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
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Abstract |
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Keywords: fusion protein/ldb1/LMO transcription factors
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Introduction |
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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 LMOldb1 interaction is specifically mediated through the N-terminal LIM domain of the LMO proteins (Jurata et al., 1996; J.E.Visvader, unpublished data) and a 38-residue region towards the C-terminus of ldb1 known as LID (LIM interaction domain) (Jurata and Gill, 1997
). The ldb1 can bind to many other nuclear LIM proteins (Agulnick et al., 1996
; Breen et al., 1998
), 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., 1997
; Jurata et al., 1998
). 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 enhancerpromoter interactions (Morcillo et al., 1997
). These findings suggest that higher order ldb1LIM 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., 1998
; Rabbitts et al., 1999
).
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 proteinprotein interactions (Gill, 1995; Dawid et al., 1998
). All LIM domains conform to the consensus sequence CX2-CX1623HX2CX2CX2CX1621CX23(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., 1998
): 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., 1994
; Konrat et al., 1997
, 1998
; Kontaxis et al., 1998
; Yao et al., 1999
) and Group 3 (Velyvis et al., 2001
), 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., 2001
).
At present little is known about the nature of the interactions between LMO proteins and ldb1. If differences exist between the LMO2ldb1 and LMO4ldb1 interactions, there may exist an opportunity to develop a reagent that could specifically inhibit the LMO2ldb1 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 LMOldb1 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 LMOldb1(LID) complexes that are amenable to structural studies by NMR.
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Materials and methods |
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The production of full-length cDNA clones corresponding to mouse LMO2 and ldb1 has been described previously (Visvader et al., 1997). 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 BamH1EcoR1 and BamH1 sites of pGEX-2T (Pharmacia), respectively, while DNA corresponding to a 40-residue region of ldb1 was cloned into the BamH1EcoR1 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 LMO2LIM1 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 416 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 TrisHCl, pH 8.0, 150 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 0.1% ß-mercaptoethanol). Small-scale preparations (10500 ml LB) were lyzed by sonication. Large-scale preparations (26 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 TrisHCl, 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 TrisHCl, 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 1050% 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 200260 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, 1999) incorporating updated versions of CDsstr (Johnson, 1999
), SELCON (Sreerama et al., 2000
) and CONTIN (Provencher and Glockner, 1981
). 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., 1981). 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., 1995
2001).
GST pulldown experiment
Both GST and GST-fusion proteins of FLIN2, FLIN4, LMO2LIM1 and LMO4LIM1 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 TrisHCl, 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 SDSPAGE.
NMR spectroscopy
Protein samples were made up in NaH2PO4 (20 mM, pH 7.0) containing NaCl (3050 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 64128 scans over a spectral width of 7200 Hz. The solvent signal was suppressed using a WATERGATE sequence (Piotto et al., 1992) immediately prior to data acquisition. The data were processed by zero-filling once and applying a LorentzianGaussian transformation prior to Fourier transformation. Spectra were referenced to d4-TSP at 0.00 p.p.m.
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Results |
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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 GSTldb1(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 SDSPAGE and electrospray ionization mass spectrometry (ESI-MS). Both GSTLMO2 and GSTLMO4 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 GSTLMO 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 1). 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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Design of FLIN2
In order to increase the binding affinity of an LMO2ldb1(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 B). The LMO2 portion of the fusion protein was chosen such that it spanned residues 2687 of mouse LMO2, including four residues on either side of the consensus LIM domain sequence. From ldb1, residues 300339 of mouse ldb1 were used; this 40-residue sequence includes the 38-residue minimal LIM-binding domain (Jurata and Gill, 1997
). 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., 1999
) 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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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 4A) are essentially identical and contain significant levels of secondary structure. Estimations of secondary structure, using the CDPro suite of programs (Sreerama, 1999
) were also identical for both proteins and fell into the following ranges: helix 2127%, ß-structure 3044% and random coil 4050%. 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 4B
), 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 (3040°C) that are accompanied by protein precipitation.
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The analogous LMO4 fusion protein, FLIN4, was also constructed, using residues 1686 of mouse LMO4 (Figure 2C). 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 2C
). 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 (1718% helical, 2741% ß-structure and 4050% 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 6B
) 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 LMOldb1(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 7, 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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Discussion |
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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 LMOldb1 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, 1998), although the extent to which this has happened for the LMOldb1(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., 1998). 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., 1996, 1997
). 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 (1215 residues) gives rise to monomers, short linkers (35 residues) give rise to dimers and directly ligated chains give rise to tetramers (Dolezal et al., 2000
). 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 LMOldb1 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 LMO2ldb1 interaction that could be utilized in the development of a specific LMO2 inhibitor.
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Notes |
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Acknowledgments |
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References |
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Bach,I., Carriere,C., Ostendorff,H.P., Andersen,B. and Rosenfeld,M.G. (1997) Genes Dev., 11, 13701380.[Abstract]
Birse,D.E., Doublie,S., Kapp,U., Strub,K., Cusack,S. and Aberg,A. (1996) FEBS Lett., 384, 215218.[ISI][Medline]
Birse,D.E., Kapp,U., Strub,K., Cusack,S. and Aberg,A. (1997) EMBO J., 16, 37573766.
Boehm,T., Foroni,L., Kaneko,Y., Perutz,M.F. and Rabbitts,T.H. (1991) Proc. Natl Acad. Sci. USA, 88, 43674371.[Abstract]
Breen,J.J., Agulnick,A.D., Westphal,H. and Dawid,I.B. (1998) J. Biol. Chem., 273, 47124717.
Dawid,I.B., Breen,J.J. and Toyama,R. (1998) Trends Genet., 14, 156162.[ISI][Medline]
Dolezal,O., Pearce,L.A., Lawrence,L.J., McCoy,A.J., Hudson,P.J. and Kortt,A.A. (2000) Protein Eng., 13, 565574.
Fersht,A.R.F. (1998) Structure and Mechanism in Protein Science: a Guide to Enzyme Catalysis and Protein Folding. Freeman, New York.
Gill,G.N. (1995) Structure, 3, 12851289.[ISI][Medline]
Grutz,G., Forster,A. and Rabbitts,T.H. (1998) Oncogene, 17, 27992803.[ISI][Medline]
Hayes,D.B., Laue,T. and Philo,J. (19952001) University of New Hampshire.
Johnson,M.L., Correia,J.J., Yphantis,D.A. and Halvorson,H.R. (1981) Biophys. J., 36, 575588.[Abstract]
Johnson,W.C. (1999) Proteins, 35, 307312.[ISI][Medline]
Jurata,L.W. and Gill,G.N. (1997) Mol. Cell. Biol., 17, 56885698.[Abstract]
Jurata,L.W., Kenny,D.A. and Gill,G.N. (1996) Proc. Natl Acad. Sci. USA, 93, 1169311698.
Jurata,L.W., Pfaff,S.L. and Gill,G.N. (1998) J. Biol. Chem., 273, 31523157.
Kenny,D.A., Jurata,L.W., Saga,Y. and Gill,G.N. (1998) Proc. Natl Acad. Sci. USA, 95, 1125711262.
Konrat,R., Weiskirchen,R., Krautler,B. and Bister,K. (1997) J. Biol. Chem., 272, 1200112007.
Konrat,R., Krautler,B., Weiskirchen,R. and Bister,K. (1998) J. Biol. Chem., 273, 2323323240.
Kontaxis,G., Konrat,R., Krautler,B., Weiskirchen,R. and Bister,K. (1998) Biochemistry, 37, 71277134.[ISI][Medline]
Larson,R.C., Osada,H., Larson,T.A., Lavenir,I. and Rabbitts,T.H. (1995) Oncogene, 11, 853862.[ISI][Medline]
Morcillo,P., Rosen,C., Baylies,M.K. and Dorsett,D. (1997) Genes Dev., 11, 27292740.
Perez-Alvarado,G.C., Miles,C., Michelsen,J.W., Louis,H.A., Winge,D.R., Beckerle,M.C. and Summers,M.F. (1994) Nature Struct. Biol., 1, 388398.[ISI][Medline]
Piotto,M., Saudek,V. and Sklenar,V. (1992) J. Biomol. NMR, 2, 661666.[ISI][Medline]
Provencher,S.W. and Glockner,J. (1981) Biochemistry, 20, 3337.[ISI][Medline]
Rabbitts,T.H., Bucher,K., Chung,G., Grutz,G., Warren,A. and Yamada,Y. (1999) Cancer Res., 59, 1794s1798s.[ISI][Medline]
Sreerama,N. (1999) University of Colorado.
Sreerama,N., Venyaminov,S.Y. and Woody,R.W. (2000) Anal. Biochem., 287, 243251.[ISI][Medline]
Velyvis,A., Yang,Y., Wu,C. and Qin,J. (2001) J. Biol. Chem., 276, 49324939.
Visvader,J.E., Mao,X., Fujiwara,Y., Hahm,K. and Orkin,S.H. (1997) Proc. Natl Acad. Sci. USA, 94, 1370713712.
Warren,A.J., Colledge,W.H., Carlton,M.B., Evans,M.J., Smith,A.J. and Rabbitts,T.H. (1994) Cell, 78, 4557.[ISI][Medline]
Yamada,Y., Warren,A.J., Dobson,C., Forster,A., Pannell,R. and Rabbitts,T.H. (1998) Proc. Natl Acad. Sci. USA, 95, 38903895.
Yao,X., Perez-Alvarado,G.C., Louis,H.A., Pomies,P., Hatt,C., Summers,M.F. and Beckerle,M.C. (1999) Biochemistry, 38, 57015713.[ISI][Medline]
Received January 29, 2001; revised April 30, 2001; accepted May 14, 2001.