terminal Region of CtIP Contains a Structured Region—Residues 45–369 of CtIP have previously been identified as mediating binding to LMO4 using a series of truncated CtIP mutants in co-immunoprecipitation experiments (5). This region contains the putative N-terminal coiled-coil domain and also spans the region of CtIP implicated in binding to the BRCT domains of BRCA1 (22). To identify a minimum structured domain within CtIP that mediates homo-oligomerization and/or interactions with other proteins, limited proteolysis of CtIP-(1–369) was performed. A recombinant fusion protein comprising MBP with CtIP-(1–369) at its C terminus was used as the substrate. MBP-CtIP-(1–369) rapidly underwent proteolysis in the presence of either trypsin or chymotrypsin to leave a stable MBP core and other moderately protease-resistant fragments corresponding to molecular masses of 18–22 kDa (Fig. 3A), suggesting that it contained significant amounts of stable structure. Similar experiments using MBP as a control showed that this protein was largely resistant to proteolysis (Fig. 3B). Three bands from a Coomassie Blue-stained gel (Fig. 3A) were excised and subjected to in-gel trypsin digestion (under conditions that lead to complete proteolysis) and MALDI-TOF analysis. A very similar array of peptides, corresponding to the same core region near the N terminus of CtIP (residues 27–142), was identified for all three bands (Fig. 3C). The sizes of the bands observed by SDS-PAGE, the constituent positively identified peptides, and the positioning of potential trypsin and chymotrypsin sites identified a minimum protease-resistant core region consisting of residues 24–168 (Fig. 3D). This corresponds to Band 1 (Fig. 3A), with the N terminus being surmised from a predicted chymotrypsin site near residue 27. Band 1 has a mass of ~18 kDa upon SDS-PAGE, and residues 24–168 have a predicted mass of 17.2 kDa. The termini of the larger trypsin fragments for Bands 2 and 3 could not be unambiguously identified due to the number of potential protease sites and lack of additional identified small peptides. However, all three bands must contain at least residues 27–141.



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  		FIG. 3.
Limited proteolysis of MBP-CtIP constructs with trypsin and {alpha}-chymotrypsin. A, partial chymotrypsin and trypsin digestion of MBP-CtIP-(1–369). Numbered bands were excised and subjected to in-gel trypsin digestion and MALDI-TOF analysis. B, partial chymotrypsin and trypsin digestion of MBP. C, CtIP-derived peptides that were identified from the trypsin digestion of Bands 1–3. The sequences of the peptides, the ranges of residues they span in CtIP, and the samples from which the peptides were identified are indicated. The peptide that was identified both with and without methionine oxidation is marked with an asterisk. Contaminants from MBP, trypsin, and keratin were also identified (not shown). D, residues 1–180 of CtIP. The predicted termini of the major polypeptide from Band 1, derived from digestion with chymotrypsin, are indicated by arrows. Tryptic peptides identified by MALDI-TOF and accounting for 86% coverage of the polypeptide are underlined. The predicted mass of residues 24–168 is 17,608 Da.

 
Design of CtIP Peptides for Biophysical Analysis—Based on the MULTICOIL predictions and our identification of a protease-resistant core in the N-terminal region of CtIP, we designed four CtIP peptides for biophysical characterization. CtIP-(22–160) comprises the entire predicted coiled-coil domain (including low probability residues). CtIP-(45–160) lacks the N-terminal residues with a low probability for forming coiled coils. CtIP-(45–92) comprises the first coiled-coil half of CtIP-(45–160), whereas CtIP-(93–160) contains the second half. These peptides were produced in a recombinant manner as GST fusion proteins. All of the GST fusion proteins tended to be insoluble when expressed at 37 °C but, in general, showed good solubility (~60%) when expressed at 25 °C. An exception was GST-CtIP-(22–160), which was moderately soluble (~30%) when expressed at 25 °C. This protein also caused problems when the peptides were cleaved from the GST fusion using thrombin. The other three proteins showed complete proteolysis (>95%), but proteolysis of GST-CtIP-(22–160) (overnight at 25 °C) reached only ~50% completion. All CtIP peptides were further purified in milligram quantities to >95% homogeneity by reversed-phase HPLC.

The N-terminal Domain of CtIP Forms Dimers with Coiled-coil Characteristics—To determine whether the CtIP peptides formed dimers (or higher order oligomers), they were subjected to size exclusion chromatography, followed by sedimentation equilibrium experiments. All of the peptides eluted from the size exclusion column as single peaks, except for CtIP-(45–92), which showed one major peak and one minor peak at a lower molecular mass. When compared with globular molecular mass standards, all of the proteins eluted at relatively low volumes, indicating the presence of elongated and/or oligomeric peptides. Sedimentation equilibrium data for all peptides gave a reasonable fit to a single species model with molecular masses approximately corresponding to dimers. These experimental estimates were slightly low for the two smaller peptides. CtIP-(45–92) had an apparent molecular mass of 11.3 kDa (with upper and lower limits of 12.2 and 10.5 kDa, respectively), compared with a theoretical dimer mass of 12.0 kDa. CtIP-(93–160) had an apparent molecular mass of 14.6 kDa (with upper and lower limits of 15.4 and 13.7 kDa, respectively), compared with a theoretical dimer mass of 16.7 kDa. Slightly better fits were obtained using monomer-dimer models (Fig. 4, B and C), suggesting that, under the conditions of the experiment, we had small but significant amounts of monomer that could interchange with dimeric species. Based on direct fits to data and estimates of percentages of monomer and dimer present, the dimerization constants of these peptides were estimated to have a lower limit of ~106 M–1. More accurate estimates of dimerization constants require higher levels of monomer in solution. Although this could be achieved by lowering protein concentrations, the low extinction coefficients of the peptides give rise to very poor signal-to-noise ratios.



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  		FIG. 4.
CtIP peptides form dimers. A, sedimentation equilibrium data for a sample set of CtIP-(93–160) at equilibrium with a loading concentration of 12 µM and a monomer model fit (——) to the data ({circ}). B, residuals from that fit (). C, residuals from a monomer-dimer model fit ({triangleup}).

 
A model for a single species dimer produced the best fits to the data obtained for CtIP-(45–160) (indicating that no significant amounts of monomer or higher order oligomers were present), with an estimated dimer molecular mass of 30 kDa (with upper and lower limits of 33 and 28 kDa, respectively). This corresponds well with a theoretical dimer mass of 28.4 kDa. Conditions for accurate determination of a dimerization constant for CtIP-(45–160) could not be established (again because of low extinction coefficients of the peptide), but must be >107 M–1 and likely to be much stronger. All models tried did not fit well to data for CtIP-(22–160). The estimated single species mass of this peptide is 40 kDa (with upper and lower limits of 42 and 37 kDa, respectively), which is larger than the theoretical dimer mass of 33.8 kDa. These data probably result from a tendency of CtIP-(22–160) to dimerize and also to aggregate in a nonspecific manner, a property that is also consistent with poor solubility upon expression and reduced efficiency of proteolysis by thrombin. This peptide was excluded from further detailed analysis.

Far-UV circular dichroism spectropolarimetry was used to determine whether these peptides could form helical structure over a range of protein concentrations. All peptides showed the double minima at 208 and 222 nm typical of {alpha}-helices at all concentrations tested (0.074–300 µM) (Fig. 5, A–E). Based on the formula (23) MRE222 nm(max) = –40,000 x (1–(2.5/n)), where n is the number of residues in the peptide, fully helical CtIP-(45–92), CtIP-(93–160), and CtIP-(45–160) would correspond to MRE222 nm = 38,000, 38,571, and 39,154, respectively. Based on these figures, the two shorter peptides contained ~50% helix over a concentration range of ~2–300 µM. Only at ~0.5 µM peptide, the lowest concentrations for which far-UV CD measurements could be made with confidence for these peptides, the helical contents dropped to slightly <50%. MRE222 nm values for CtIP-(45–160) corresponded to ~100% helix at all peptide concentrations (0.074–74 µM). At the higher concentrations used, all three peptides had an MRE208 nm: MRE222 nm ratio that was <1, which is often cited as a marker of coiled-coil conformation. Notably, this ratio was evident at lower concentrations of CtIP-(45–160) than the two shorter peptides, indicating that the coiled-coil structure is more stable in this peptide. The presence of TFE is known to enhance helical conformation, but to disrupt coiled-coil conformation. The addition of 50% TFE to CtIP-(45–160) at moderate protein concentration (~1 µM) had little effect on helical content, but brought the MRE208 nm:MRE222 nm ratio closer to 1 (from 0.85 in the absence of TFE to 0.96 in the presence of TFE) (Fig. 5D). The presence of 50% TFE increased the helical content of the CtIP-(45–92) and CtIP-(93–160) peptides to ~95 and 88%, respectively (Fig. 5E). When these peptides were subjected to thermal denaturation, CtIP-(45–92) showed a progressive decrease of signal at 222 nm, with no signs of cooperative unfolding. CtIP-(93–160) and CtIP-(45–160) both showed sigmoidal unfolding curves above 28 °C, indicating cooperative unfolding (Fig. 5F). The midpoints of the major unfolding phases were 52.5 ± 0.3 °C for CtIP-(45–160) and 47.3 ± 0.1 °C for CtIP-(93–160). Both peptides may have an additional unfolding phase at lower temperatures that could not be fitted by equations for three-state unfolding.



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  		FIG. 5.
Far-UV circular dichroism analysis of CtIP peptides. A, far-UV CD spectrum of CtIP-(45–92) at 305 µM (——), 23 µM (–––), and 0.46 µM (····); B, far-UV CD spectrum of CtIP-(93–160) at 290 µM (——), 29 µM (–––), and 0.58 µM (····); C, far-UV CD spectrum of CtIP-(45–160) at 74 µM (——), 3.7 µM (-----), 0.37 µM (····), and 0.46 µM (–––); D, CtIP-(45–160) at 1.1 µM (–––) and at 1.1 µM in the presence of 50% TFE (——); E, CtIP-(45–92) at 7.3 µM (–·–) and at 7.3 µM in the presence of 50% TFE (——) and CtIP-(93–160) at 6.4 µM (····) and at 6.4 µM in the presence of 50% TFE (–––); F, temperature denaturation profiles of CtIP-(45–92) at 22 µM ({square}), CtIP-(93–160) at 14 µM ({circ}), and CtIP-(45–160) at 3.4 µM ({blacktriangleup}). The temperature was increased at a rate of 1 °C/min, and the raw CD signal was monitored at 222 nm. Every eighth point is shown for clarity. Note that the scales on the y axes differ and that the x axis in F is temperature. deg, degrees.

 
Models of the CtIP-(45–160) Structure—The sedimentation equilibrium and CD data indicate that CtIP-(45–160) forms a stable homodimeric coiled coil with a very high degree of helicity and that CtIP-(45–92) and CtIP-(93–160) form homodimers with some coiled-coiled characteristics, but of lower stability and helical content than the larger peptide. These data support three simple models for the structure of CtIP-(45–160). First, the longer peptide makes a single long stable parallel coiled coil that would measure ~17 nm along its long axis (Model A) (Fig. 6A). Second, the two halves form two independent parallel coiled coils joined by a flexible hinge region, and stability is enhanced by the chelate effect, i.e. reduced losses of entropy upon the binding of two sites on a single molecule versus two separate molecules will increase the overall stability (Model B) (Fig. 6B). Third, the two halves fold back and interact, possibly to form a four-helix bundle comprising two coiled coils (Model C) (Fig. 6C).



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  		FIG. 6.
Models for the folding topology of CtIP-(45–160). A, Model A is one coiled coil ~17 nm in length. B, Model B comprises two shorter coiled coils linked by a flexible region. C, Model C comprises two coiled coils that interact to form a more compact structure, such as a four-helix bundle. D, shown is the structure of the RAD50 dimerization domain, which forms a novel zinc hook fold (Protein Data Bank code 1L8D [PDB] ) (26). Models were created using MolMol (41) with the structure of the actin-bundling protein cortexillin-I (Protein Data Bank code 1D7M [PDB] ) (30) as the basis structure for a long parallel coiled coil.

 
To help distinguish between these models, dynamic light scattering was used to estimate the hydrodynamic radii (rH) and frictional ratios (f/f0) of CtIP-(45–92) (rH = 2.9 nm, f/f0 = 1.93), CtIP-(93–160) (rH = 3.3 nm, f/f0 = 1.95), and CtIP-(45–160) (rH = 3.2 nm, f/f0 = 1.57). Sedimentation velocity experiments were also used to determine the same values for CtIP-(45–160) (rH = 3.27 nm, f/f0 = 1.62), as well as the experimental sedimentation coefficient (sT,b = 1.6) and the extrapolated sedimentation coefficient at 20 °C in water (s20,w = 2.1).

For a globular protein with a mass of 28.4 kDa, s20,w ~ 3.3. The much lower value determined for CtIP-(45–160) indicates that the protein is elongated. Although experimentally determined values of rH and s20,w can be used to make estimates for axial ratios of molecules, these estimates break down if the shape of the molecules substantially deviates from an ideal sphere (24). This may be due in part to inaccurate estimations of hydration constants for rod-like protein domains such as coiled coils (25). However, we would expect that if CtIP-(45–160) were to form one single extended coiled-coil dimer, it should have an increased rH relative to the shorter peptides. No increase in rH for CtIP-(45–160) was observed, suggesting that this peptide adopts a relatively compact conformation and is unlikely to resemble Model A. It would be expected that a flexible linker (Model B) between the two halves would be susceptible to proteolytic cleavage. There are several potential trypsin and chymotrypsin sites in the linker region that are protected from proteolysis, notably Arg-91 in the center of the hinge. Since CtIP was not sensitive to trypsin at this site (Fig. 3), it is likely not to be solvent-exposed. Therefore, Model B is highly unlikely. CtIP-(45–92) and CtIP-(93–160) have pI values of 8.1 and 4.6, respectively, suggesting that, at physiological pH, complementary charges on the two coiled coils could mediate an interaction as in Model C. Using size exclusion chromatography, we could not detect any interaction between these two peptides (data not shown). However, there are inherent differences in entropy contributions and small differences in sequence (e.g. a primary amine and additional glycine and serine residues at the N terminus of CtIP-(93–160)), which might preclude an intermolecular interaction.

The presence of a CXXC motif (residues 89–92) in the hinge that lies between the sequences of the two shorter peptides raises another possibility. A similar CXXC motif in RAD50 lies at the apex of two hook-shaped antiparallel coiled coils and mediates dimerization through ligation of a single zinc ion to form a "zinc hook" (Fig. 6D) (26). In RAD50, dimerization though the zinc hook is absolutely dependent on the presence of zinc; in the absence of zinc, the RAD50 peptides form monomeric intramolecular antiparallel coiled coils. It is unlikely that CtIP forms a similar structure. Most experiments on CtIP were performed on reversed-phase HPLC-purified protein, which precludes the presence of zinc ions. However, the addition of Zn(II) did not change the far-UV CD spectrum of CtIP-(45–160) and did not affect the hydrodynamic radius (rH = 3.05 nm) or association properties (dimer mass of 29.5 kDa by sedimentation equilibrium) of CtIP-(45–160). Furthermore, both halves (CtIP-(45–92) and CtIP-(93–160)) and the whole dimerization domain (CtIP-(45–160)) formed dimers in the absence of added Zn(II). It seems likely that dimer formation in the shorter peptides is mediated by at least partial formation of parallel coiled coils and that this type of pairing is maintained within the whole domain. Interestingly, it does appear that CtIP-(45–160) can bind Zn(II). The addition of Co(II) to the peptide caused changes in UV-visible spectra that are characteristic of metal binding in zinc finger domains (data not shown). However, rather than leveling out at a 1:1 Co(II):dimer ratio (as might be expected for a zinc hook-like structure), the spectra continued to change, suggesting that metal binding is weak and/or nonspecific. Thus, the CXXC motif in CtIP-(45–160) does not affect dimerization. Overall, the data presented here imply that CtIP-(45–160) assumes the approximate conformation of a four-helix bundle comprising two coiled coils (Model C).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The data presented above show that CtIP forms a homodimer and that dimerization is mediated by an N-terminal coiled-coil domain, encompassed by residues 45–160. The sequences of coiled-coil motifs are characterized by heptad repeats, designated a–g, of which positions a and d are usually hydrophobic. The structures of many designed and naturally occurring coiled coils have been determined, most of which contain fewer than six continuous heptad repeats (for example, the Mad homodimer (27) and the Fos/Jun heterodimer (28)). Notable exceptions are actin-associated proteins such as tropomyosin (29) and the dimerization domain of cortexillin-I (30), which have 40 and 18 continuous heptad repeats, respectively. Dynamic light scattering and sedimentation velocity data suggest that the dimerization domain of CtIP (CtIP-(45–160)) does not form a similarly long extended coiled-coil structure, but folds back upon itself to form a four-helix bundle or other compact topology, such as the heterodimer interface of colicin E3 with its immunity protein (31). That interface is made largely by the association of two antiparallel dimeric coiled coils forming a long stalk corresponding to ~10 heptad repeats. The size of the CtIP dimerization domain would be similar; however, it is expected to form parallel rather than antiparallel coiled coils.

Many long heptad repeat segments taken from proteins predicted to form extended coil coils do not form coiled coils in solution. This led to the hunt for a coiled-coil trigger site, a region that nucleates protein folding within a coiled coil. The consensus sequence XXLEXchXcXccX, where L is leucine, E is glutamate, c is any charged residue, h is any hydrophobic residue, and X is any residue, was identified as a trigger site in cortexillin-1 and was found to be present in a large number of coiled-coil proteins (32). Although segments of CtIP-(45–160) correspond to part of this consensus sequence, no full trigger site can be identified in the peptide sequence. Rather, coiled-coil formation in this domain is likely to be mediated by a number of different stabilizing effects within the domain (33).

Dimerization and oligomerization are very common properties of proteins and make significant contributions to the function of proteins. For example, in prokaryotic systems, many proteins that regulate gene expression are dimeric, reflecting the palindromic nature of their DNA recognition sequences. Events that promote or prevent dimerization (such as post-translational modification and small ligand or DNA binding) can add extra layers of control to processes that are critical to the survival of the organism (for a recent review, see Ref. 34).

In eukaryotic organisms, regulatory processes are much more complex, and control of cellular events appears to rely heavily on synergy between multiple proteins. Many regulatory proteins can make interactions with many different proteins and form multiprotein complexes. The activity of a multiprotein complex depends on the composition of the complex, so, for example, a protein can act as a transcriptional repressor in one complex or a transcriptional activator in another. Dimerization could contribute to the formation of multiprotein complexes in a number of different ways. Many individual interactions (i.e. between protein domains or between protein and DNA) are weak; increasing the number of binding sites by dimerization of one or more members could increase overall binding affinities. Proteins that have the potential to make multiple interactions may use similar or overlapping regions of the polypeptide chain to bind different partners. Two halves of a homodimer could bind alternative partners, altering the specificity or activity of a complex. This may be the case for the LIM-binding protein Ldb1. Homodimerization of Ldb1 by its N-terminal domain allows the protein to bind alternative LIM homeodomain proteins through its C-terminal LIM interaction domains (35). Alternatively, this multiple mode of binding could provide a means for extracting a protein from one complex and introducing it into a second complex that might be required for a successive stage of the cell cycle.

CtIP can form a multimeric complex with BRCA1, LMO4, and Ldb1 (5). It is not yet established whether these are the only members of such a complex; but of the known members, all have the potential to interact with other partners, and three of the four members (BRCA1, Ldb1, and now CtIP) have been shown to homodimerize (36, 37), although BRCA1 preferentially forms a stable heterodimer with BARD1 (38). The potential for making further interactions by this complex is enormous (Fig. 7).



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  		FIG. 7.
Schematic diagram of a complex formed by CtIP, BRCA1, LMO4, and Ldb1. CtIP homodimerizes through the N-terminal coiled coil. BRCA1 can homodimerize through the N-terminal RING domain. Ldb1 homodimerizes via the N-terminal dimerization domain. Other potential interacting partners are indicated. HD, homeodomain; TF, transcription factor; RNApol-II, RNA polymerase II; RLIM, Ring finger LIM domain-binding protein; DEAF, deformed epidermal autoregulatory factor; NMI, n-Myc-interacting protein.

 
The dimerization domain for CtIP lies close to currently identified binding domains for LMO4 (CtIP-(45–371)) and BRCA1 (CtIP-(133–369)) and contains the Rb-binding motif (CtIP-(153–157)). However, CtIP-(45–160) is not sufficient to bind either LMO4 or BRCA1. The dimerization of CtIP appears to be very strong, suggesting that the native form of the protein is a dimer, but the affinity and stoichiometry of these other interactions has yet to be determined. LXCXE motifs have been shown to bind Rb as extended polypeptide chains (39, 40), so it might be expected that Rb binds CtIP with a 2:2 stoichiometry (i.e. two Rb monomers binding a single CtIP dimer). It is not yet clear if the domains of CtIP that interact with LMO4 and/or BRCA1 lie fully outside region 45–160 (in which case, 2:2 stoichiometry would be expected) or if the binding domains overlap the dimerization domain (in which case, 1:2 stoichiometry would be expected). It is also possible that the binding domains for LMO4 and BRCA1 overlap, in which case, a different protein could bind to each half of the CtIP homodimer (i.e. 1:1:2 stoichiometry). The interaction of CtIP with these proteins may involve competitive or cooperative binding, which could moderate the function of CtIP-containing complexes.

The presence of a CXXC motif in CtIP-(45–160) is intriguing. Although it is highly unlikely that this motif contributes to a RAD50-like zinc hook structure within the domain, it may have implications for the further binding of this region to other proteins via a Zn(II) (or other metal) ligand. For example, a second CXXC motif can be found near the second putative coiled-coil domain in CtIP (residues 813–816). An interaction resulting from the ligation of Zn(II) by both CXXC motifs could bring the two termini of CtIP in close proximity. Alternatively, zinc ligation via similar motifs in other proteins could facilitate additional interactions with CtIP.

The identification and investigation of the dimerization domain of CtIP represents the first definition of a structural and functional feature of this protein. Further studies must include the detailed characterization of known protein-binding domains of CtIP, the identification of additional binding partners, and a greater understanding of how the different domains and their interacting residues contribute to the regulation of cellular processes. It will be interesting to see how CtIP dimerization contributes to these properties and to the activities of this corepressor.


   FOOTNOTES
 
* This work was supported in part by grants from the National Health and Medical Research Council of Australia and the Australian Research Council. 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

§ Both authors contributed equally to this work. Back

Supported by Australian Postgraduate Research Awards. Back

§§ Supported by the Victorian Breast Cancer Research Consortium. Back

¶¶ Australian Research Council Research Fellow. To whom correspondence should be addressed: School of Molecular and Microbial Biosciences, Bldg. G08, University of Sydney, NSW 2006, Australia. Tel.: 61-2-9351-6025; Fax: 61-2-9351-4726; E-mail: j.matthews{at}mmb.usyd.edu.au.

1 The abbreviations used are: MBP, maltose-binding protein; GST, glutathione S-transferase; HPLC, high pressure liquid chromatography; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TFE, trifluoroethanol; HA, hemagglutinin; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MRE, mean residue ellipticity. Back

2 Available at multicoil.lcs.mit.edu/cgi-bin/multicoil. Back


   ACKNOWLEDGMENTS
 
We thank Dr. David Gell for assistance with sedimentation velocity experiments.



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 ABSTRACT
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 RESULTS
 DISCUSSION
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