ABSTRACT LMO2 was discovered via chromosomal translocations in T-cell leukaemia and shown normally to be essential for haematopoiesis. LMO2 is made up of two LIM only domains (thus it is a

LIM-only protein) and forms a bridge in a multi-protein complex. We have studied the mechanism of formation of this complex using a single domain antibody fragment that inhibits LMO2 by

sequestering it in a non-functional form. The crystal structure of LMO2 with this antibody fragment has been solved revealing a conformational difference in the positioning and angle between

the two LIM domains compared with its normal binding. This contortion occurs by bending at a central helical region of LMO2. This is a unique mechanism for inhibiting an intracellular

FUNCTION OF HUR Article Open access 18 December 2024 AN ALYREF-MYCN COACTIVATOR COMPLEX DRIVES NEUROBLASTOMA TUMORIGENESIS THROUGH EFFECTS ON USP3 AND MYCN STABILITY Article Open access 25

March 2021 RNA-BINDING PROTEINS OF KHDRBS AND IGF2BP FAMILIES CONTROL THE ONCOGENIC ACTIVITY OF MLL-AF4 Article Open access 05 November 2022 INTRODUCTION The LIM-only family of proteins has

four members (LMO1-4) and are implicated in a spectrum of cancers1, including T cell leukaemia (LMO1, 2, 3)2,3,4,5,6,7, diffuse large B cell lymphoma (LMO2)8,9, prostate cancer (LMO2)10,11,

neuroblastoma (LMO1 and LMO3)12,13,14 and breast carcinoma (LMO4)15. _LMO2_ was originally cloned from a chromosomal translocation breakpoint in T-cell acute leukaemia (T-ALL)3,5 that

involved T-cell receptor loci (either _TCRδ_ at 14q11 or _TCRβ_ 7q35 in respectively t(11;14)(p13;q11) or t(7;11)(q35;p13)) and the _LMO2_ gene on chromosome 11 at band p13. Aberrant LMO2

expression is oncogenic in T cells16,17,18, while its normal role is in haematopoiesis, being required for both primitive and definitive haematopoiesis19,20, as well as a role in endothelial

cell remodeling21. The _LMO2_ gene encodes a cysteine-rich protein, comprising two zinc-binding LIM domains22, each with two LIM fingers, that acts as a protein binding module interacting

with a number of different proteins to form bipartite DNA-binding multi-protein complexes23. In the normal development of T cells, LMO2 expression is down regulated during the initial stages

of T-cell development24 and is not required for normal development of this lineage25. T-ALL is characterized by an accumulation of immature T-cells in the bone marrow. The effect of

enforced LMO2 expression in T-cells was analysed with a transgenic mouse model in which _Lmo2_ was expressed in thymocytes using the _CD2_ promoter. Analysis of thymocytes from these mice

showed an asymptomatic (pre-leukaemic) phase associated with an increase in the proportion of CD4-;CD8- double negative cells18,26 with a majority block at the double negative 3 (DN3;

CD44-;CD25+) stage of development preceding the appearance of clonal T cell tumours27. Transplantation studies using the T cells from a _CD2-Lmo2_ transgenic mouse showed that the DN3 cells

(but not DN1, DN2 or DN4) could be transplanted into irradiated recipients and had properties of self-renewal27, further supporting the concept that the initial function of LMO2 in T cell

neoplasia is to cause a population cells (DN3) to expand as the site of secondary mutations to cause overt neoplasia. The _Lmo2_-transgenic mice develop clonal thymic tumours from around 6–9

months18,26 implying that LMO2 is necessary but not sufficient for T cell neoplasia. Conversely, it was shown that intracellular antibody fragments and peptide aptamers that interfere with

LMO2 in tumour cells are effective in suppressing tumour growth28,29,30, indicating that sustained expression is required to maintain T cell tumourigenesis. Since LMO2 functions as a

transcription factor that assembles multi-protein complexes in normal settings (e.g. erythropoiesis) or abnormal settings (e.g. T cell cancer where chromosomal translocations cause aberrant

LMO2 expression), approaches are needed to interrogate the mechanisms by which these protein complexes form. This will lead to an understanding of the biochemistry of this transcription

factor complex assembly and the controls that are exerted in aberrant settings such as tumours and will potentially add to understanding of how protein complexes form in normal and abnormal

cell metabolism. We previously compared the DNA binding properties of the LMO2-complex in erythroid cells with an LMO2-complex in T cell tumours from a strain of _CD2-Lmo2_ transgenic mice

using the CASTing methodology31. We observed that in erythroid cells (a normal LMO2 expression setting) the LMO2 complex comprised LDB1, TAL1/SCL, E47 and GATA1 with the TAL1/SCL-E47

basic-helix-loop-helix (bHLH) heterodimer binding an E-box DNA motif and the GATA1 protein binding the GATA site23. Conversely, the T cell complex could comprise LMO2, LDB1 with two

TAL1/SCL-E47 heterodimers, each binding to E-box motifs in T cells from _Lmo2_-transgenic mice32. Further binding studies with LMO2 showed it has the capability to bind to other bHLH

proteins such as LYL1 and TAL233 both of which have been implicated in T cell tumours34 suggesting a possible inter-connectivity of LMO2 and bHLH proteins, dictated by aberrant expression of

LMO2 in human cancer. LMO2 is down regulated by the DN3 stage in mice18,26,27 and evidently it is enforced expression of LMO2 that results in a block in T-cell development. In addition to

the intricacies of the protein-protein interactions (PPIs), a central question about LMO2 is the way in which it and other specific proteins, find each other in the cellular milieu and

locate the DNA binding sites. As a corollary, there are currently no clear mechanisms that could be exploited to develop therapies based on interfering with these LMO2-dependent PPI. The

central role of LMO2 prompted us to develop an anti-LMO2 single VH domain intracellular antibody fragment (referred to as VH#576) to interfere with LMO2 protein function35. Antibodies and

derivative fragments have the capacity to bind a huge variety of antigens with high specificity and affinity. Antibody binding sites comprise heavy chain variable domain (VH) and light chain

variable domains (VL), each with three complementarity determining regions (CDRs), directly contacting antigen and involved in binding affinity and specificity. Single domain antibody

fragments encompass a minimal antigen recognition and can also be used for intracellular applications. The single domain comprising specific V region framework scaffolds have excellent

properties of solubility, stability and high expression levels within the cell. VH#576 moderates the tumourigenic function of LMO2 _in vivo_ as it prevents Lmo2-dependent tumour growth in a

transplantation assay. We have determined the crystal structure of human LMO2 in complex with VH#576 at 2.9 Å resolution (PDB ID 4KFZ) and found that LMO2 adopts a stable but contorted

structure whose conformation is altered around central, short alpha helical region between the two LIM domains36. All three complementarity determining regions (CDRs) of the VH#576 are

involved in direct interaction with LMO2. This suggests a mechanism for the normal role of LMO2 in protein complex formation and a mechanism for the perturbation of LMO2 structure-function

by the VH. Our work locates specific interacting residues on the surface of both LMO2 and VH#576 that could inform the design of small molecules to disrupt leukaemia-associated LMO2

complexes. RESULTS INTERACTION WITH THE SINGLE ANTIBODY DOMAIN STABILISES LMO2 STRUCTURE We previously described a single domain (VH) antibody fragment (VH#57635), identified by the

Intracellular Antibody Capture procedure37,38, that displays inhibitory function when binding to LMO2 in T cell neoplasias or erythroid cells. As an approach to determine the mechanism of

LMO2 inhibition by VH#576, we co-expressed LMO2 with VH#576 as a recombinant protein complex in _E. coli_ for structural studies. Whereas previous attempts to make soluble LMO2 recombinant

protein have been unsuccessful, co-expression with VH#576 in _E. coli_ drastically increases the solubility, due in part to shielding of hydrophobic regions. Parenthetically, this property

of single antibody domains, or scFv, to assist in deriving soluble recombinant proteins, should be a generally useful property for structural studies of otherwise intransigent proteins.

Crystal trials were carried out with the purified heterodimer and diffracting crystals were obtained (Supplementary Fig. 1). This resulted in a 2.9 Å structure of human LMO2 in complex with

VH#576 (Fig. 1 and data collection statistics in Supplementary Table 1). The architecture of VH#576 is that of the common immunoglobulin fold and was solved by molecular replacement. The

LIM1 and LIM2 domains of LMO2 were positioned according to experimentally determined zinc positions. Structure refinement was completed using PHENIX-REFINE and AUTOBUSTER software. The LIM1

domain of LMO2 possesses the characteristic LIM domain structure of four β-strands and this is followed by a short α-helix, encompassing the hinge region residue Phe8836. Each zinc atom was

positioned between a pair of anti-parallel β-strands. All four zinc ions are present in the structure and each is coordinated in a tetrahedral geometry by the side chains of three cysteine

residues and one histidine or aspartic acid residue. Furthermore, zinc ions are stable in a cellular environment due to the lack of redox activity39. LMO2 has a long N-terminal tail (residue

8–26, see Supplementary Fig. 2B) with no defined secondary structure in the crystal. The first 20 residues of LMO2 are predicted to be disordered by regional order neural network

software40. This region has a high content of charged/hydrophilic residues and few bulky hydrophobic residues characteristic of intrinsic disordered proteins41. The function of this

N-terminal region may be in transcriptional control, as we have shown that it can act as a transcriptional trans-activator in two-hybrid reporter assays42. Each of the VH#576 CDR loops is

involved in the binding to LMO2 (Fig. 1) and these span amino-acid contacts in both LIM domains (contact residues are listed in Supplementary Table 2). In addition, there is an interacting

loop from a framework region that binds to the LIM2 domain (Fig. 1B); this region of hydrophobic contacts lies at the former VH-VL interface of VH#576 spanning Val37 to Tyr50 (see

Supplementary Fig. 2A). The close proximity of residues Leu45 and Tryp47 of VH#576 to Met108 and Met106 of Lmo2 indicate the hydrophobic effect to be a driving force in the LMO2-VH

interaction. Both the methionine residues of LMO2 are located in the finger 3 region. Other hydrophobic amino acids interactions are in finger 4 and between the zinc-coordination residues of

the second zinc atom of finger 3 (Supplementary Table 2). The potency of interaction of the individual residues in the VH#576 CDRs was elucidated by their mutagenesis to Gly and/or Ala and

the effect on interaction between the mutant VH#576 and LMO2 was determined by a mammalian two-hybrid luciferase reporter assay (Fig. 2A–C). To assess effects of the CDR mutations on

expression levels, each mutant VH made as a fusion with VP16 at the C-terminal and expression assessed by Western blotting using anti-VP16 antibody (shown at the bottom of each panel of Fig.

2A–C and in Supplementary Fig. 3 with controls of VH#576 and/or VHY#6). These data show that residues in all three CDRs are critical for VH binding as specific mutations ablate interaction

of the VH and LMO2 in the mammalian intracellular assay. The critical CDR residues are indicated in Supplementary Fig. 2A. Residues of CDR3 are particularly important (8 residues in this

region are hot spots) when analysed in the two hybrid assay, as it has the highest proportion of residues involved in the interaction with LMO2. This concurs with the crystal structure (Fig.

2D, E). A hydrogen bonding network occurs between the LIM finger three of LIM2 and CDR3 of VH#576 residues Ser103, Glu105 and Thr107 (Fig. 2E, Supplementary Table 2). Arg109, a residue that

is critical in LMO2-LDB1 LID interaction, forms a salt bridge with Glu105 of VH#576. In effect the VH#576 exploits naturally used loops (CDRs) as well as a region generally masked by VH-VL

interaction to perform its role in inhibiting LMO2 function. LMO2 STRUCTURE DIFFERS WHEN BOUND TO THE VH OR TO LDB1 LID We generated an _in silico_ structure of the LMO2 protein using mouse

Lmo4 and partial mouse Lmo2 structural data43,44 that identified a central helical region between the two LIM domains28. This included the hinge region residue Phe88 that was identified when

the structure of LMO2 was solved in complex with LDB1-LID36. LDB1 is a widely expressed nuclear adaptor protein that binds LMO proteins through its C-terminal LID (LIM-interacting domain)

and forms part of the LMO2 protein complex first identified in erythroid cells23 and later shown to be of similar composition in T cell neoplastic cells32. Superimposition of the respective

shapes of LMO2 was carried out for our current structural analysis of LMO2-VH complex with the LMO2:LDB1-LID fusion protein36 and using the first LIM domain of LMO2 in both crystal

structures for alignment (Fig. 3A). This shows that the LIM domains of both dimeric structures have close structural similarity but the relative orientation of the LIM1 and LIM2 domain

varies markedly (Fig. 3A, B). The LIM2 is mis-oriented compared with LMO2:LDB1-LID allowing interactions between VH#576 and the conserved β-strand, b7 of LMO2 (see Supplementary Fig. 2 for β

strand nomenclature). In figure 3C, hydrophobic amino acids are represented in ball and stick form at the former VH/VL interface of anti-LMO2 and residues Met106 and Leu117 indicating the

hydrophobic contribution to binding at the protein-protein interface. Specifically, the angle between LIM1 and LIM2 varies by 23° between the two structures. The zinc positions within the

electron density map were located using PHENIX AutoSol and are shown in Supplementary figure 4. Although an angle of motion between the LIM domains of LMO2 was observed between two

independently determined crystal structures of LMO2:LDB1-LID fusion protein45,36 when comparing LMO2 in complex with LID or with VH#576 an overall root mean square deviation (RMSD) of 5.3 Å

was calculated. The conformational change in LMO2 bound by the VH is more profound than that between LMO2:LDB1-LID structures where there is a maximum overall RMSD of 2.6 Å. In essence,

VH#576 acts to contort LMO2 through a series of high affinity interactions, particularly with LIM2 of LMO2. Thus we conclude that the VH#576 interaction with LMO2 results in contortion of

the LMO2 protein because LMO2 is highly dynamic and flexible such that its stable configuration is dependent upon its interaction partner. This may reflect a mechanism of allosteric control

mediating associations with a broad range of natural protein partners. THE ANTI-LMO2 VH SEQUESTERS LMO2 FROM NORMAL BINDING PARTNERS IN VIVO The alternative conformations that the LMO2

flexibility allows suggests that protein-protein interactions can be influenced by this and that the mechanism by which the anti-LMO2 VH impairs function is by removing newly synthesized

LMO2 into a separate complex. This hypothesis was tested using transfection studies in CHO cells that assessed individual interactions. It has been shown that LMO2 binds directly to a number

of T-ALL associated bHLH proteins, including TAL1/SCL, TAL2 and LYL118,23. LMO2 does not bind directly to the E2A bHLH E12 or E4733 but forms a complex with E47 via LMO2-TAL1/SCL

interaction in which the latter hetero-dimerises with E47. Luciferase assays were performed in CHO cells that were transfected with two LMO2 baits comprising the Gal4DBD fused to LMO2 or the

LMO2-LDB1 LID fusion and a TAL1/SCL-VP16 fusion prey, with or without a vector expressing E47 bHLH (Fig. 4A, B). TAL1/SCL binds to LMO2 but does so relatively poorly compared with the

LMO2-LID fusion (Fig. 4A). In addition, the binding has more efficacy when the TAL1/SCL partner E47 is present (Fig. 4A) although again the LMO2-LID bait is more efficacious. Little evidence

of interaction was found with the LMO2-VH#576 when the LMO2 was expressed as the fusion with the LDB1 LID (Fig. 4B) since the luciferase activation was reduced to control levels (e.g. when

LMO2-LID was co-expressed with an anti-RAS VH#Y6). Pull-down experiments were carried out to further analyse the mutually exclusive _in vivo_ protein binding. COS-7 cells were co-transfected

with LMO2, LDB1, TAL1, E47 and GATA-1 and either Flag-tagged VH#576 or Flag-tagged VH#Y6 (a non-relevant VH). Protein complexes associated with the VH proteins were analysed by capturing

protein with anti-Flag antibody beads, fractionating on SDS-PAGE and Western blotting with the antibodies shown in Fig. 4C. The pull-down data mirror those obtained in the two-hybrid

reporter systems since the Western shows that expressed VH#576 associates with predominant amounts of LMO2 (Fig. 4C), only a very small, proportional amount of LDB1 but we could not detect

associated TAL1/SCL or GATA1 in this assay. Finally, it does not appear that the binding of LMO2 by VH#576 simply results in removal of LMO2 by proteolysis as MEL cells transfected with

VH#576 expression vectors display approximately similar levels of LMO2 protein as do untransfected cells (Fig. 4D). This means that LMO2-VH complex is located in the cells as a stable dimer

that is not _per se_ subject to increased instability. These data support the concept that binding of LMO2 by the VH#576 limits its natural interactions by sequestration and that LDB1

presents the LIM domains in such a way to enable further native protein complex assembly. DISCUSSION The _LMO2_ gene is a paradigm of a chromosomal translocation master gene46 as it encodes

a developmental regulator whose function is subverted by the contextual alteration of expression following translocation to T cell receptor loci. This property, manifest in transgenic mouse

models by a blockade of differentiation of thymocytes18,26 and is exerted by LMO2 mediating the formation multi-protein DNA-binding complexes23. Our data show that the anti-LMO2

intracellular VH antibody fragment substantially interferes with the formation of this protein complex by a mechanism that does not simply involve enhanced degradation of the LMO2 protein

itself47. Rather, structural determination of LMO2 in complex with the VH suggests a mechanism of functional inhibition whereby the conformation of LMO2, when in complex with VH, is distinct

from that normally found and that this impairs binding of LMO2 to its natural partners. While the first LIM domain seems to be structurally unaltered by interaction with the VH compared to

interaction with LDB1 (Figs. 1, 3), the second LIM domain lies at an angle compared to LMO2-LDB1. The degree of distortion of the second LIM domain is reminiscent of the inhibitory effect a

peptide aptamer that we discovered that also interacts with the finger 4 of LMO228. In this case, the peptide seems to bind competitively to the fourth zinc atom. These observations suggest

that targeting this region of the LMO2 protein would be an effective drug strategy. In normal circumstances, interaction of LMO2 with LDB1 confers a structure on LMO2 that enables the

assembly of an active transcription factor complex. The conserved hinge region between the two LIM domains of LMO2 has been identified which enables interaction with other proteins, such as

bHLH that may occur in contact with DNA. The anti-LMO2 VH exploits this flexible region to allow contortion of LMO2 into a conformation that impairs the binding of natural partners such as

TAL1/E47 and GATA-1, thereby inhibiting formation of a functional transcription factor complex. Mammalian two-hybrid data also show that VH#576 binds poorly to LMO2:LDB1-LID (Fig. 4) and

cannot compete with LDB1 for LMO2 binding whereas LDB1 can compete VH#576 binding to LMO235. This is consistent with the higher affinity of LDB1 than VH#576 for LMO2 (the former being kd =

2.0×10−8 M and latter kd = 9.4×10−8 M35. Therefore, inhibition of LMO2 by VH#576 presumably occurs by sequestering newly synthesized protein. Further, the small size of LMO proteins suggests

they should be capable of freely moving between the cytoplasm and the nucleus)30,48 however they are found predominantly in the nucleus, possibly through nuclear retention by LDB1. The

interaction between VH#576 and LMO2 may occur in the cytoplasm following protein synthesis, preventing the interaction between LMO2 and LDB1. These studies into anti-LMO2 VH suggest a model

(Fig. 5) in which alternative LMO2 structures dictate functional outcome. Our studies suggest that initially synthesized LMO2 protein is intrinsically disordered and that binding to a

partner protein confers a configuration that nucleates subsequent protein complex formation. The structure of LMO2:LDB1-LID36,49, demonstrates a relatively flat, extended rod structure, able

to “present” the LIM fingers to allow for other protein-protein interactions such as with GATA150. By contrast, interaction with VH confers a distinct structure that renders LMO2 inactive

due to inefficient binding of natural partners. Recent data reveal the N-terminal zinc finger of GATA1 mediates the interaction between GATA1 and LMO251. These data and our _in silico_ model

of the pentameric complex generated from individual structural information (Fig. 5), indicate that a single molecule of LMO2 could bridge the DNA binding proteins GATA1 and TAL1/SCL-E47, in

keeping with our original model of the LMO2 complex23. These data suggest a model in which LMO2 binds LDB1 in the cytoplasm and is then transported to the nucleus. The LDB1 interaction

“tethers” the LIM domains in such a confirmation to allow TAL1/SCL-E47 to interact with the LMO2 LIM1 domain and for GATA-1 to interact with the LIM 2 domain and also other protein-protein

interactions. The data described in this paper have implications for both the mechanism of LMO2 transcription factor complex formation and how the structure of LMO2 may be exploited in a

therapeutic approach. All our previous attempts to produce recombinant LMO2 protein have proved unsuccessful due to the intransigent insolubility of the protein in induction systems

(unpublished). However, we found that co-expressing LMO2 with the anti-LMO2 single domain resulted in efficient production of soluble protein that was readily purified and crystallized in

the dimeric state35. This has two main implications. First, the use of single domain VH (or indeed scFv) to facilitate co-expression of otherwise intransigent proteins is likely to be a

useful general strategy. Molecules like LMO2, that show significant intrinsic flexibility52,49, could be stabilized by binding with antibody fragments aided by the masking of hydrophobicity

in the interaction with the VH interface. Keeping in mind that the VH#576 antibody single domain was isolated from a diverse library of VH segments merely by its binding property with an

LMO2 bait, the mechanism of functioning is significant. The manifest stabilization of the LMO2 structure by interaction with the VH#576 and the sequestration of the bound LMO2 into a state

that reduces the interaction of LMO2 with its natural partners such as LDB1 and TAL1/SCL has implications for the natural formation of the LMO2 multi-protein complex. The noted hinge region

between the two LIM domains provides the motional flexibility that locks LMO2-LDB1 interactions and places LMO2 in a configuration that facilitates the surrounding protein complex formation

involving variously TAL1/SCL, E47 and GATA1. This final complex may require contact with DNA binding sites. In similar vein, when the anti-LMO2 VH#576 binds to the unstructured LMO2, the

dimer complex adopts a configuration that distorts the LMO2, particularly the LIM2 domain is bent around Phe88 and the fourth zinc atom is misaligned from the normal context. This

structurally serves to sequester LMO2 protein away from the natural complex. The LMO2-VH dimer is not wholly degraded but rather is isolated. The nature of the VH#576 single domain binding

to LMO2 is unusual as there is binding from framework residues (listed in Supplementary Table 2), some of which come from former VH-VL interface residues. The region of VH#576 binds to the

LIM2 domain of LMO2 assisting in the stable configuration of LMO2. Residues from all three VH CDRs are involved in binding LMO2 such that there are contacts on both LIM1 and LIM2 domains

across the Phe88 hinge. The imposition of this ‘four-handed’ binding by VH#576 is sufficient to impose functional ablation by sequestration. VH#576 is a macromolecule that could be used as a

drug _per se_ if methods for efficient delivery into cells _in vivo_ can be achieved53. There are several options whereby this might be achievable for the use of macromolecules as drugs

(macrodrugs)54. The use of chemical emulators of macrodrug binding is an enticing, but not yet achieved goal, of this field of work. It has been thought that obtaining small molecule drugs

that prevent protein-protein interaction would not be possible but more examples of this are becoming known55. One issue will be the relatively low affinity that can be achieved with a small

molecule to bind at a PPI interface and prevent interaction of two larger proteins. Our findings with the LMO2-VH#576 complex offers an possible strategy that would be based on obtaining

two compounds to emulate, respectively, the binding sites of VH#576 on LIM1 and LIM2. If these can be isolated and chemically joined56, the affinity of the final compound would be the

product of the two affinities, potentially giving a high affinity compound that can emulate the LMO2 bending and effective inactivation. METHODS PROTEIN EXPRESSION AND PURIFICATION For

co-expression of recombinant LMO2 and the anti-LMO2 VH576 single domain, an initial bicistronic expression vector (pRK-HIS-TEV-VH576-LMO2) was constructed by sub-cloning the anti-LMO2 VH#576

in-frame35. Truncated _LMO2_ (_LMO2Δ8NΔ11C_ residues 9–147) cDNA was used to replace LMO2 in the pRK-HIS-TEV-VH576-LMO2 construct using restriction enzyme sites _NheI_ and _EcoRI_.

pRK-HIS-TEV-VH576-LMO2 was subsequently engineered to create expression vector pRK-HIS-TEV-VH576-LMO2 (_LMO2Δ8N_ residues 9-158). The construct was transformed into _E. coli_ C41(DE3) and

cultured in 1 L of LB plus ampicillin (100 μg/ml) at 37°C, aerated at 225 rpm until the absorbance at 600 nm reached 0.6. ZnSO4 was added prior to induction to a final concentration of 0.1 

mM. Protein expression was induced by adding Isopropyl β-D-1-thiogalactopyranoside to a final concentration of 0.5 mM and incubated at 16°C for 14 hours. The VH#576-LMO2 protein complex was

extracted after cell disruption (Constant cell disruption system) in 20 mM Tris-HCl pH 8, 250 mM NaCl, 20 mM imidazole, 0.1 mM ZnSO4, 10 mM β-mercaptoethanol and EDTA-free protease inhibitor

cocktail (Roche Diagnostics, Mannheim, Germany). LMO2 and VH#576 were co-purified using a Ni2+ charged HiTrap chelating HP column (5 ml, GE Healthcare) using a gradient elution from 20 to

300 mM imidazole. To remove His-tag from VH#576, the purified proteins were digested with his-tagged Tobacco Etch virus protease at 4°C overnight during dialysis against the buffer without

imidazole. The proteins were passed through a nickel-nitrilotriacetic acid agarose column (Qiagen), the bound material eluted and concentrated using an Amicon Ultra-15 centrifugal filter

device, 10 KDa cut-off (Millipore, MA, USA). For further purification of LMO2-VH#576 complex, the proteins were size-excluded by gel filtration chromatography with a Hi-Load Superdex 75

column (GE Healthcare, Uppsala, Sweden) in 20 mM Tris-HCl pH 8, 150 mM NaCl, 1 mM DTT. Fractions containing VH#576/LMO2 complex were pooled and concentrated to 8 mg/ml and used for

crystallisation (Supplementary Fig. 1A). CRYSTALLIZATION AND STRUCTURE DETERMINATION OF LMO2 IN COMPLEX WITH VH#576 VH#576-LMO2_Δ8NΔ11C_ was crystallised using the sitting-drop

vapour-diffusion method at 294 K. Precipitant solutions (100 μl per well) were dispensed into reservoirs of a Greiner 96 well plate. Using a robot (Cartesian) 100 nl from each reservoir was

dispensed onto each platform along with 100 nl of protein to form a single droplet. Each plate was sealed manually, using self adhesive transparent film (Viewseal, Greiner). Crystals

appeared after a day and further optimization was performed in 24-well Cryschem plates (Hampton Research, CA, USA), 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 100 mM MES monohydrate pH 6.0, 0.8 M ammonium sulfate and additive 1,

6 hexanediol. Crystals typically measured 100 μm by 40 μm (Supplementary Fig. 1B). For data collection, the crystals were flash-cooled to 100 K in a cryo-protectant solution consisting of

mother liquor and 30% glycerol. A three wavelengths MAD data set was collected on the Zn K-edge using an ADSC Q315 detector on beamline I02 at the Diamond Light Source (Didcot, UK). Analysis

of the diffraction data using HKL200057 showed that the crystals belonged to space group _P_6 (unit cell dimensions _a_, _b_ = 124.3, _c_ = 81.4 Å and α, β = 90.0°, γ = 120.0°) with two

molecules in the asymmetric unit (Matthews coefficient, _V__M_ = 2.87 Å3/Da−1 with a solvent content of 57%58. The structure of VH#576/LMO2 was solved by a combination of molecular

replacement and SAD phasing. A molecular replacement solution for the VH#576 was determined with the program PHASER59 using the anti-RAS VH#Y6 heavy chain as a search model (PDB 2UZI).

PHENIX AUTOSOL60 was used to locate the LMO2 intrinsic zinc positions using a three-wavelength anomalous dispersion experiment at the peak, inflection and remote wavelengths of the Zn X-ray

absorption edge. The molecular replacement program MOLREP61 was used to search within this map for two LIM1 domains (using the isolated LIM1 from LMO2:LDB1-LID as a search model (PDB 2XJY)36

and successfully positioned the two molecules. The LIM2 domain (from LMO2:LDB1-LID) was manually positioned in the electron density map using the determined PHENIX zinc positions as anchor

points. Manual model building of the structure was done using the COOT (Crystallographic Object-Oriented Toolkit) software62 and restrained refinement was performed with PHENIX-REFINE and

then AUTOBUSTER63,64 taking care of keeping the same Rfree test set in both programs. Non-crystallographic symmetry (NCS) restrains were imposed on the 2 VH#576/LMO2 heterodimers. Towards

the end of refinement, TLS (Translation/Libration/Screw) vibrational motion refinement was used. At a later date diffraction data was collected from crystals made up of VH#576-LMO2Δ_8N_,

formed using the same conditions. Data was processed and refined to give a final Rwork/Rfree of 23.9/25.8%. Refinement statistics are listed in Supplementary Table I. MUTATION OF THE

ANTI-LMO2 VH AND MAMMALIAN TWO-HYBRID ANALYSIS Transient mammalian two-hybrid assays were carried out as described elsewhere65. Selected residues in the CDR regions of VH#576 were mutated,

from the base vector template pEF-VH#576-VP16 to either glycine or alanine by point mutation followed by assembly PCR. The final PCR product was cloned back into pEF-VP16 using _SfiI_ and

_NotI_ as a fusion with the VP16 activation domain. Truncated LMO2 bait (amino acids 28-150, see Supplementary Fig. 2) was cloned into the pM3 vector66 as a fusion with the GAL4 DNA-binding

domain. CHO cells were co-transfected using Lipofectamine 2000 (Invitrogen) as previously described67 with appropriate plasmids pM-LMO2 bait, pEFVP16-prey, pG5-Fluc and pRL-CML. Forty-eight

hours after transfection, the cells were lysed and assayed with the Dual-Luciferase reporter assay system (Promega) according to the manufacturer's instructions. All luminescence values

by _firefly_ luciferase were normalized using _Renilla_ luciferase levels. Expression of VH#576 was analysed by Western blotting using anti-VP16 antibody, (14–5 Santa Cruz biotechnology).

An LMO2-LDB1-LID construct was made as a fusion encoding LMO2 (residues 26 to 156) fused to LDB1-LID domain residues 336 to 368; the LMO2 was coupled C-terminally to LID via a ten residue

linker (GSGGSGGSGG). The insert was amplified by PCR from pGBT9-LMO2:LDB1-LID (a kind gift from Dr Jacqueline Matthews) and cloned into SalI/NotI sites of pM3. To compare the interaction

between LMO2 or LMO2:LDB1-LID with TAL1 or VH#576, pM3-bait (pM3-LMO2 or pM3-LDB1-LID:LMO2), pEF-VP16-prey (pEF-TAL1-VP16, pEF-VH#576-VP16 or pEF-VH#6-VP16), pG5-luc, pEF-BOS-E4723 and

pRL-CML plasmids were transiently co-transfected into COS-7 cells using Lipofectamine 2000 (Invitrogen). Forty-eight hours post-transfection, Firefly and Renilla luciferase enzyme activities

were assayed with the Dual-Luciferase reporter assay system. TRANSFECTION OF MEL CELLS WITH VH#576 pFUW-3XFlag-VH576-GFP and pFUW-3XFlag-VHY6-GFP vectors were prepared through a series of

sub-cloning steps. Assembly PCR was used to generate BamHI-P2A-AgeI-GFP-NheI-STOP-EcoRI. The final PCR product was digested with BamHI-EcoRI and cloned into the same sites of the parent

vector pFUW68 3XFlag-VH#576 or 3XFlag-VH#Y6 were amplified and the PCR products were cloned into restriction enzyme sites _SalI_ and _XbaI_. The vectors expressing the Flag-tagged anti-LMO2

VH#576 or anti-RAS VH#Y6 into murine erythroleukaemia MEL(585) cells were transiently transfected using an Amaxa Nucleofector™ apparatus (Amaxa), according to the manufacturer's

instructions. 1×107 cells were re-suspended in the specified Amaxa electroporation buffer (buffer R) plus 5 μg of the plasmid. GFP-positive cells were separated by sorting using Cytomation

MOFLOW cytometer 24 hours after transfection. Equal number of cells were lysed by 30minute incubation on ice with RIPA buffer (150 mM sodium chloride 1.0% NP-40, 0.5% sodium deoxycholate,

0.1% SDS, 50 mM Tris, pH 8.0) and equivalent amounts of protein extracts were separated by SDS-PAGE prior to transfer to membranes. Western analysis used anti-FLAG (clone M2, Sigma catalog

number F3165), anti-LMO2 (clone 1A9-1, AbD SeroTec) or anti-tubulin (clone AA13, Sigma). PULL-DOWN WITH ANTI-FLAG BEADS COS-7 cells were seeded in a 12 well plate and co-transfected, using

Lipofectamine 2000 (Invitrogen) with pGL3-ElbLUC-(Ebox-GATA)2, pEF-LDB1, pEF-LMO2, pEF-TAL1, pEF-E47 and either pEF-3XFLAG-VH576-NLS-neo or pEF-3XFLAG-VHY6-NLS-neo. Each transfection was

done in triplicate. Forty-eight hours post-transfection, cells were removed from the wells using trypsin and triplicate wells pooled. Cells were pelleted by centrifugation at 1000Xg for 5

minutes and the supernatant removed. The cells were re-suspended in 200 μl lysis buffer (50 mM Tris HCL, pH7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton 100, protease inhibitor) and rotated at 4°C

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LMO2 and FOG1/ZFPM1 by the transcription factor GATA1. Proc. Natl Acad Sci USA 108, 14443–8 (2011). Article  CAS  ADS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS This work

was supported by grants from the Leukaemia and Lymphoma Research and the Medical Research Council. E.J.M. is a Royal Society University Research Fellow. We would like to thank Dr.

Jacqueline Matthews for the expression clone pGBT9-LMO2:LDB1-LID. AUTHOR INFORMATION Author notes * H. Sewell Present address: Fujifilm Diosynth Biotechnologies Belasis Avenue Billingham,

TS23 1LH, UK * T. Tanaka Present address: Dainippon Sumitomo Pharma Co., Ltd. Protein Analysis Group Genomic Science Laboratories, 3-1-98 Kasugade-naka, Konohana-ku Osaka, 544–0022, Japan *

N. Fernandez-Fuentes Present address: Institute of Biological, Environmental and Rural Science Aberystwyth University Aberystwyth, SY23 3EB, United Kingdom * Sewell H., Tanaka T. and Omari

K. El contributed equally to this work. AUTHORS AND AFFILIATIONS * Weatherall Institute of Molecular Medicine MRC Molecular Haematology Unit University of Oxford John Radcliffe Hospital

Oxford OX3 9DS, UK H. Sewell, A. Cruz, J. Chambers & T. H. Rabbitts * Leeds Institute of Molecular Medicine Wellcome Trust Brenner Building St. James's University Hospital

University of Leeds Leeds, LS9 7TF, UK H. Sewell, T. Tanaka, A. Cruz, N. Fernandez-Fuentes, J. Chambers & T. H. Rabbitts * Wellcome Trust Centre for Human Genetics Division of Structural

Biology University of Oxford Headington, Oxford, OX3 7BN, UK K. El Omari & E. J. Mancini Authors * H. Sewell View author publications You can also search for this author inPubMed Google

Scholar * T. Tanaka View author publications You can also search for this author inPubMed Google Scholar * K. El Omari View author publications You can also search for this author inPubMed 

Google Scholar * E. J. Mancini View author publications You can also search for this author inPubMed Google Scholar * A. Cruz View author publications You can also search for this author

inPubMed Google Scholar * N. Fernandez-Fuentes View author publications You can also search for this author inPubMed Google Scholar * J. Chambers View author publications You can also search

for this author inPubMed Google Scholar * T. H. Rabbitts View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS H.S., T.T. and T.H.R. designed

experiments. H.S., T.T., J.C. carried out the work. H.S., T.T., K.E.O., E.M., A.C. solved the crystal structure. N.F.F. carried out computational biology, T.H.R. designed, supervised the

project and interpreted data. All authors have reviewed the manuscript and H.S., T.T. and T.H.R. wrote the paper. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing

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CITE THIS ARTICLE Sewell, H., Tanaka, T., Omari, K. _et al._ Conformational flexibility of the oncogenic protein LMO2 primes the formation of the multi-protein transcription complex. _Sci

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