Intermolecular Interactions of Cardiac Transcription Factors NKX2.5

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Intermolecular interactions of cardiac transcription factors NKX2.5 and TBX5 Lagnajeet Pradhan, Sunil Gopal, Shichang Li, Shayan Ashur, Saai Suryanarayanan, Hideko Kasahara, and Hyun-Joo Nam Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00171 • Publication Date (Web): 01 Mar 2016 Downloaded from http://pubs.acs.org on March 5, 2016

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Intermolecular interactions of cardiac transcription factors NKX2.5 and TBX5 Lagnajeet Pradhan1, Sunil Gopal1, Shichang Li1, Shayan Ashur1, Saai Suryanarayanan1, Hideko Kasahara2 and Hyun-Joo Nam1*

1

Department of Bioengineering, University of Texas at Dallas, Richardson, TX 75080 2

Department of Functional Genomics, University of Florida, Gainesville, FL 32610

Running Title: Intermolecular interactions of NKX2.5 and TBX5

Correspondence to: Hyun-Joo Nam, PhD University of Texas at Dallas. 800 W Campbell Road, RL10, Richardson, TX 75080. Telephone: (972) 883-5786, E-mail: [email protected]

Funding Source: This work is supported by a grant to H.-J. N. from the American Heart Association (13BGIA13960001).

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Abbreviations: A Adenine, ANF atrial natriuretic factor, C Cytosine, CHD congenital heart disease, EMSA electrophoretic mobility shift assay, G Guanine, HD homeodomain, T Thymidine

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Abstract: Heart development in mammalian systems is controlled by combinatorial interactions of master cardiac transcription factors such as TBX5 and NKX2.5. They bind to promoters/enhancers of downstream targets as homo- or hetero-multimeric complexes. They physically interact and synergistically regulate their target genes. To elucidate the molecular basis of the intermolecular interactions, a heterodimer and a homodimer of NKX2.5 and TBX5 were studied using X-ray crystallography. Here we report a crystal structure of human NKX2.5 and TBX5 DNA binding domains in a complex with a 19 base pair target DNA and a crystal structure of TBX5 homodimer. The ternary complex structure of NKX2.5 and TBX5 with the target DNA shows physical interactions between the two proteins through Lys158 (NKX2.5), Asp140 (TBX5) and Pro142 (TBX5), residues that are highly conserved in TBX and NKX families across species. Extensive homodimeric interactions were observed between the TBX5 proteins in both crystal structures. In particular, in the crystal structure of TBX5 protein which includes the N-terminal and DNA binding domains, intermolecular interactions were mediated by the N-terminal domain of the protein. The N-terminal domain of the TBX5 was predicted as “intrinsically unstructured”, and in one of the two molecules in an asymmetric unit, the Nterminal domain assumes a β-strand conformation bridging two β-sheets from the two molecules. The structures reported here may represent general mechanisms for combinatorial interactions among transcription factors regulating developmental processes.

Key words: NKX2.5, TBX5, X-ray crystallography, Atrial natriuretic factor promoter, proteinDNA interaction 3


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In mammals, the complex and sophisticated process of heart morphogenesis is managed by master cardiac transcription factors (TFs) such as NKX2.5, TBX5, GATA4, HAND2, MEC2FC, and MESP1 1. Through combinatorial interactions, a handful of the cardiac TFs can orchestrate the complex spacio-temporal regulation of heart development2, 3. For example, NKX2.5, GATA4 and TBX5 interact physically, and synergistically activate downstream targets4, 5. Being master regulators of heart development, mutations in these genes are linked to congenital heart diseases (CHDs), the most common birth defect

6-10

. Specifically, mutations in NKX2.5 are linked to

diverse CHDs such as atrial and ventral septal defects, atrioventricular block, and Tetralogy of Fallot as well as progressive cardiac disorders appearing in adulthood 10, 11. Holt-Oram syndrome is the main congenital disease associated with TBX5 mutations. It displays pathological phenotypes such as upper-limb deformation and cardiac malformation3, 8. Since the core cardiac TFs function through interactions with multiple partners, their mutations can be manifested in various phenotypes, and the same phenotypes can be attributed to different mutations12-14. Thus, functions of the core cardiac TFs are determined by the context of interactions with other transcription regulators. NKX2.5 and TBX5 bind to the specific target DNA motifs through their centrally located DNA binding domains, the homeodomain (HD: amino acid residues 138-197) in NKX2.5 and TBox domain (TBD: amino acid residues 58-239) in TBX5. NKX2.5 binds to a consensus motif containing T(C/G)AAGTG, and TBX5 recognizes a T-Box sequence of T/CA/G/CACACCT/C15, 16

. The amino- and carboxy-terminal domains contain regulatory elements involved in

interactions with other transcription factors or transcription machinery17, 4


18

. They interact as

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homo- or hetero-multimeric complexes and cooperatively bind to their target DNAs5, 19. Through the combinatorial interactions, a set of cardiac TFs can recognize low frequency targets leading to specific downstream cascades distinct from those of an alternative complex5. To successfully perform this, the TFs are expected to have mechanisms for multi-partner interactions, and each interaction should be substantial enough to induce cooperative DNA binding. Details and modes of mechanisms enabling such multi-partner interactions are not clearly understood. The NKX2.5 HD and the TBX5 protein containing TBD and the N-terminal domain (TBX1239

: amino acid residues 1-239) were shown to be necessary and sufficient for physical and

functional interactions between the two proteins5. With only DNA binding domains of both proteins, partial activities were observed, implying that DNA binding domains are important components for physical interactions and the synergistic activation5.

Since the N-terminal

domain of the TBX5 is necessary for the full interaction with NKX2.5, the N-terminal domain is expected to play an important role in inter-molecular interactions. To understand the mechanisms of combinatorial interactions among the cardiac transcription factors, we determined a series of TBX5 and NKX2.5 complex structures using X-ray crystallography methods. One of the downstream targets, atrial natriuretic factor (ANF), contains multiple TBX5 and NKX2.5 binding motifs at its proximal promoter occupied by the two proteins4, 5. The -252 site (ANF252) of the proximal promoter contains closely-spaced NKX2.5 and TBX5 binding sites and the -242 region (ANF-242) contains two NKX2.5 recognition motifs in palindromic arrangement. They provide convenient platforms for physical interactions among the cardiac factors19. The binary complex structures of TBX5 TBD with DNA were previously described, and we have reported the crystal structure of NKX2.5 HDs bound to the ANF-242 motif20, 21. Here we report

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the crystal structure of a ternary complex, NKX2.5 HD and TBX5 TBD bound to ANF-252 DNA, and a crystal structure of TBX51-239 homodimer. Materials and methods: Protein expression and purification Cloning of the NKX2.5 HD and TBX5 TBD expression constructs and purification of the proteins were previously reported21-23. The DNA coding amino acids 1-239 of TBX5 protein (TBX51-239) was cloned between the Nde I and Bam HI sites of pET28 vector (Novagene). The expressed protein contains an N-terminal 6-Histidine tag and a thrombin cleavage site derived from the vector. TBX51-239 was produced based on a previously described protocol with minor modifications20, 23. Briefly, Escherichia coli BL21 (RIPL) cells were used for protein expression with protein induction by adding 0.5 mM IPTG and growing the cells overnight at 24°C. The cells were lysed by sonication, and proteins in the soluble fraction were purified by standard affinity chromatography using a Cobalt-sepharose column (New England Biolabs). It was further purified by size-exclusion chromatography using Superdex 75 column (GE Healthcare) with a buffer containing 20 mM HEPES pH 7.5, 0.15 M NaCl, 5 mM MgCl2 and 2 mM DTT. Oligonucleotides for co-crystallization experiments were synthesized (Sigma Genosys) and annealed in 10 mM Tris pH 8.0, 100 mM NaCl, and 10 mM MgCl2 first by heating the solution to 95°C for 10 minutes and cooling down slowly to room temperature in a water bath. For protein-DNA complex purification, the double-stranded DNA and the purified proteins, NKX2.5 HD and TBX5 TBD or TBX51-239, were mixed roughly in the molar ratio of 1:2:1.5 to insure full occupancy of the DNA by the proteins. The protein-DNA complex was purified by 6


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size-exclusion chromatography using Superdex 200 column (GE Healthcare) in Gel Filtration Buffer containing 20 mM Tris pH 8.0, 0.15 M NaCl and 10 mM DTT. The purified ternary complex was analyzed by SDS-PAGE and concentrated to the final protein concentration of ~10 mg/ml, and stored at –80°C. Crystallization, X-ray data collection, and structure determination Crystallization of the ternary complex NKX2.5 HD, TBX5 TBD and DNA were discussed previously23. For crystallization of TBX51-239, the purified complex of NKX2.5 HD, TBX51-239 and ANF-252 DNA were screened by the sparse matrix with crystallization drops containing 1 µl of protein/DNA sample and 1 µl of crystallization solution at room temperature. Hexagonal crystals were obtained from well solutions containing 0.1 M Sodium Cacodylate, pH 6.4, 10 mM MgSO4 and 1.8 M (NH4)2SO4. Data collection and structure determination of the NKX2.5 HD, TBX5 TBD and ANF-252 DNA ternary complex were discussed elsewhere23. The TBX51-239 crystals were cryoprotected in HEPES pH 7.0, 100 mM KCl, 10 mM CaCl2, 30% PEG 400 and 10 mM DTT, and flash-frozen in liquid nitrogen. The X-ray diffraction data were collected at the A1 beamline of the Cornell High Energy Synchrotron Source (CHESS). The data were indexed and processed using HKL2000 Suite24. Molecular replacement search was performed using the TBX5 TBD (PDB ID: 2X6V) as search models20, 21. The program Phaser was used for the search and the structure was refined using the PHENIX suite 25, 26. The data and refinement statistics of both crystal structures are shown in Table 1. ITC Measurement 7


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To prepare the sample for ITC experiments, the purified TBX51-239 protein was dialyzed against 10 mM PBS pH 7.3 buffer containing 1 mM β–mercaptoethanol, and concentrated by ultrafiltration using Amicon concentrator 10K MWCO (Millipore) to the final concentration of 4 mM. The ITC data was collected using Microcal iTC200 calorimeter (Malvern, Worcestershire, UK). For each titration experiment, 1.6 µl of concentrated TBX51-239 solution (4 mM) was injected into the 200 µl of diluted solution (20 µM) with a 5 minute time interval. For the control experiment, 20 µM protein solution was injected into the cell containing the solution with the same protein concentration. For analyses of the data, ITC peaks were first integrated using the Microcal Origin v7 or alternatively by using automated peak-shape analysis program NITPIC27. To obtain dimer dissociation constant ( ) and enthalpy ( ) from the integrated peaks, we have implemented a web-based analysis program based on a simple monomer-dimer dilution model (http://nuproplot.com/fitc). Using this on-line tool, the ITC data were fit into a standard nonlinear regression (least-square) model in order to obtain estimates of and values. Results and Discussion: Structural studies of the NKX2.5 and TBX5 complex bound to the composite target DNA (ANF252) were carried out using NKX2.5 HD, TBX5 TBD58-239, and TBX51-239 containing both Nterminal domain and the TBD. To produce ternary complexes of NKX2.5, TBX5 and DNA, the proteins were co-purified with the target DNA. In solution, the ternary complexes, NKX2.5HD/TBX5-TBD/ANF-252 and NKX2.5-HD/TBX51-239/ANF-252, were readily formed and purified23. The crystals of the ternary complex, NKX2.5 HD and TBX5 TBD in complex with 8


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ANF-252 DNA, were obtained from initial crystallization trials. However, crystals for NKX2.5 HD, TBX51-239 and DNA complex were not produced from repeated trials. Instead, multiple forms of TBX51-239 dimeric crystals were obtained from the solutions containing all three components. We have determined the crystal structure of the NKX2.5 HD/ TBX5 TBD/ ANF252 DNA ternary complex and the TBX51-239 homodimeric structure. Overview of the NKX2.5 HD/TBX5 TBD/ANF-252 ternary complex structure The crystal structure of NKX2.5 HD (residues 138-197) and TBX5 TBD (residues 58-239) with blunt-ended

19

base

pair

ANF-252

DNA

(TCTCACACCTTTGAAGTGG

/CCACTTCAAAGGTGTGAGA) was determined at the 2.9 Å resolution with current R-free and R-work values of 0.189 and 0.249, respectively (Table 1). Two sets of ternary complexes are present in an asymmetric unit. The NKX2.5 HD and TBX TBD, respectively, showed identical folding in both complexes. In the ternary complex, electron densities for amino residues 142-197 of NKX2.5, corresponding to residues 5-60 in the HD numbering scheme, were clearly defined and modeled accordingly 21. NKX2.5 HD forms a typical HD structure with three helices and an N-terminal extension (Figure 1a and b). It is similar to the previously reported NKX2.5 HD structure (PDB ID: 3rkq) with the Cα atom rmsd value of 0.55 Å21. The TBX5 TBD of the ternary complex shows an identical folding to the previously reported TBX5 TBD bound to its target DNA (PDB ID: 2x6v) with the Cα atom rmsd value of 0.73 Å 20. The TBD is composed of seven-stranded β-barrel (βA-C, βC’, and βE-G) closed by a smaller βpleated sheet (Figure 1c) 20, 28-30. The TBX5 TBD model in the ternary complex includes residues

9


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53-238 excluding the disordered loop between the βF and βG (FG loop; amino acid residues 189-199) (Figure 1a and c). Between the two ternary complexes in an asymmetric unit, slight differences were observed in DNA conformation and relative positions of the NKX2.5 and TBX5 (Figure 2a and b). When two TBX TBDs in an asymmetric unit were superimposed, the Cα rmsd of the two NKX2.5 HDs is 2.87 Å, indicating a small displacement of the relative positions. In one of the two complexes (Complex 1), NKX2.5 HD is making close contacts with TBX5, and the DNA is slightly bent to accommodate this configuration (Figure 2b). In Complex 2, a loop from TBX5 of Complex 1 is inserted at the interface of NKX2.5 and TBX5, separating the two proteins. Both NKX2.5 and TBX5 proteins interact with the 19mer ANF-252 DNA containing composite binding sites for TBX5 and NKX2.5, 5’-TCTCACACCTTTGAAGTGG-3’ comprising a TBX5 binding site at the 5’ segment and an NKX2.5 binding site near the 3’ end with one base pair spacer between the two. All the DNA residues could be assigned into their corresponding electron densities without ambiguity. In both complexes, the 19-mer ANF-252 DNA segment shows overall standard B-form configurations. Deviations from the standard Bform DNA were observed where close interactions with the proteins were observed20, 21, 31. NKX2.5 recognizes the DNA mostly using Helix 3 and the N-terminal extension as previously reported21. NKX2.5 mutations with clinical implication have been previously analyzed using the structural model21, 32. Major interactions between TBX5 TBD and the DNA are mediated by residues from Helix 3 and the C-terminal 310 helix. Additional interactions are observed between the DNA and R81, R82, K159, N162, S175, T215 and Y217 of TBX5 (Figure 2c) 20. Many pathological missense mutations are located in the TBD impairing DNA binding20, 10


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33, 34

. While this manuscript was in preparation, NKX2.5 HD-TBX5 TBD fusion protein and

ANF DNA structure was reported. Due to crystallization difficulties, NKX2.5 and TBX5 proteins were produced as a fusion protein, and the structure of the binary complex was determined at a lower resolution than the structure reported here. The protein structure shows identical structural arrangement as our ternary structure35. Intermolecular interactions between NKX2.5 HD and TBX5 TBD bound to ANF-252 DNA The TBX5 and NKX2.5 bound to the 19-mer ANF-252 DNA with composite binding sites interact with each other using a relatively small contact surface (~100 Å2). Within this small interaction surface several hydrogen bond interactions were observed between the two proteins: the terminal amide group of TBX5 R150 hydrogen bonds with the carbonyl group of NKX2.5 C193 (S193 in our structure); the terminal amide of NKX2.5 K158 makes a double hydrogen bond with the carbonyl groups of TBX5 D140 and P142 (Figure 3). In TBX family transcription factors, basic amino acids are highly conserved at the R150 equivalent or neighboring positions. In NK2 family proteins, Lysine at the K158 position is also conserved (Figure 1b and c). Basic amino acids at both positions allow hydrogen bonding with main chain carbonyl groups of the respective binding partners. These basic residues protrude outside of the proteins but are not involved in DNA binding. This suggests that these well-conserved charged residues may function as a versatile tool for the inter-molecular interactions commonly observed between the transcription factors. D140 and P142 of TBX5 are a part of a highly conserved loop (amino acid 137-143) with two Proline residues at the 139 and 142 positions. This loop makes a small acidic patch composed of carbonyl groups of P139, D140 and P142 and the terminal carboxylic group of 11


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D140. This acidic patch is ideal for binding with terminal groups of basic amino acids such as K158 of NKX2.5 (Figure 3b and c). A recently identified missense mutation in TBX5, A143T, located at this loop is associated with sporadic dilated cardiomyopathy36. The A143T mutation may change the conformation of the loop and the charged nature of the contact surface. Interestingly, A143T mutation is reported to impair synergistic activation of ANF promoter with the GATA4 transcription factor36. GATA4 may employ a similar interaction mode as NKX2.5 using basic amino acid side chains, and the A143T mutation in TBX5 may disrupt the physical interactions and synergistic activation. Multiple combinatorial interaction partners of TBX5 may share a common interaction mode for physical interactions and the ternary complex structure reported here shows an example of such interaction mechanisms. In Complex 2, a loop from TBX5 of Complex 1 (amino acids 129-131) is wedged at the interface of NKX2.5 and TBX5 preventing the inter-molecular interactions observed in Complex 1. In the ternary crystal structure of NKX2.5-HD /TBX5-TBD/ANF-252, additional intermolecular interactions were observed between the two TBX5 TBDs bound to the two different DNA molecules. The interface area was over 900 Å2, and the interaction was mediated by one side of the β barrel composed of βC, βF and βG (Figure 2a). Hydrophobic residues located at the surface of the protein (L101, L103, V107, L135, V137 and F201) are involved in the interactions. Dimeric interactions between TBX5 TBD domains were also observed in previous TBX5 structures. Although a different face of the β-barrel was used, β-strands C, F and G comprise the dimeric interfaces in both structures20. In solution, TBX5 TBD behaves as a monomer so the extensive interactions observed in the crystal structure may not be the correct reflections of this protein in a diluted solution20. However, with the exposed hydrophobic

12


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residues, these β-strands are likely to be involved in additional interactions. The TBX51-239 structure described in the next section shows an example of β-strands C, F and G involved in inter- and intra-molecular interactions. Overview of TBX51-239 structure Purified solutions of NKX2.5, TBX51-239 and ANF-252 DNA failed to produce a ternary complex crystal. Instead, crystals of TBX5 in dimeric interactions were readily produced in multiple crystallization conditions. One of the crystals diffracted to 2.6 Å and the model was refined to the final Rwork and Rfree of 0.237 and 0.284, respectively (Table 1). Two TBX5 proteins were present in an asymmetric unit, and amino acids 36-231 in one of the two TBX5 proteins (Mol1) and residues 50-232 of the second molecule (Mol2) are modeled into well-defined electron densities (Figure 4). The individual TBX5 TBD structure is similar to the unliganded TBX5 TBD structures (PDB ID: 2x6v) with Cα rmsd of 0.62 Å 20. Compared to the TBX5 TBD of the NKX2.5-HD/TBX5-TBD/DNA ternary complex, major differences were observed in the C-terminal 310 helix and in the loop between the βF and βG (FG loop). The C-terminal 310 helix is disordered in the TBX51-239 as was reported in the TBX5 TBD DNA-free structure 20. This helix is a major contact interface between the protein and DNA and is well ordered only in the presence of a target DNA20, 28. The FG loop disordered in the ternary complex is better defined in the TBX51-239 dimer and shows a similar conformation to that of the DNA-free TBX5 TBD structure20. In the TBX3 TBD crystal structure, the FG loop adopts a 310 helix conformation28, but in TBX5, it forms a tightly packed loop without any canonical

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secondary structures. The TBX5 FG loop contains two Gly residues and contains a two-residue insertion compared to TBX3 which may contribute to the flexibility of the TBX5 FG loop. The structure of N-terminal residues (amino acids 1-50) has not been previously reported in any TBX proteins, but in the TBX51-239 structure, a portion of the N-terminal residues forms a βstrand (Figure 5a and b). Of the 50 residues preceding the TBX TBD, only amino acid 36-50 could be modeled in Mol1 of the TBX51-239 dimer, and among them amino acids 43-48 form a well-ordered β-strand (Figure 4b). In Mol2 most of the N-terminal residues were disordered. Dimeric interactions of the TBX51-239 When the N-terminal residues (1-50) of TBX5 were analyzed for secondary structural motifs, it was predicted to be “instrinsically disordered”37, 38. However, in the TBX51-239 structures, the Nterminal amino acids 42-48 form a β-strand (labeled βN) in one of the two monomers in an asymmetric unit (Figure 1c). This N-terminal β-strand (βN) functions as a bridge connecting the βG strands from each monomer extending the two sets of β-sheets composed of βC, βF and βG (Figure 4). Amino acids 36-42 of Mol1 are also located at the interface making additional intermolecular interactions. The interface area expands to 990 Å2 with ~30 residues from each protein located at the interface. In addition to main chain hydrogen bonds of the β-sheet structure, multiple side chain interactions stabilize the dimeric interaction. Pro42 and Phe46 are involved in several hydrophobic interactions including aromatic stacking (Figure 4c). One of the TBX5 germline missense mutations, Q49K, is positioned at the dimeric interface

39

. In monomeric

TBX5, Gln49 located at the surface of the protein is not involved in any obvious functions such as structural stability or DNA binding. In the dimeric context, however, Gln49 plays key roles. In 14


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Mol1, Gln49 is part of a β-turn structure connecting βN and βA. In Mol2, Gln49 makes dimeric interactions through van der Waals interactions with Phe46 of Mol1 (Figure 4c). Substitution to Lys at this position would create less than optimal interactions between the two molecules. We further analyzed the interface using the PISA server (Protein, Interface, Surface and Assembly), and it yielded the complex formation significance score (CSS) of 0.371 indicating an auxiliary role of the interface for complex formation

40

. It also predicted that the TBX51-239

dimer is stable in solution. This dimeric interface does not overlap with any known DNA interaction surface on TBX5, and the dimeric interaction is unlikely to interfere with DNA binding (Figure 4d). To investigate the oligomeric state of the TBX51-239 in solution and its binding affinity, we determined the equilibrium dissociation constant (Kd) and enthalpy change (∆Hdim) for oligomeric interactions of the TBX51-239 using isothermal titration calorimetry (ITC). The ITC data showed a characteristic endothermic process, and the integrated data profile is consistent with a simple monomer-dimer dissociation model (Figure 5)

41

. The TBX51-239 proteins show

weak dimeric interactions with a value of 434± 39 µM (Figure 5b). Compared to the TBX5 TBD and DNA interaction with reported Kd values in the hundreds nanomolar range, dimeric protein interaction is multiple orders of magnitude weaker. The observed dimeric interaction between the two TBX5 proteins would only be possible with high local concentrations of TBX5. The protein-protein interactions using an “instrisically disordered protein” (IDP) region is an ideal strategy for transcription factors such as TBX5. Combinatorial interactions employed by cardiac transcription factors during developmental regulation require multi-partner interactions by each transcription factor. Flexibility of IDP enables binding onto diverse surfaces and to 15


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different partners42. Using a single binding motif, significant interactions can be achieved with multiple partners. The transition of disordered to ordered structures upon protein-protein interactions have been previously documented43. Our structure demonstrates an example of an IDP in both disordered and structured conformations upon protein-protein interaction. The crystal structures of TBX proteins were previously determined while bound to in vitro selected DNA targets in a palindromic arrangement28-30, and such an arrangement would not allow the dimeric interactions observed here since the dimeric interaction interface is on the opposite side of the DNA contact region (Figure 4d). All TBX5 targets identified to date only contain a single binding site, and no palindromic or tandem dual binding sites have been reported4. In the promoter of ANF and Connexin 40, downstream targets of TBX5, multiple TBX5 target sites are present with ~100–1000 base pair spaces between the two sites. Based on the TBX51-239 structure and ITC analysis reported here, two TBX5 proteins bound within a promoter can form a dimer by forming a DNA loop. High local concentration generated by binding within the promoter may enable dimerization despite the low binding affinity of TBX5. Conclusions The crystal structures of the NKX2.5-HD/TBX5-TBD/DNA ternary complex and TBX51-239 dimer described here demonstrate examples of inter-molecular interactions employed by cardiac transcription factors during combinatorial interactions. In the ternary complex structure, hydrogen bonds among conserved residues mediate the interactions between the proteins bound to the dual binding sites. Small binding interfaces are used among the conserved residues. The TBX51-239 dimer structure shows inter-molecular interactions employing an “Intrinsically Disordered Protein” motif. By using an IDP which conforms to a rigid structure upon binding to 16


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a partner, cardiac transcription factors can interact with multiple partners. The structures reported here may represent general mechanisms for combinatorial interactions among cardiac transcription factors. Acknowledgements: We are grateful to the staff at the A1 beamline of Cornell High Energy Synchrotron Source and 19-ID beamline of Advance Photon Source for assistance during X-ray data collection. We thank Dr. Alan Cooper for the discussion on the mathematical model of dimerization and Mr. Kevin Fortin for critical reading of the manuscript. Atomic coordinates and the structural factors are deposited in the Protein Data Bank with accession numbers 4S0H (NKX2.5-HD/TBX5TBD/DNA) and 5BQD (TBX51-239 dimer).

Author contributions: The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

.

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FIGURE LEGEND: Figure 1. Overall structure of TBX5-TBD/NKX2.5-HD/ANF-252 DNA ternary complex and sequence alignments of NK and TBX family proteins. (a) Structural features of the ternary complex. The NKX2.5 HD is shown in cyan (helices) and orange (coils), and TBX5 TBD in yellow (strands), red (helices) and green (coils). The ANF-252 DNA containing dual binding sites of NKX2.5 and TBX5 is also shown in cartoon representation. Secondary structure elements are labeled. (b) Sequence alignment of NK family HD domains are shown with amino acid residue numbers of NKX2.5 on top. (c) Sequence alignment of TBX family protein segments equivalent to TBX5 residues 1-239. In both (b) and (c), secondary structure elements are represented using red zigzag lines for α- and 310-helices, red arrows for β-strands and blue lines for coils on top of the sequences. Residues involved in inter-molecular interactions between NKX2.5 and TBX5 are shown in red. The sequence was aligned using the T-Coffee alignment server 44 and the secondary structure was calculated using ESPript 45. Figure 2. Two ternary complexes in an asymmetric unit and protein-DNA interactions. (a) Two ternary complexes are shown with NKX2.5 HD in green and TBX5 TBD in magenta in Complex 1, and NKX2.5 HD in yellow and TBX5 TBD in cyan in Complex 2, respectively. (b) Superposition of both complexes is shown following the color scheme in panel (a) except that the DNA backbone of Complex 2 is shown in blue. Closer contacts between NKX2.5 and TBX5 were observed in Complex 1 compared to Complex 2. A loop from the TBX5 of Complex 1 wedged between the two proteins in Complex 2 is marked with an arrow in panel (a). (c) Schematic diagram of protein-DNA interaction in the ternary complex. The figure was generated using NuProPlot 46.


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Figure 3. Inter-molecular interactions between NKX2.5 and TBX5 bound to ANF-252 DNA. Interactions between TBX5 and NKX2.5 were observed in the circled area in panel (a). (b) Detailed view of the contact interface was shown with side chains of the contact residues in stick representation. Hydrogen bonds are illustrated by dotted lines. (c) Molecular surface of the contact interface in the same view as in (b) is shown. Figure 4. Dimeric interactions in the TBX51-239 structure. (a) Two molecules in an asymmetric unit of TBX51-239 structure are shown. Two TBDs are shown in blue and orange, respectively, and the N-terminal domain in cyan. Secondary structure elements are labeled. (b) N-terminal domain residues 36-49 are shown in stick representation with 2Fo-Fc map (1 σ level). (c) Closeup view of the TBX51-239 dimeric interface. Contributing residues in homodimeric interactions are shown in stick representation with hydrogen bonds illustrated as dotted lines. (d) The NKX2.5-HD/TBX5-TBD/DNA ternary complex model is superimposed onto the TBX51-239 dimer. The dimeric interface does not overlap with the DNA contact region of TBX5. They are on the opposite side of the molecule. Figure 5. ITC measurement of TBX51-239 dimeric interactions. (a) ITC endothermic peaks (green) related to dissociation of dimers to monomers upon injection of concentrated samples (4 mM) into the diluted sample solution (20 µM) are shown. The negative control peaks are shown in red. (b) Nonlinear fitting of the integrated areas (open circles) of the peaks in panel (a) is shown with

calculated

Kd

and

∆H.

The

plot

was

generated

using

fITC

program

(http://www.nuproplot.com/fitc).


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Table 1. Crystallographic data and refinement statistics

TBX51-239 Dimer

NKX2.5 HD /TBX5 TBD/DNA Complex Crystallographic data statistics Space group

P 21

P6122

Cell Parameters

a=69.30, b= 77.78, c= 77.60 Å, β=108.31º

a=b=69.30, c=77.78,

X-ray source

A1, CHESS

A1, CHESS

Resolution (Å)

47.06-2.81 (2.99-2.81)

50.00-2.60 (2.69-2.60)

No. Unique reflection

17,571 (966)

22,021 (2075)

Completeness (%)

98.8 (89.0)

99.41 (96.51)

a

0.083 (0.494)

0.106 (0.681)

Redundancy

3.5 (3.6)

5.0 (4.9)

< I/σ(I) >

11.4 (2.3)

8.88 (2.08)

Rsym

Refinement statistics of current models Protein atoms (n)

3802

3053

Resolution

38.16-2.81 Å

49.23-2.60 Å

DNA atoms (n)

1546

0

Solvent atoms (n)

2

13

Rcryst (bwork / cfree)

0.188/0.247

0.238/0.287

d

rmsd bond lengths

0.01 Å

0.01 Å

d

rmsd bond angles

1.39°

1.39°

Ramachandran plot Favored (%) 92 95.1 Disallowed (%) 0.45 1.36 a Rsym = ∑hkl∑i |Ii(hkl)-|/∑hkl∑iIi(hkl) where Ii(hkl) is the intensity of an individual hkl reflection and is the mean intensity for all measured values of this reflection. b Rwork= ||Fo|─|Fc||/|Fo|, where Fo and Fc represents the observed structure factor amplitudes and the structure factor amplitudes calculated from the atomic model, respectively. c Rfree was calculated with the 5% of randomly selected reflections excluded from the data set during refinement. d rmsd, root mean square deviation.


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Intermolecular Interactions of Cardiac Transcription Factors NKX2.5

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