Structure of the C-terminal Heme-binding domain of THAP domain containing protein 4 from Homo sapiens

Introduction

The Thanatos (the Greek god of death)-associated protein (THAP) domain is a sequence specific DNA binding domain that contains a C2-CH (Cys-Xaa_2-4-Cys-Xaa_35-50-Cys-Xaa₂-His) zinc finger that is similar to the DNA domain of the P element transposase from Drosophila¹. THAP containing proteins have been observed in the proteome of humans, pigs, cows, chickens, zebrafish, Drosophila, C. elegans, and Xenopus¹. To date there are no known THAP domain proteins in plants, yeast, or bacteria. There are 12 indentified human THAP domain-containing proteins (THAP0 – 11). In all human THAP protein, the THAP domain is located at the N-terminus and is approximately 90 residues in length. While all of the human THAP containing proteins have a homologous N-terminus, there is extensive variation in both the predicted structure and length of the remaining protein¹. Even though the exact function of these THAP proteins is not well defined, there is evidence they play a role in cell proliferation², apoptosis³, cell cycle modulation⁴, chromatin modification⁵, and transcriptional regulation⁶. THAP containing proteins have also been implicated in a number of human disease states including heart disease⁴, neurological defects⁷ and several types of cancers^6,8,9.

Human THAP4 is a 577 residue protein of unknown function that is proposed to bind DNA in a sequence specific manner similar to THAP1^10,11 and has been found to be upregulated in response to heat shock¹². THAP4 is expressed in a relatively uniform manner in a broad range of tissues and appears to be upregulated in lymphoma cells and highly expressed in heart cells¹³. The C-terminal domain of THAP4 (residues 415-577), designated here as cTHAP4, is evolutionarily conserved and is observed in all know THAP4 orthologues. Several single domain proteins lacking a THAP domain are found in plants and bacteria, and show significant levels of homology to cTHAP4. It appears that cTHAP4 belongs to a large class of proteins that have yet to be fully functionally characterized.

On the basis of prior work, we predicted that cTHAP4 is composed of a heme-binding nitrobindin domain¹⁴, making THAP4 the only human THAP protein predicted to bind a cofactor. Nitrobindin, a recently characterized protein from Arabidopsis thaliana¹⁴, is structurally similar and exhibits nitric oxide (NO) binding properties that resemble the heme-binding nitrophorins. Nitrophorins utilize a heme moiety to store, transport, and release NO in a pH specific manner¹⁵. While the exact function of nitrobindin is not fully known, the similarities between the well characterized NPs implies a role in NO transport, sensing, or metabolism¹⁴. In order to better elucidate the possible function of THAP4, we solved the heme-bound structure of cTHAP4 to a resolution of 1.79 Å.

Material and Methods

Protein purification

The selenomethionine labeled protein was generated utilizing the standard Center for Eukaryotic Structural Genomics (CESG) pipeline protocols for cloning, protein expression, protein purification, and overall information management^16-19. Incorporation of the hemin group was completed as previously described¹⁴.

Crystallization, Diffraction Data Collection, and Structure Solution

Crystals utilized for data collection were grown at 4° C by hanging drop vapor diffusion by mixing 2 μL of an 10 mg•ml^-1 cTHAP4 solution (5 mM HEPES buffer, pH 5.0 containing NaCl (50 mM) and NaN3 (0.3 mM)) with 2 μL of reservoir solution (100 mM Triethanolamine buffer at pH 7.5 containing polypropylene glycol 400 (55%)).

X-ray diffraction data were collected at the Life Sciences Collaborative Access Team (LS-CAT) 21-ID-G beamline at the Advanced Photon Source, Argonne National Laboratories. Diffraction images were indexed, integrated, and scaled using HKL2000 ²⁰. The cTHAP4 selenium substructure was determined using Hyss ²¹. The results from Hyss indicated there were fourteen heavy atom sites per asymmetric unit corresponding to twelve selenomethionine and two iron sites. cTHAP4 was phased to 1.79 Å using a single wavelength (0.97857 Å) with autoSharp ²². The experimental electron density map was of excellent quality, and the bound heme moites were apparent in the density modified map. The structure was completed with alternate cycles of model building in Coot ²³ and refinement in Phenix ²⁴. All refinement steps were monitored using an R_free value based on 5.0% of the independent reflections.

The final model of cTHAP4 was refined to a resolution of 1.79 Å with an R_cryst of 0.165 and an R_free of 0.201. All pertinent information on data collection, refinement, phasing, and model statistics are summarized in Table I. Model quality was assessed using MolProbity²⁵. cTHAP4 renderings were generated using PyMOL ²⁶.

Table I.

Summary of crystal parameters, data collection, structure phasing and refinement statistics. Values in parentheses are for the highest resolution shell.

Crystal parameters
Space group	P212121
Unit-cell parameters (Å)	a = 41.933, b = 74.694, c = 103.064
Data collection statistics
Wavelength (Å)	0.97857
Resolution range (Å)	35.12 – 1.79 (1.84 – 1.79)
No. of reflections (measured / unique)	173378 / 29830
Completeness (%)	95.9 (76.2)
Rmerge*	0.093 (0.457)
Redundancy	5.8 (4.8)
Mean I / sigma (I)	18.16 (2.92)
Phasing statistics‡
Mean FOM (centris / acentric)	0.306 / 0.116
Phasing power (anomalous)	0.963
Cullis R-factor (anomalous)	0.846
Refinement and model statistics
Resolution range (Å)	35.12 – 1.79
No. of reflections (work / test)	29830 / 1510
Rcryst§	0.165 (0.192)
Rfree¶	0.201 (0.248)
RMSD bonds (Å)	0.009
RMSD angles (°)	1.449
B factor (protein / solvent / Heme) (Å2)	23.43 / 23.14 / 24.94
No. of protein atoms	2636
No. of waters	273
No. of auxiliary molecules	2 Heme
Ramachandran plot (%)
Favorable region	98.44
Additional allowed region	1.56
Disallowed region	0.00
PDB ID	3IA8

^*R_merge = Σ_h Σ_i | I_i (h) − <I(h)>| / Σ_hΣ_i I_i(h), where I_i(h) is the intensity of an individual measurement of the reflection and <I(h)> is the mean intensity of the reflection.

^‡Phasing in autoSharp

^§R_cryst = Σ_h ‖F_obs| − |F_calc‖ / Σ_h |F_obs|, where F_obs and F_calc are the observed and calculated structure-factor amplitudes,

^¶R_free was calculated as R_cryst using 5.0 % of randomly selected unique reflections that were omitted from the structure refinement.

Results and Discussion

Overall structure and structure quality

The cTHAP4 structure had well-defined electron density corresponding to residues 415-576. There was not sufficient electron density to definitively place residues 413, 414, and 577, and subsequently, these residues were not modeled in the cTHAP4 structure. Two molecules of cTHAP4 were observed per asymmetric unit and the structure belonged to the space group P2₁2₁2₁.

cTHAP4 is composed almost exclusively of β-strands. Ten antiparallel strands form a compact β-barrel, which is capped on one end by ten residue long 3₁₀ α-helix. The other end of the β-barrel is exposed to the solvent (Figure 1A). The overall fold of cTHAP4 is identical to the fold observed in heme-binding protein nitrobindin¹⁴, which is not surprising, given that cTHAP4 and nitrobindin share 59% sequence similarity. A large cavity composed of primarily hydrophobic residues is formed in the interior of the β-barrel. In the cTHAP4 structure, a heme molecule is bound in this hydrophobic cavity and is coordinated by His567 (Figure 1A). Similar to Nitrobindin, there is no distal histidine within bonding distance of the heme iron. However, His480 is positioned in such a way that the imizidazole ring in parallel to the heme plane. One of the two heme propionate groups forms a hydrogen bond with Thr444, while the other propionate group is solvent-exposed and does not hydrogen bond with any amino acid side chains. The heme pocket is relatively open when compared to the globins with the heme primarily solvent-exposed.

Figure 1.

A. Cartoon representation of cTHAP4 is shown going from blue at the N-terminus to red at the C-terminus. The bound heme is shown as sticks with carbon in white, nitrogen in blue, oxygen in red, and iron in orange. Looking down the barrel shows the heme coordinated by His567 and the extended hydrophobic cavity formed by the β-barrel. B. Structural alignment of the cTHAP4 dimer in green and the Nitrobindin dimer 14 (PDB ID 3EMM) in brown. Conserved hydrophobic residues that compose the dimer interface are shown as black sticks.

The two monomers of cTHAP4 in the asymmetric unit form an interface of approximately 1312 Ų as calculated by the PISA server²⁷, suggesting that cTHAP4 forms a dimer in the crystal lattice. The cTHAP4 dimer is identical to the dimer observed in the nitrobindin structure ¹⁴ (Figure 1B). A number of hydrophobic residues that are conserved between cTHAP4 and nitrobindin compose the dimer interface (Figure 1B). Nitrobindin likely forms a dimer in solution, further suggesting that the cTHAP4 dimmer is biologically relevant and perhaps plays a functional role.

cTHAP4 Homologues

A BLAST search utilizing the cTHAP4 sequence revealed the closest cTHAP4 homologues to be THAP4 proteins from other mammalian species. In addition to the mammalian THAP4 proteins, a number of hypothetical proteins or proteins of unknown function from plant and bacteria were found to be 38% to 34% identical to cTHAP4. Of these additional proteins the only one that has been characterized is the NO binding nitrobindin. A sequence alignment of cTHAP4 and its closest homologues reveals that the heme-binding histidine (His567) and the residues within 4 Å of the bound heme are highly conserved (Figure 2). Given the conservation of the heme-binding histidine it is extremely likely that all THAP4 proteins coordinate a heme.

Figure 2.

Sequence alignment of THAP4 from Mus musculus (Q6P3Z3), THAP4 from Rattus norvegicus (Q642B6), THAP4 from Homo sapiens (Q8WY91), THAP4 from Callithrix jacchus (A6MKW1), THAP4 from Bos taurus (Q2TBI2), Nitrobindin from Arabidopsis thaliana (O64527), uncharacterized protein from Vitis vinifera (A5BBZ0), fatty acid-binding like protein from Mycobacterium smegmatis (A0R6J8), and fatty acid-binding like protein from Mycobacterium vanbaalenii (A1T297). Residues that are within 4 A of the bound heme of cTHAP4 are highlighted in green. The strictly conserved histidine that coordinates the heme is highlighted in red.

A number of cTHAP4 structural homologues were identified by Dali²⁸. The top structure indentified was nitorbindin (PDB ID 3EMM Z = 32.8). cTAHP4 and nitrobindin are structurally nearly identical and have an overall C_α RMS deviation of 0.68 Å (Figure 1B). Two uncharacterized proteins from Mycobacterium tuberculosis (PDB ID 2FR2 Z = 24.3 and PDB ID 2FWV Z = 23.4) were close structural homologues to cTHAP4. Despite having a histidine in a structurally similar position as cTHAP4 both 2FR2 and 2FWV were solved without a bound heme ²⁹. Several additional proteins were identified as possible structural homologues that were either hypothetical proteins or functionally annotated as fatty acid binding proteins.

Possible Function

Given the structural similarities between cTHAP4 and nitrobindin, and to a lesser extent the nitophorins¹⁵, it is probable that cTHAP4 binds NO and has similar NO binding properties to nitrobindin¹⁴. Perhaps cTHAP4 acts as a NO sensor, which utilizes the heme coordinated to His567 as a NO binding platform that can modulate the DNA binding activity of the N-terminal THAP domain. This would allow THAP4 to act as a NO dependent transcriptional regulator. While none of the THAP domain containing proteins are known to interact with NO, THAP7 and THAP11 have been shown to be transcriptional regulators ^5,6. In addition to transcriptional regulation, THAP proteins has also been implicated in mediating cell death^3,4. It is possible that THAP4 functions in a similar manner and is involved in NO regulated apoptosis³⁰. While the function of THAP4 still remains unknown, the structure of cTHAP4 provides not only insight into a possible function, but also a direction for future research on the role of THAP4.