Purification, crystallization and preliminary X-ray analysis of a fusion of the LIM domains of LMO2 and the LID domain of Ldb1.
ABSTRACT LMO2 (LIM domain only 2), also known as rhombotin-2, is a transcriptional regulator that is essential for normal haematopoietic development. In malignant haematopoiesis, its ectopic expression in T cells is involved in the pathogenesis of leukaemia. LMO2 contains four zinc-finger domains and binds to the ubiquitous nuclear adaptor protein Ldb1 via the LIM-interaction domain (LID). Together, they act as scaffolding proteins and bridge important haematopoietic transcription factors such as SCL/Tal1, E2A and GATA-1. Solving the structure of the LMO2:Ldb1-LID complex would therefore be a first step towards understanding how haematopoietic specific protein complexes form and would also provide an attractive target for drug development in anticancer therapy, especially for T-cell leukaemia. Here, the expression, purification, crystallization and data collection of a fusion protein consisting of the two LIM domains of LMO2 linked to the LID domain of Ldb1 via a flexible linker is reported. The crystals belonged to space group C2, with unit-cell parameters a = 179.9, b = 51.5, c = 114.7 Å, β = 90.1°, and contained five molecules in the asymmetric unit. Multiple-wavelength anomalous dispersion (MAD) data have been collected at the zinc X-ray absorption edge to a resolution of 2.8 Å and the data were used to solve the structure of the LMO2:Ldb1-LID complex. Refinement and analysis of the electron-density map is in progress.
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ABSTRACT: Cell fate is governed by combinatorial actions of transcriptional regulators assembling into multiprotein complexes. However, the molecular details of how these complexes form are poorly understood. One such complex, which contains the basic-helix-loop-helix heterodimer SCL:E47 and bridging proteins LMO2:LDB1, critically regulates hematopoiesis and induces T cell leukemia. Here, we report the crystal structure of (SCL:E47)bHLH:LMO2:LDB1LID bound to DNA, providing a molecular account of the network of interactions assembling this complex. This reveals an unexpected role for LMO2. Upon binding to SCL, LMO2 induces new hydrogen bonds in SCL:E47, thereby strengthening heterodimer formation. This imposes a rotation movement onto E47 that weakens the heterodimer:DNA interaction, shifting the main DNA-binding activity onto additional protein partners. Along with biochemical analyses, this illustrates, at an atomic level, how hematopoietic-specific SCL sequesters ubiquitous E47 and associated cofactors and supports SCL's reported DNA-binding-independent functions. Importantly, this work will drive the design of small molecules inhibiting leukemogenic processes.Cell Reports 07/2013; · 7.21 Impact Factor
Acta Cryst. (2010). F66, 1466–1469
Acta Crystallographica Section F
Purification, crystallization and preliminary X-ray
analysis of a fusion of the LIM domains of LMO2
and the LID domain of Ldb1
Kamel El Omari,aCatherine
Porcherband Erika J. Mancinia*
aDivision of Structural Biology, The Wellcome
Trust Centre for Human Genetics, University of
Oxford, Roosevelt Drive, Oxford OX3 7BN,
England, andbMRC Molecular Haematology
Unit, Weatherall Institute of Molecular
Medicine, John Radcliffe Hospital, University of
Oxford, Oxford OX3 9DS, England
Correspondence e-mail: firstname.lastname@example.org
Received 5 July 2010
Accepted 16 August 2010
LMO2 (LIM domain only 2), also known as rhombotin-2, is a transcriptional
regulator that is essential for normal haematopoietic development. In malignant
haematopoiesis, its ectopic expression in T cells is involved in the pathogenesis
of leukaemia. LMO2 contains four zinc-finger domains and binds to the
ubiquitousnuclear adaptor protein Ldb1 via the LIM-interaction domain (LID).
Together, they act as scaffolding proteins and bridge important haematopoietic
transcription factors such as SCL/Tal1, E2A and GATA-1. Solving the structure
of the LMO2:Ldb1-LID complex would therefore be a first step towards
understanding how haematopoietic specific protein complexes form and would
also provide an attractive target for drug development in anticancer therapy,
especially for T-cell leukaemia. Here, the expression, purification, crystallization
and data collection of a fusion protein consisting of the two LIM domains of
LMO2 linked to the LID domain of Ldb1 via a flexible linker is reported. The
crystals belonged to space group C2, with unit-cell parameters a= 179.9, b= 51.5,
c = 114.7 A˚, ? = 90.1?, and contained five molecules in the asymmetric unit.
Multiple-wavelength anomalous dispersion (MAD) data have been collected at
the zinc X-ray absorption edge to a resolution of 2.8 A˚and the data were used to
solve the structure of the LMO2:Ldb1-LID complex. Refinement and analysis of
the electron-density map is in progress.
LMO2 (LIM domain only 2) is a member of the LIM-only (LMO)
family of LIM domain-containing transcriptional cofactors. LIM
domains are 55-residue cysteine-rich structural units composed of
two zinc fingers linked by a two amino-acid residue hydrophobic
linker. LIM domain-containing proteins are believed to play crucial
roles in many essential cellular processes such as cell growth,
trafficking, cytoskeletal organization, differentiation and apoptosis
(Zheng & Zhao, 2007; Bach, 2000) by mediating protein–protein
interactions through their zinc-finger domains. Specifically, the
presence of a tandem of LIM domains in the LMO proteins confers
on them the potential to engage in multiple protein–protein inter-
LMO2 is a 158-amino-acid nuclear protein composed of two LIM
domains and a small N-terminal transactivation domain. LMO2 plays
a central role in haematopoietic stem-cell development, erythro-
poiesis and angiogenesis (Warren et al., 1994; Yamada et al., 2000;
Yamada et al., 1998). Upon chromosomal translocations or biallelic
transcriptional activation, its ectopic expression is involved in the
pathogenesis of T-cell acute lymphoblastic leukaemia (T-ALL;
Boehm et al., 1991; Ferrando et al., 2004). In normal haematopoiesis,
LMO2 interacts with the ubiquitously expressed protein Ldb1 [also
known as CLIM (LIM homeobox protein cofactor) or NLI (nuclear
LIM-domain interactor)]. Ldb1 comprises a 39-amino-acid C-term-
inal LIM-interaction domain (LID) that mediates interaction with all
LMO proteins and LIM homeodomains (Jurata & Gill, 1997;
Kadrmas & Beckerle, 2004) and an N-terminal dimerization domain
(Jurata & Gill, 1997) that allows the formation of higher order
protein complexes. Indeed, the LMO2:Ldb1 complex acts as a scaf-
folding protein and participates in the assembly of a DNA-binding
multiprotein complex that includes transcriptional regulators such as
SCL, E2A and GATA-1 (Lecuyer et al., 2007; Schlaeger et al., 2004;
Wadman et al., 1997). When abnormally expressed, similar protein
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complexes are believed to be involved in tumorigenesis in T-ALL
(Grutz et al., 1998; Herblot et al., 2000; Ono et al., 1998). Interestingly,
a role for LMO2 in B-cell lymphomas (Natkunam et al., 2007) and
prostate cancer (Ma et al., 2007) has also been reported. Conse-
quently, LMO2 has become a very attractive anticancer drug target.
Efforts are currently focused on designing peptides and/or intra-
bodies that are able to disrupt transcriptional complexes containing
LMO2 (Nam et al., 2008; Appert et al., 2009).
NMR structures of the N-terminal LIM domains of LMO4 and
LMO2 in complex with the Ldb1 LID domain (Ldb1-LID; PDB
codes 1j2o and 1m3v, respectively; Deane et al., 2003) provided the
first examples of LIM:Ldb1-LID complexes and highlighted the
residues responsible for the interactions of the N-terminal LIM
domains. The structure of both LIM domains of LMO4 fused to
Ldb1-LID has alsobeen solved
(LMO4:Ldb1-LID; PDB code 1rut; Deane et al., 2004); however, the
structure of the two-LIM-domain LMO2 in complex with Ldb1-LID
has not been reported to date. Despite their 46% sequence identity,
LMO2 and LMO4 have different functions and binding partners and
bind Ldb1 with different affinities (Ryan et al., 2006). In order to
obtain insights into the mechanism of action of LMO2 and to detail
its binding interface with the Ldb1-LID domain, we set out to solve
the structure of the LMO2:Ldb1-LID complex. Here, we report its
production, crystallization and preliminary diffraction analysis.
2. Material and methods
2.1. Cloning and purification
The LMO2:Ldb1-LID construct consists of human LMO2 (resi-
dues 26–153) fused to the Ldb1-LID fragment (residues 336–368)
via an 11-residue linker (GGSGGHMGSGG; Fig. 1). The sequence
coding for the fusion protein was obtained as described previously
(Deane et al., 2001). Briefly, the LMO2-linker insert was produced by
PCR using primers 1 (50-CGG GAT CCATCC CTG CTG ACATGC
GGC GG) and 2 (50-GCC ACC GGA ACC CAT ATG ACC GCC
GCT GCC ACC CCC ATT GAT CTT AGT CCA CTC) and linker-
Ldb1-LID was produced using primers 3 (50-GGT GGC AGC GGC
GGT CATATG GGT TCC GGT GGC GAT GTG ATG GTG GTG
GGG GA) and 4 (50-GGA ATT CTC ACT ATT ACT CGT CGT
CAA TGC CGT TGG). The full-length construct was obtained by a
third PCR using primers 1 and 4. The insert encoding LMO2:Ldb1-
LID was cloned into the pETduet vector (Novagen) between BamHI
and EcoRI restriction sites.
The plasmid pETduet-LMO2:Ldb1-LID encoding N-terminally
6?His-tagged protein was transformed into Rosetta (DE3) pLysS.
Growth was carried out at 310 K in Luria broth (LB) supplemented
with 50 mg ml?1ampicillin until the absorbance at 600 nm reached
0.7. Expression of the fusion protein was induced by the addition of
1 mM isopropyl ?-d-1-thiogalactopyranoside (IPTG) and growth was
continued for 16 h at 303 K. The cells were harvested by centrifu-
gation and resuspended in 50 mM sodium phosphate pH 7.4, 500 mM
NaCl, 10 mM imidazole and 0.2%(v/v) Tween-20. The cells were then
disrupted by sonication on ice and the lysate was clarified by
The supernatant was applied onto an Ni2+-charged chelating
column equilibrated with lysis buffer without detergent. The protein
was eluted with a gradient of imidazole. Fractions containing
LMO2:Ldb1-LID were pooled for additional purification using a S75
gel-filtration column (GE Healthcare) with 20 mM Tris pH 7.4,
300 mM NaCl and 1 mM DTT. Fractions containing LMO2:Ldb1-
LID were pooled and concentrated to 20 mg ml?1using Amicon
2.2. Crystallization and data collection
All crystallization experiments were performed at 295 K using the
sitting-drop vapour-diffusion method. Initial screening of 768 condi-
tions belonging to various kits from Hampton Research and Emerald
BioStructures was carried out using Cartesian Honeybee X8
dispensing robots to pipette 100 nl protein solution and 100 nl
precipitant solution into single drops in 96-well Greiner plates
(Walter et al., 2005). Crystals appeared after a day and further opti-
mization was performed in 24-well Linbro plates, mixing 2 ml protein
solution with 1 ml reservoir solution and equilibrating the drop
against 500 ml reservoir solution. The best diffracting crystals grew
within one week of setup in 1.6 M NaCl, 100 mM citrate pH 5 and
1 mM DTT (Fig. 2). For data collection, crystals were briefly
immersed in 4 M sodium malonate pH 5.0 prior to flash-cooling in a
nitrogen-gas stream. The Zn atoms of the LIM zinc-finger domains
were used as anomalous scatterers for structure determination using
the multiple-wavelength anomalous dispersion (MAD) method. Data
sets were collected from a single crystal of LMO2:Ldb1-LID on
beamline BM14 at the ESRF, Grenoble, France using a MAR CCD
225 detector with an oscillation of 1?, an exposure time of 60 s and a
crystal-to-detector distance of 242.4 mm. Following a broad X-ray
excitation scan of the crystal, data were collected at three wave-
lengths near the zinc absorption edge (peak ?1= 1.28226 A˚with
f0= ?7.5, f00= 5.4, inflection ?2= 1.28267 A˚with f0= ?10.2, f00= 2.9
and high-energy remote ?3= 1.27565 A˚; Table 1). Indexing, inte-
gration and scaling were carried out using HKL-2000 (Otwinowski &
Acta Cryst. (2010). F66, 1466–1469El Omari et al.
? LMO2:Ldb1-LID complex
Schematic representation of the LMO2:Ldb1-LID fusion protein. In the figure,
LMO2 is shown in light blue with its LIM domains in dark blue and Ldb1 is shown
in yellow with its LID domain in red.
LMO2:Ldb1-LID crystals. Crystal dimensions are approximately 70 ? 50 ? 50 mm.
3. Results and discussion
LMO2:Ldb1-LID was expressed as a soluble 6?His-tagged protein
with a yield of about 5 mg of pure protein per litre of LB culture.
Analysis of the data from size-exclusion chromatography indicated
that LMO2:Ldb1-LID behaves as a monomer in solution (data not
shown). LMO2:Ldb1-LID crystallized in a variety of different con-
ditions, all with a high salt concentration (>1.5 M) and a low pH (<6).
The LMO2:Ldb1-LID crystals (Fig. 2) used for data collection had
typical dimensions of 50 ? 50 ? 50 mm and appeared to be multiple
diffracted poorly (<4.0 A˚) and analysis of the diffraction data indi-
cated the presence of more than one lattice. Consecutive rounds of
annealing greatly improved the diffraction quality and resolution
limits of the crystals. Following this strategy, it was possible to collect
data from a single crystal which diffracted to 2.8 A˚resolution and
presented only two predominant lattices. A three-wavelength MAD
data collection at the Znabsorption edge was carried out on beamline
BM14 using this crystal. Despite the presence of two crystal lattices,
the diffraction spots could be indexed and scaled in either crystal
orientation (Fig. 3), resulting in two data sets of equal quality (as
judged by the data-collection statistics; Table 1). The two data sets
could also be scaled together into a single data set belonging to the
monoclinic space group C2, with unit-cell parameters a = 179.9,
El Omari et al.
? LMO2:Ldb1-LID complex
Acta Cryst. (2010). F66, 1466–1469
The figure shows two possible indexings of the same diffraction image created by the superimposition of two crystal lattices. Indexing was performed using the program
b = 51.5, c= 114.7 A˚, ?= 90.1?(Table 1). Despite a ?-angle value very
close to 90?, it was not possible to scale the data in orthorhombic
LMO2:Ldb1-LID molecules per asymmetric unit of the crystal, with a
VMvalue of 2.4 A˚3Da?1and a solvent content of 49% (Matthews,
Molecular-replacement algorithms (MOLREP and Phaser; Vagin
& Teplyakov, 1997; McCoy, 2007) were unable to solve the structure
of LMO2:Ldb1-LID despite the availability of a 50% identical model
(LMO4:Ldb1-LID; PDB code 1rut; Deane et al., 2004). As well as
both LIM domains together, the individual LIM domains of LMO4
were unsuccessfully used as search models, suggesting a difference in
the configuration of the LMO2 and LMO4 proteins. Despite the
strong anomalous signal from the naturally occurring Zn atoms in
LMO2:Ldb1-LID (four zincs per LMO2:Ldb1-LID, 20 zincs per
asymmetric unit) as judged by the anomalous signal of 6.9–8 A˚
observed in SHELXC (Pape & Schneider, 2004), it was initially not
possible to find the positions of the Zn atoms when using either of the
two individual data sets. Only when the two data sets were scaled and
merged together (Table 1) was it possible to solve the anomalous
scatterer substructure by determining and refining the position of the
20 Zn atoms per asymmetric unit with an overall figure of merit of 0.7.
The serendipitous presence of the two lattices aided structure solu-
tion by providing us with the possibility of collecting twice the
number of reflections from the outset, minimizing the radiation
damage and improving the recorded anomalous signal. Zinc-site
determination, phasing, density modification and initial model
building were performed using PHENIX AutoSol and AutoBuild
(Adams et al., 2002). More recently, crystals of LMO2:Ldb1-LID
displaying a different morphology (plates) and belonging to space
P21were obtained that contained one molecule in the asymmetric
unit and diffracted to a resolution of 2.4 A˚. Structure refinement and
analysis of the two crystal forms is under way.
We are grateful to Martin Walsh, Hassan Belrhali and Mario
Bumann at the BM14 UK MAD beamline (ESRF, Grenoble, France)
for assistance with data collection. This work was supported by
Leukaemia and Lymphoma Research. EJM is a Royal Society
University Research Fellow and CP is supported by the Medical
Adams, P. D., Grosse-Kunstleve, R. W., Hung, L.-W., Ioerger, T. R., McCoy,
A. J., Moriarty, N. W., Read, R. J., Sacchettini, J. C., Sauter, N. K. &
Terwilliger, T. C. (2002). Acta Cryst. D58, 1948–1954.
Appert, A., Nam, C.-H., Lobato, N., Priego, E., Miguel, R. N., Blundell, T.,
Drynan, L., Sewell, H., Tanaka, T. & Rabbitts, T. (2009). Cancer Res. 69,
Bach, I. (2000). Mech. Dev. 91, 5–17.
Boehm, T., Foroni, L., Kaneko, Y., Perutz, M. F. & Rabbitts, T. H. (1991). Proc.
Natl Acad. Sci. USA, 88, 4367–4371.
Deane, J. E., Mackay, J. P., Kwan, A. H., Sum, E. Y., Visvader, J. E. &
Matthews, J. M. (2003). EMBO J. 22, 2224–2233.
Deane, J. E., Ryan, D. P., Sunde, M., Maher, M. J., Guss, J. M., Visvader, J. E. &
Matthews, J. M. (2004). EMBO J. 23, 3589–3598.
Deane, J. E., Sum, E., Mackay, J. P., Lindeman, G. J., Visvader, J. E. &
Matthews, J. M. (2001). Protein Eng. 14, 493–499.
Ferrando, A. A., Herblot, S., Palomero, T., Hansen, M., Hoang, T., Fox, E. A.
& Look, A. T. (2004). Blood, 103, 1909–1911.
Grutz, G. G., Bucher, K., Lavenir, I., Larson, T., Larson, R. & Rabbitts, T. H.
(1998). EMBO J. 17, 4594–4605.
Herblot, S., Steff, A. M., Hugo, P., Aplan, P. D. & Hoang, T. (2000). Nature
Immunol. 1, 138–144.
Jurata, L. W. & Gill, G. N. (1997). Mol. Cell. Biol. 17, 5688–5698.
Kadrmas, J. L. & Beckerle, M. C. (2004). Nature Rev. Mol. Cell Biol. 5,
Lecuyer, E., Lariviere, S., Sincennes, M. C., Haman, A., Lahlil, R., Todorova,
M., Tremblay, M., Wilkes, B. C. & Hoang, T. (2007). J. Biol. Chem. 282,
Ma, S., Guan, X. Y., Beh, P. S., Wong, K. Y.,Chan, Y. P.,Yuen, H. F., Vielkind, J.
& Chan, K. W. (2007). J. Pathol. 211, 278–285.
Matthews, B. W. (1968). J. Mol. Biol. 33, 491–497.
McCoy, A. J. (2007). Acta Cryst. D63, 32–41.
Nam, C.-H., Lobato, M. N., Appert, A., Drynan, L. F., Tanaka, T. & Rabbitts,
T. H. (2008). Oncogene, 27, 4962–4968.
Natkunam, Y., Zhao, S., Mason, D. Y., Chen, J., Taidi, B., Jones, M., Hammer,
A. S., Hamilton Dutoit, S., Lossos, I. S. & Levy, R. (2007). Blood, 109, 1636–
Ono, Y., Fukuhara, N. & Yoshie, O. (1998). Mol. Cell. Biol. 18, 6939–
Otwinowski, Z. & Minor, W. (1996). Methods Enzymol. 276, 307–
Pape, T. & Schneider, T. R. (2004). J. Appl. Cryst. 37, 843–844.
Ryan, D. P., Sunde, M., Kwan, A. H., Marianayagam, N. J., Nancarrow, A. L.,
Vanden Hoven, R. N., Thompson, L. S., Baca, M., Mackay, J. P., Visvader,
J. E. & Matthews, J. M. (2006). J. Mol. Biol. 359, 66–75.
Schlaeger, T. M., Schuh, A., Flitter, S., Fisher, A., Mikkola, H., Orkin, S. H.,
Vyas, P. & Porcher, C. (2004). Mol. Cell. Biol. 24, 7491–7502.
Vagin, A. & Teplyakov, A. (1997). J. Appl. Cryst. 30, 1022–1025.
Wadman, I. A.,Osada,H., Grutz, G.G., Agulnick, A.D., Westphal,H., Forster,
A. & Rabbitts, T. H. (1997). EMBO J. 16, 3145–3157.
Walter, T. S. et al. (2005). Acta Cryst. D61, 651–657.
Warren, A. J., Colledge, W. H., Carlton, M. B., Evans, M. J., Smith, A. J. &
Rabbitts, T. H. (1994). Cell, 78, 45–57.
Yamada, Y., Pannell, R., Forster, A. & Rabbitts, T. H. (2000). Proc. Natl Acad.
Sci. USA, 97, 320–324.
Yamada, Y., Warren, A. J., Dobson, C., Forster, A., Pannell, R. & Rabbitts,
T. H. (1998). Proc. Natl Acad. Sci. USA, 95, 3890–3895.
Zheng, Q. & Zhao, Y. (2007). Biol. Cell, 99, 489–502.
Acta Cryst. (2010). F66, 1466–1469El Omari et al.
? LMO2:Ldb1-LID complex
Values in parentheses are for the outer shell.
Peak Inflection Remote
DataLattice 1Lattice 2MergedLattice 1Lattice 2MergedLattice 1Lattice 2Merged