Design, production and characterization of FLIN2 and FLIN4: the engineering of intramolecular ldb1:LMO complexes

Janet E. Deane1, Eleanor Sum2, Joel P. Mackay1, Geoffrey J. Lindeman2, Jane E. Visvader2 and Jacqueline M. Matthews1,3

1 Department of Biochemistry, University of Sydney, Sydney, NSW 2006 and 2 Walter and Eliza Hall Institute of Medical Research, Royal Melbourne Hospital, Melbourne, VIC 3050, Australia


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The nuclear LIM-only (LMO) transcription factors LMO2 and LMO4 play important roles in both normal and leukemic T-cell development. LIM domains are cysteine/histidine-rich domains that contain two structural zinc ions and that function as protein–protein adaptors; members of the LMO family each contain two closely spaced LIM domains. These LMO proteins all bind with high affinity to the nuclear protein LIM domain binding protein 1 (ldb1). The LMO–ldb1 interaction is mediated through the N-terminal LIM domain (LIM1) of LMO proteins and a 38-residue region towards the C-terminus of ldb1 [ldb1(LID)]. Unfortunately, recombinant forms of LMO2 and LMO4 have limited solubility and stability, effectively preventing structural analysis. Therefore, we have designed and constructed a fusion protein in which ldb1(LID) and LIM1 of LMO2 can form an intramolecular complex. The engineered protein, FLIN2 (fusion of the LIM interacting domain of ldb1 and the N-terminal LIM domain of LMO2) has been expressed and purified in milligram quantities. FLIN2 is monomeric, contains significant levels of secondary structure and yields a sharp and well-dispersed one-dimensional 1H NMR spectrum. The analogous LMO4 protein, FLIN4, has almost identical properties. These data suggest that we will be able to obtain high-resolution structural information about the LMO–ldb1 interactions.

Keywords: fusion protein/ldb1/LMO transcription factors


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Three nuclear LIM-only (LMO) transcription factors, LMO1, -2 and -4 play a major role in normal and leukemic T-cell development. LMO1 and LMO2 (also known as rhombotin 1 and 2) were discovered in T-cell acute lymphoblastic leukemia (T-ALL) patients who carried the chromosomal translocations t(11;14)(p15;q11) and t(11;14)(p13;q11), respectively (Boehm et al., 1991Go). Both proteins are expressed in immature T-cells and are thought to prevent further differentiation and maturation (Visvader et al., 1997Go; Grutz et al., 1998Go; Kenny et al., 1998Go). Expression of the LMO1 and LMO2 genes is down-regulated during maturation, but over-expression as a result of the above chromosomal translocations leads directly to T-ALL in children or tumors with similar properties in mice containing an LMO2 transgene (Larson et al., 1995Go). The recently discovered LMO4 is expressed in adult T-cells and is also thought to play a significant role in allowing T-cells to differentiate and reach maturation (Grutz et al., 1998Go; Kenny et al., 1998Go). In addition, the function of LMO proteins is not confined to T-cells; LMO1, -2 and -3 are all involved in neuronal development, while the targeted disruption of LMO2 showed that it is essential for early hematopoiesis (Warren et al., 1994Go; Yamada et al., 1998Go).

All members of the LMO family bind with high affinity to the widely expressed nuclear protein ldb1 (LIM domain binding protein 1; also known as NLI and CLIM2). The LMO–ldb1 interaction is specifically mediated through the N-terminal LIM domain of the LMO proteins (Jurata et al., 1996Go; J.E.Visvader, unpublished data) and a 38-residue region towards the C-terminus of ldb1 known as LID (LIM interaction domain) (Jurata and Gill, 1997Go). The ldb1 can bind to many other nuclear LIM proteins (Agulnick et al., 1996Go; Breen et al., 1998Go), but binding to cytoplasmic LIM proteins has not been reported. The ldb1 protein also contains an N-terminal homodimerization domain, allowing the formation of tetrameric complexes comprising an ldb1 dimer and two separate LIM proteins (Bach et al., 1997Go; Jurata et al., 1998Go). In the case of LIM homeodomain proteins, such a tetrameric complex also has the potential to bind to DNA. There is also some evidence to suggest that a recently isolated Drosophila homolog of the ldb family, Chip, has a role in altering chromatin structure to facilitate remote enhancer–promoter interactions (Morcillo et al., 1997Go). These findings suggest that higher order ldb1–LIM complexes may modulate gene expression by participating in the formation of DNA-binding multi-protein complexes. In particular, it has been proposed that in T-ALL over-expressed LMO1 and -2 displace LMO4 as the binding partner for ldb1 in adult T-cells (Grutz et al., 1998Go; Rabbitts et al., 1999Go).

The sequences of LMO proteins are composed almost entirely of two LIM domains, the primary function of which is believed to be the ability to mediate specific protein–protein interactions (Gill, 1995Go; Dawid et al., 1998Go). All LIM domains conform to the consensus sequence C–X2-C–X16–23–H–X2–C–X2–C–X2–C–X16–21–C–X2–3–(C,H,D), where the eight conserved residues form two sequential zinc-ligating modules. LIM-containing proteins are functionally diverse and are grouped according to sequence similarities (Dawid et al., 1998Go): LMO proteins are combined with LIM homeodomain and LIM kinase subfamilies in Group 1; Group 2 contains the CRP and CRIP subfamilies; and the heterogeneous Group 3 contains most remaining LIM proteins. Three-dimensional structures have been determined for members of Group 2 (Perez-Alvarado et al., 1994Go; Konrat et al., 1997Go, 1998Go; Kontaxis et al., 1998Go; Yao et al., 1999Go) and Group 3 (Velyvis et al., 2001Go), revealing a highly conserved fold. However the observation of differences between the surface features of representatives from the different groups suggests that the mode of interaction of LIM proteins from different classes with their partner proteins may differ markedly (Velyvis et al., 2001Go).

At present little is known about the nature of the interactions between LMO proteins and ldb1. If differences exist between the LMO2–ldb1 and LMO4–ldb1 interactions, there may exist an opportunity to develop a reagent that could specifically inhibit the LMO2–ldb1 interaction in the T-cells of patients with T-ALLs. We have therefore investigated the interaction of LMO proteins with ldb1, with that ultimate aim. While it has been straightforward to produce recombinant forms of ldb1 for study, the production of stable forms of LMO proteins has been problematic. In order to alleviate this problem and facilitate the determination of LMO–ldb1 structures, we have designed, produced and characterized the fusion proteins FLIN2 and FLIN4. These two engineered proteins comprise a LIM domain (from LMO2 and LMO4, respectively), a linker region and ldb1(LID). Both proteins exist as stable monomers and appear to form intramolecular LMO–ldb1(LID) complexes that are amenable to structural studies by NMR.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Molecular biology

The production of full-length cDNA clones corresponding to mouse LMO2 and ldb1 has been described previously (Visvader et al., 1997Go). A mouse LMO4 cDNA clone in pSP72 (Promega) was a gift from Dr K.Hahm. The complete coding sequences of LMO2 and LMO4 were subcloned into the BamH1–EcoR1 and BamH1 sites of pGEX-2T (Pharmacia), respectively, while DNA corresponding to a 40-residue region of ldb1 was cloned into the BamH1–EcoR1 sites of pGEX-2T. The insert encoding FLIN2 was produced using two PCR steps with Pfu-polymerase, template DNA from above and the following oligonucleotide primers: (1) 5'-CGG GAT CCC TGC TGA CAT GTG GTG G; (2) 5'-GCC ACC GGA ACC CAT ATG ACC GCC GCT GCC ACC AAG CCT GA; (3) 5'-GGT GGC AGC GGC GGT CAT ATG GGT TCC GGT GGC GAT GTG ATG GTG GTG GGG GA; and (4) 5'-GGA ATT CTC ACT ATT ACT CGT CGT CAA TGC CGT TGG. In the first step two separate reactions, using oligonucleotide pairs (1)–(3) and (2)–(4), were carried out in order to generate fragments corresponding to LMO2–LIM1 fused to the linker and ldb1(LID) fused the linker region. These overlapping fragments were combined with oligonucleotides (1) and (4) in the second step to generate the full-length fusion construct. The insert encoding FLIN4 was produced in a similar fashion using the oligonucleotide primers (5) 5'-CGG GAT CCC TCT CCT GGA AGC GCT GC and (6) 5'-GCC ACC GGA ACC CAT ATG ACC GCC GCT GCC ACC AGC ACC GCT ATT CCC AAA in place of (1) and (3). These inserts were subcloned into the pGEX-2T vector using BamH1 and EcoR1 restriction sites and then sequenced to confirm their identities.

Protein expression and purification

Overnight cultures from freshly transformed Escherichia coli BL21(DE3) cells were used to inoculate Luria broth (LB) containing 50 µg/ml ampicillin. The cells were grown at 37°C with shaking until an A280 nm of ~0.6 was reached, whereupon the solutions were cooled to 25°C and protein over-expression was induced by the addition of IPTG (0.4 mM). After 4–16 h, cells were harvested by centrifugation (15 min, 5000 g, 4°C) and the resultant cell pellets were either used immediately or frozen and stored at -20°C.

The cell pellets were resuspended in lysis buffer (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.1% ß-mercaptoethanol). Small-scale preparations (10–500 ml LB) were lyzed by sonication. Large-scale preparations (2–6 l LB) were made into a homogeneous suspension using a hand-held homogenizer, then passed five times through a Rannie Mini-lab 8.30H homogenizer at 500 kPa. The resultant cell suspension was centrifuged (15 000 g, 4°C, 20 min) and the soluble fraction was applied to a column containing glutathione-Sepharose 4B (Pharmacia flow rate 1 ml/min) that had been pre-equilibrated in lysis buffer (30 ml). The column was washed with three column volumes of wash buffer (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.5 mM PMSF, 0.1% ß-mercaptoethanol, 10% glycerol, 1 mM ZnSO4), then four column volumes of thrombin cleavage buffer (20 mM Tris–HCl, pH 8.0, 50 mM NaCl, 1 mM CaCl, 0.1% ß-mercaptoethanol). The column was incubated with thrombin (80 U) overnight at room temperature. The cleaved protein was eluted with thrombin buffer containing 5 µM PMSF and 0.1% ß-mercaptoethanol) in four 25 ml fractions that were snap-frozen and either stored at -20°C or further purified immediately.

The samples from GSH affinity chromatography were subjected to anion-exchange chromatography using a Mono Q HR 5/5 column (Amersham Pharmacia Biotech, Castle Hill, NSW, Australia). The column was run at 1 ml/min in 20 mM NaH2PO4 buffer (pH 7.0) containing 1 mM dithiothreitol (DTT). FLIN2 was eluted using a linear gradient of 10–50% NaCl (1 M). Fractions containing purified protein were pooled and stored at 4°C.

Circular dichroism spectropolarimetry (CD)

CD spectra were recorded on a Jasco J-720 spectropolarimeter equipped with a Neslab RTE-111 temperature controller. CD data were collected over the wavelength range 200–260 nm and 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 baseline corrected. The concentration of this sample was determined by amino acid analysis (Australian Proteome Analysis Facility, Macquarie University, Sydney, Australia). Estimates of secondary structural content were made using the CDPro suite of programs (Sreerama, 1999Go) incorporating updated versions of CDsstr (Johnson, 1999Go), SELCON (Sreerama et al., 2000Go) and CONTIN (Provencher and Glockner, 1981Go). Thermal unfolding experiments were controlled with a water-bath equipped with a Peltier system employing a heating rate of 1°C/min.

Sedimentation equilibrium experiments

Sedimentation equilibrium experiments were carried out in an Optima XL-A analytical ultracentrifuge (Beckman Instruments, Palo Alto, CA, USA) using an An-60ti rotor at 25°C. Data were collected at three different speeds (20 000, 25 000 and 35 000 r.p.m.) for three different loading concentrations in a solvent containing 20 mM NaH2PO4 (pH 7.0), 50 mM NaCl and 1 mM DTT. Absorbance versus radius scans (0.001 cm increments) were collected at 3 h intervals until the samples had reached equilibrium, as indicated by an exact overlay of subsequent scans. Data were recorded at 280 nm and baseline corrected using data recorded at 360 nm. Analysis of the data was carried out using NONLIN (Johnson et al., 1981Go). The density of the solvent and the partial specific volume of the protein (adjusted to include the contribution of Zn(II); p = 0.7187 g/ml) were estimated using Sednterp (Hayes et al., 1995Go–2001).

GST pulldown experiment

Both GST and GST-fusion proteins of FLIN2, FLIN4, LMO2–LIM1 and LMO4–LIM1 were immobilized on GSH-Sepharose (Pharmacia). To ~2 µg of each were added 30 µl of the soluble fraction of an ldb1(LID)–MBP preparation in 265 µl of pulldown buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.5% IGEPAL CA-630, 0.1% ß-mercaptoethanol, 0.5 mM PMSF, 10 µM ZnSO4). The samples were incubated with gentle mixing for 1 h at 4°C and the beads were washed five times with 1 ml of PBS. The relative amounts of ldb1(LID)–MBP bound were determined by SDS–PAGE.

NMR spectroscopy

Protein samples were made up in NaH2PO4 (20 mM, pH 7.0) containing NaCl (30–50 mM), DTT (1 mM), D2O (5%, v/v) and 20 µM d4-(trimethylsilyl)propionic acid (d4-TSP). NMR spectra were recorded at 25°C on a Bruker DRX600 NMR spectrometer that was equipped with a triple resonance (HCN) probe and three-axis pulsed-field gradients. One-dimensional (1D) 1H spectra were acquired with 8K complex data points and 64–128 scans over a spectral width of 7200 Hz. The solvent signal was suppressed using a WATERGATE sequence (Piotto et al., 1992Go) immediately prior to data acquisition. The data were processed by zero-filling once and applying a Lorentzian–Gaussian transformation prior to Fourier transformation. Spectra were referenced to d4-TSP at 0.00 p.p.m.


    Results
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 Abstract
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 Results
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 References
 
Ldb1(LID) stabilizes LMO2 and LMO4

In order to produce the milligram quantities of LMO proteins and ldb1 required for structural studies, we first subcloned the DNA encoding the full-length LMO proteins and the ldb1(LID) peptide into pGEX-2T. This vector enables proteins to be over-expressed as glutathione-S-transferase (GST) fusions under the control of an IPTG-inducible promoter. All proteins were expressed in E.coli BL21(DE3) cells, but only GST–ldb1(LID) expressed in the soluble fraction at 37°C. This protein was purified by GST affinity chromatography and thrombin cleavage on GSH-Sepharose, followed by reversed-phase (RP) HPLC. This protocol yielded ldb1(LID) at >95% purity, as judged by SDS–PAGE and electrospray ionization mass spectrometry (ESI-MS). Both GST–LMO2 and GST–LMO4 were largely insoluble when over-expression was induced at 37°C and up to ~30% soluble at 25°C. Levels of soluble protein were increased to ~70% when sarkosyl (1%) and EDTA (5 mM) were added to the cell pellet prior to sonication (EDTA is needed to prevent the precipitation of sarkosyl). However, because the detergent and EDTA might strip the two zinc ions from the LIM portion of the fusion, a refolding step, in which ZnCl2 (1 mM) was added to the wash buffer, was included after binding GST–LMO proteins to GSH-Sepharose. These proteins were relatively stable when bound to the beads, but upon treatment with thrombin a large percentage of the resultant LMO protein remained bound to the GSH-Sepharose beads. This bound protein could only be removed by treatment with high concentrations of denaturants (e.g. 6 M urea) and not by gentler methods such as incubation with glycerol (10%) or non-denaturing detergents.

By incubating ldb1(LID) with the immobilized GST-fusion proteins prior to thrombin treatment, we were able to recover a large percentage of LMO2 or LMO4. Similar treatment with BSA was not successful, suggesting that the cleaved proteins were being eluted as part of an ldb1(LID)–LMO complex (Figure 1Go). We collected sufficient material to confirm the identity of these proteins by ESI-MS, but were not able to purify more than microgram quantities of LMO2 and LMO4 using this method. We surmised that a gradual loss of protein with time resulted from dissociation of the ldb1(LID)–LMO complex and the subsequent aggregation of LMO2 and LMO4.



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Fig. 1. Purification of LMO4 using ldb1(LID). SDS–PAGE showing: lane 1, GST–LMO4 bound to GSH-Sepharose beads; lanes 2–4, elution fractions after incubation with ldb1(LID) and subsequent cleavage with thrombin; lanes 5 and 6, proteins bound to beads after digestion with thrombin and elution of free protein (digestion for 1.5 h and overnight, respectively); lane 7, elution fractions after incubation with BSA and subsequent cleavage with thrombin; lanes 8 and 9, proteins bound to beads after digestion with thrombin and elution of free protein (digestion for 1.5 h and overnight).

 
In order to determine whether the isolated LIM1 domains of LMO2 and LMO4 (which are sufficient to interact with ldb1) would be more amenable to purification, we made several LIM1 constructs as either GST fusions or His-tagged proteins. In addition, we made cysteine-to-serine mutations at non-conserved cysteine residues in LMO4. Although the cysteine-to-serine mutants had marginally higher solubility, none of the proteins could be purified in yields that were sufficient for biophysical analysis. We concluded that LMO2 and LMO4 have limited stability in isolation, but can be stabilized by the formation of ldb1(LID)–LMO complexes. However, the strength of those interactions appeared not to be sufficiently high to permit large-scale purification of ldb1(LID):LMO complexes.

Design of FLIN2

In order to increase the binding affinity of an LMO2–ldb1(LID) complex that would enable us to purify LMO2, we designed a fusion protein comprising the LIM1 domain of LMO2 and ldb1(LID) joined by a flexible linker (Figure 2A and BGo). The LMO2 portion of the fusion protein was chosen such that it spanned residues 26–87 of mouse LMO2, including four residues on either side of the consensus LIM domain sequence. From ldb1, residues 300–339 of mouse ldb1 were used; this 40-residue sequence includes the 38-residue minimal LIM-binding domain (Jurata and Gill, 1997Go). Because the orientation of the two halves of the complex is unknown, the length of the linker region was chosen such that its extended conformation could span the breadth of a LIM domain. We determined, using the PDB coordinates for CRP1 (Yao et al., 1999Go) and the `build' function in Swiss-3D (available from http://expasy.proteome.org.au/sw3d/index.html), that an 11-residue linker should be satisfactory for this purpose. Glycine residues were included in the linker for flexibility and serine residues were added to improve solubility. A variety of codons were used for the repeated G and S residues to ensure that the primers would anneal to unique sites during PCR. Because the production of the insert involved multiple PCR steps, it was possible that one or more mutations could be generated and an NdeI restriction site was designed into the linker region to allow the splicing of DNA fragments if required. This introduced two non-G/S residues (histidine and methionine) in the center of the linker. The final linker sequence was GGSGGHMGSGG. This protein was named FLIN2 (fusion of the LIM interacting domain of ldb1 and the N-terminal LIM domain of LMO2).




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Fig. 2. Origins and sequence of FLIN2. (A) Schematic showing the regions of LMO2 and ldb1 that were used in the design of FLIN2. (B) Sequence of FLIN2. Gly 1 is derived from the thrombin cleavage site in pGEX-2T, the region corresponding to LMO2–LIM1 (residues 26–87 of LMO2) is underlined, the linker region is shown in italics and the remaining residues correspond to ldb1(LID) (residues 300–339). (C) Sequence of FLIN4. The region corresponding to LMO4–LIM1 (residues 16–86 of LMO4) is underlined, the linker region is shown in italics and remaining residues correspond to ldb1(LID). In both cases consensus zinc-ligating residues are shown in bold and the numbering shown corresponds to the FLIN proteins.

 
GST-FLIN2 was over-expressed and purified by affinity chromatography on GSH-Sepharose beads and then treated with thrombin and eluted to yield >80% pure protein (Figure 3AGo). The protein was further purified (>95% purity) using anion-exchange chromatography where a main peak eluted at ~250 mM NaCl (Figure 3BGo). A sample of this peak was subjected to RP-HPLC followed by ESI-MS giving a mass corresponding to that predicted for FLIN2 (MWtheor = 12 589.0 Da, MWexp = 12 588.6±0.5 Da). A minor peak that eluted at slightly higher salt concentrations contained protein corresponding to the same mass and was thought to be misfolded and/or oxidized FLIN2. To prevent the possibility of misfolding due to oxidation of a non-zinc-ligating cysteine residue, we introduced a cysteine-to-serine mutation at position 38 of the fusion protein (FLIN2_C38S). However, no significant improvement was seen in the anion-exchange profile of the mutant protein.



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Fig. 3. Over-expression and purification of FLIN2. (A) SDS–PAGE showing: lane 1, insoluble fraction; lane 2, soluble fraction; lane 3, flow-through after loading on to GSH-Sepharose beads; lane 4, GST–FLIN2 bound to GSH beads; lane 5, beads after thrombin cleavage and elution of free protein; lanes 6–9, post-thrombin cleavage elution fractions; lanes 10–12, FLIN2 after purification by anion-exchange chromatography; lane 13, GST. (B) Purification of FLIN2 by anion-exchange chromatography. The column was run in buffer at pH 7.0 containing NaH2PO4 (20 mM), DTT (1 mM) and eluted in the same buffer containing 1 M NaCl (buffer B). The chromatogram shows absorbance at 280 nm (solid line, left ordinate) and the salt gradient used to elute the protein (dashed line, right ordinate).

 
Preliminary characterization of FLIN2

To determine whether or not FLIN2 was folded and to estimate the proportions of different secondary structural elements present, both FLIN2 and FLIN2_C38S were subjected to far-ultraviolet (UV) circular dichroism spectropolarimetry (CD). The spectra for both proteins (Figure 4AGo) are essentially identical and contain significant levels of secondary structure. Estimations of secondary structure, using the CDPro suite of programs (Sreerama, 1999Go) were also identical for both proteins and fell into the following ranges: helix 21–27%, ß-structure 30–44% and random coil 40–50%. Preliminary thermal denaturation experiments were carried out on FLIN2 and FLIN2_C38S to investigate the relative thermal stabilities of the proteins and to gauge whether unfolding transitions could indicate anything about the relative affinities of LMO proteins for ldb1. Although the proteins appeared to undergo a major unfolding transition with a TM of ~65°C (Figure 4BGo), the unfolding was not reversible, as judged by significant changes in spectra recorded before and after the temperature scans. Temperature scans on more concentrated samples displayed additional transitions at much lower temperatures (30–40°C) that are accompanied by protein precipitation.



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Fig. 4. Far-UV CD spectra of FLIN2 and FLIN2_C38S. (A) Spectra of FLIN2 ({square}) and FLIN2_C38S (•) at 25°C are the average of three scans and are baseline corrected. (B) Temperature scans of FLIN2 ({square}) and FLIN2_C38S (•) monitored at 200 nm. The heating rate was 1°C/min. In all cases peptides (10 µM) were buffered in a solution containing NaCl (150 mM) and NaH2PO4 (20 mM), DTT (1 mM) at pH 7.0.

 
Sedimentation equilibrium experiments were used to determine the solution properties of FLIN2. Nine data sets were collected over three speeds and three different loading concentrations of FLIN2 and these data were fitted simultaneously to a single species model (Figure 5Go). The small absolute values and random scatter of residuals to the curve fit demonstrate that this model is a good fit to the data. The estimated solution molecular mass was 13 000 Da (with 95% confidence intervals of 12 500 and 13 500 Da), which agrees well with the theoretical mass of the protein (12 719 Da including two zinc ions) and shows that FLIN2 is monomeric in solution.



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Fig. 5. Main panel: a representative dataset from the sedimentation equilibrium analysis of FLIN2 (50 mM NaCl, 20 mM NaH2PO4, pH 7.0, 1 mM DTT), shown as a plot of A280 nm against radial position. A total of nine datasets were recorded and fitted globally. A fit of the data to a single species model is shown (solid line). Inset: residuals from the fit of this dataset are shown. The small size and random scatter of the residual plot indicate a good fit to the single species model.

 
Finally, we recorded a 1D 1H NMR spectrum of FLIN 2 (Figure 6AGo). The peaks within the spectra are sharp and well dispersed, confirming that the protein is folded and not undergoing any appreciable aggregation.



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Fig. 6. 1D 1H NMR spectra of FLIN proteins. (A) FLIN2 and (B) FLIN4. Proteins (~300 µM) were buffered in a solution containing NaCl (30 mM), NaH2PO4 (20 mM), DTT (1 mM), D2O (5% v/v) and d4-TSP (20 µM). Spectra were collected at 25°C.

 
FLIN4 resembles FLIN2

The analogous LMO4 fusion protein, FLIN4, was also constructed, using residues 16–86 of mouse LMO4 (Figure 2CGo). This sequence includes the seven residues before the first consensus cysteine of LIM1 (and thereby incorporates a natural GS sequence at the thrombin site of the GST linker) and 10 residues beyond the consensus aspartate (the last LIM consensus residue). This construct also contains cysteine-to-serine mutations at position 37 and 49 of FLIN4 (Figure 2CGo). ESI-MS gave a mass corresponding to that predicted for FLIN4 (MWtheor = 13 004.4 Da, MWexp = 13 002.7±0.7 Da). The properties of FLIN4 are very similar to those of FLIN2. Far-UV CD spectra indicate that FLIN4 is folded (17–18% helical, 27–41% ß-structure and 40–50% random coil, according to CDstr), while sedimentation equilibrium data show that FLIN4 is monomeric with an estimated solution molecular weight of 13 100 Da (95% confidence limits are 12 400 and 13 800 Da; Mtheor = 13 135 Da including two zinc ions). A 1D 1H NMR spectrum (Figure 6BGo) displays signals that are similarly sharp and well dispersed.

FLIN2 and FLIN4 form intramolecular interactions

Unlike isolated LMO2 and LMO4, both FLIN2 and FLIN4 appear to be folded and well behaved in solution. This suggests that the fusion proteins do form stable intramolecular LMO–ldb1(LID) complexes. In order to investigate further whether it is the formation of a specific complex that allows the LMO proteins to be purified as fusions and not non-specific changes in the properties of the polypeptide (e.g. a change in pI), we performed a GST pulldown experiment. Proteins corresponding to LIM1 of LMO2 and LMO4, and also FLIN2 and FLIN4, were produced as GST fusions and immobilized on GSH-Sepharose, while ldb1(LID) was produced as a maltose-binding protein (MBP) fusion. As can be seen in Figure 7Go, ldb1(LID)–MBP bound to the LIM1 domains of both LMO2 and LMO4, but not to GST alone, FLIN2 or FLIN4. This result indicates that the ldb1(LID)-binding sites of LMO2 and LMO4 in the FLIN proteins are not available for binding to intermolecular ldb1(LID).



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Fig. 7. The binding site for ldb1 is no longer available in FLIN2 and FLIN4. SDS–PAGE analysis shows the relative binding of MBP–ldb1(LID) to immobilized GST fusions. Lane 1, molecular weight standards; lane 2, GST; lane 3, GST–LMO2(LIM1); lane 4, GST–LMO4(LIM1); lane 5, GST–FLIN2; lane 6, GST–FLIN4; lane 7, 1/10 soluble fraction containing MBP–ldb1(LID).

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
In order to develop a stable LMO–ldb1 complex we engineered a protein, FLIN2, comprising LIM1 of LMO2 and the LIM-binding domain of ldb1 fused via a flexible linker region. Whereas both the LMO2 and LMO4 proteins and their isolated LIM1 domains are difficult to purify, FLIN2 and its LMO4 counterpart, FLIN4, are readily produced and purified. FLIN2 and FLIN4 are folded and monomeric and the quality of their NMR spectra suggests that these proteins will enable us to obtain high-resolution structural information.

The rationale for designing this protein came from the observation that, in the presence of ldb1(LID), both LMO2 and LMO4 could be isolated as an LMO–ldb1 complex. These complexes had limited stability that may reflect the strength of the interactions. Thus the complex may dissociate during purification, thereby releasing the unstable isolated LIM domains. The construction of an intramolecular rather than an intermolecular complex has allowed us to increase the effective association constant of the interaction by reducing the translational and rotational entropy losses associated with the binding of two separate molecules. This phenomenon is well known as the chelate effect in inorganic chemistry and can increase association constants by several orders of magnitude (Fersht, 1998Go), although the extent to which this has happened for the LMO–ldb1(LID) interactions cannot currently be estimated.

It is likely that the binding of ldb1 stabilizes the LMO proteins by specifically binding to a hydrophobic patch on the surface of the LMO protein. In the unbound state, this hydrophobic patch could give the LMO proteins a tendency to bind non-specifically to other surfaces. 15N-relaxation studies on a LIM1 domain from CRP2 revealed a flexible hydrophobic core that permits some relative motion between the two zinc-binding modules of the LIM domain. It was proposed that this flexibility might be important in the optimization of the LIM1 interface for interaction with its physiological binding partner (i.e. an induced fit model) and as a potential enthalpic compensation mechanism for entropy loss upon binding (Kontaxis et al., 1998Go). If such a mechanism exists for the LIM1 domains of the nuclear LMO proteins, then binding to ldb1 could additionally stabilize the LIM domain by reducing flexibility and any associated tendencies to expose local hydrophobic regions.

The use of an artificial linker to join interacting proteins or fragments of proteins covalently is well established (Birse et al., 1996Go, 1997Go). A major consideration in the design of such fusion proteins is the length of the artificial linker, which can be important for stoichiometry. A clear example of this has been shown with the development of single variable chain fragments of antibodies. These fusion proteins comprise the VH and VL regions of antibodies, which interact to form minimal antigen-binding domains, separated by a flexible linker. The use of long linkers (12–15 residues) gives rise to monomers, short linkers (3–5 residues) give rise to dimers and directly ligated chains give rise to tetramers (Dolezal et al., 2000Go). Clearly, the two components of an interaction must be correctly oriented for an intramolecular interaction to occur and a short linker may prevent this, leaving the opportunity for multiple intermolecular interactions to form. In the case of the FLIN proteins, the linkers [and any unstructured regions proximal to the linker in either or both LIM1 or ldb1(LID)] are sufficiently long to permit only monomer formation, as shown by sedimentation equilibrium experiments. While proteins are generally tolerant of additional or alternative linkages at termini (e.g. the use of fusion proteins used as recombinant protein expression systems), a different problem might arise if the termini of LIM1 or ldb1(LID) were involved in the interaction interface or if the linker prevented complex formation. This issue cannot be fully addressed until more structural information is available, although the ability of ldb1(LID) to rescue LMO proteins combined with the relative stability of the FLIN proteins strongly suggests that a native-like interaction is taking place in FLIN2 and FLIN4.

In summary, the design and production of FLIN2 and FLIN4 have overcome the problems experienced with insolubility, aggregation and instability of recombinant LMO2 and LMO4. The fusion proteins are folded, monomeric, stable, appear to undergo an intramolecular LMO–ldb1 interaction and are suitable for structural studies. The determination of these structures will provide us with valuable information about the interaction of LIM proteins with ldb1 and should allow the identification of any specific features of the LMO2–ldb1 interaction that could be utilized in the development of a specific LMO2 inhibitor.


    Notes
 
3 To whom correspondence should be addressed Back


    Acknowledgments
 
This project was supported by the Victorian Breast Cancer Research Consortium Australia and grants from the Leo and Jenny Leukemia and Cancer Foundation of Australia and the Australian Research Council (ARC). J.M.M. and J.P.M. are ARC Australian Research Fellows.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
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Received January 29, 2001; revised April 30, 2001; accepted May 14, 2001.





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