Crystal structure of the methyltransferase domain of human TARBP1

HIV-1 is the causative agent of acquired immunodeficiency syndrome (AIDS). The virus contains a RNA genome that produces a chromosomally integrated DNA during the replicative cycle. Regulation of HIV-1 gene expression requires the transactivator protein Tat,1 the cis-acting regulatory elements in the long terminal repeat (LTR),2 and the downstream regulatory element TAR.3 The upstream regulatory elements including SP1 binding sites and the TATA element are critical for both basal and Tat-induced gene expression. Tat is a RNA-binding protein4, 5 that plays a significant role in transcription elongation,6 as well as in transcription initiation.7, 8 Tat activity requires the transactivation-responsive region, TAR, which is the regulatory region located downstream of the HIV-1 transcriptional initiation site.3 TAR functions as a RNA regulatory signal rather than as a DNA element.9 It forms a stable stem-loop structure where Tat binds.10 Studies showed that Tat activates the LTR less efficiently in rodent than in human cells, suggesting that cellular RNA-binding proteins are also involved in the regulation of HIV replication.11-13 Searching for cellular cofactor which interacts with the TAR RNA identified one binding partner of TAR: TAR (HIV-1) RNA binding protein 1(TARBP1).12, 14 TARBP1 and RNA polymerase II compete for binding to TAR RNA. It is believed that TARBP1 protein acts to disengage RNA polymerase II from TAR during transcriptional elongation.15, 16 TARBP1 is a protein of 1621 amino acids. This protein does not contain any classical RNA binding motifs such as zinc fingers or ribonucleoprotein binding domains. A Leucine zipper domain was identified in the middle of the protein (amino acids 535–556). The most conserved part of the protein is the C-terminus of TARBP1. It contains a methyltransferase domain, which belongs to the SPOUT (SpoU-TrmD) methyltransferase superfamily.17 This family of methytransferases is responsible for m1G, m3U, and 2′-O-methylation of the ribose sugar of both tRNA and rRNA.18 RNAs involved in translation and post-transcriptional process contain a lot of modified nucleotides.19, 20 Methylation in RNAs plays an important role in pre-RNA processing and RNA functions.21-23 In bacteria, RNA modifications are involved in antibiotics resistance and the most common modes of resistance is through methylation of the 23S rRNA.24, 25 To date, a number of structures of SPOUT family members from bacteria have been solved. However, no structural information is available for the eukaryotic members of this family. Here we report the crystal structure of methyltransferase domain of human TARBP1 protein in complex with S-adenosyl-L-homocysteine (AdoHcy) at 1.6-Å resolution. Our structure reveals a structure fold that is different from the classical AdoMet-dependent methyltransferase fold26 and a co-factor (AdoHcy) binding mode that is distinct from that in the classical methyltransferases. These structural features are the hallmarks of the SPOUT methyltransferase superfamily. DNA fragment encoding the methyltransferase domain of human TARBP1 (amino acids 1438–1621) was amplified by PCR and sub-cloned into pET28a-LIC vector (http://www.sgc.utoronto.ca/SGC-WebPages/toronto-vectors.php), downstream of the poly-histidine coding region. Human TARBP1 methyltransferase domain was over expressed in E. coli BL21 (DE3) codon plus RIL strain (Stratagene) by the addition of 1 mM isopropyl-1-thio-D-galactopyranoside and incubated overnight at 15°C. Harvested cells were resuspended in 50 mM sodium phosphate buffer, pH 7.4, supplemented with 250 mM NaCl, 5 mM imidazol, 2 mM β-mercaptoethanol and 5% glycerol. The cells were lysed by passing through a microfluidizer (Microfluidics Corporation) at 20,000 psi. After clarification of the crude extract by high-speed centrifugation, the lysate was loaded onto a 5 mL HiTrap Chelating column (Amersham Biosciences), charged with Ni2+. Column was washed with 10 column volumes of 20 mM Tris-HCl buffer, pH 8.0, containing 250 mM NaCl, 50 mM imidazole and 5% glycerol, the protein was then eluted with 20 mM Tris-HCl buffer, pH 8.0, 250 mM NaCl, 250 mM imidazole, 5% glycerol. The protein was loaded on a Superdex200 column (Amersham Biosciences), equilibrated with 20 mM Tris-HCl buffer, pH 8.0, and 150 mM NaCl. Thrombin (Sigma) was added to combined fractions containing TARBP1 to remove His-tag. The protein was further purified to homogeneity by ion-exchange chromatography. The protein purification yield was 25.4 mg protein from 1 L of culture. Purified TARBP1 protein (11 mg/mL) was crystallized in the presence of S-adenosyl-L-homocysteine (Sigma), using the hanging drop vapor diffusion method at 20°C by mixing equal volume of the protein solution with the reservoir solution containing 20% PEG 5000 MME, 0.1 M Bis-Tris pH 6.5. Crystals were soaked in the corresponding mother liquor supplemented with 20% glycerol as cryoprotectant before freezing in liquid nitrogen. X-ray diffraction data were collected at 100 K at beamline 17ID of advanced photon source (APS) at Argonne National Laboratory. Data were processed using the HKL2000 suite.27 The structure of methyltransferase domain of TARBP1 was solved by molecular replacement using the program MOLREP.28 The PDB 1GZ0 was used as the search model. ARP/wARP was used for automatic model building.29 Graphics program COOT30 was used for model building and visualization. Crystal diffraction data and refinement statistics for the structure are displayed in Table I. The methyltransferase domain of TARBP1 was expressed in E. coli cells and purified to homogeneity by Ni chelating column, followed by gel filtration chromatography and ion exchange chromatography. The protein exists as a dimer in solution as was judged by gel-filtration chromatography (data not shown). The purified protein was crystallized in complex with S-adenosyl-L-homocysteine (AdoHcy). The crystals belong to space group P21 and contain two subunits of TARBP1 per crystallographic asymmetric unit [Fig. 1(A)]. The two molecules are identical and can be superimposed with root-mean-square deviation (RMSD) of 0.287 Å for all Cα atoms. Overall strucuture of human TARBP1 methyltransferase domain. (A) Dimerization of TARBP1 methyltransferase domain. The structure is shown in cartoon representation. The two chains are colored in blue and red. The bound cofactor, AdoHcy, is shown in stick-and-ball representation, with the carbon atoms colored in gray. (B) Overall fold of TARBP1 monomer. The structure is shown in cartoon representation. All secondary structure elements are labeled. The N- and C-terminus are labeled as NT and CT, respectively. (C) The knot structure near the C-terminus of TARBP1. The knot structure is formed by threading the β6 strand through the β4–β5 loop. The β4–β5 loop is colored in blue, the β6 strand is colored in red. The final model of TARBP1 consists of residues 1438–1621, one AdoHcy molecule for each subunit. The three-dimentional structure of TARBP1 is shown in Figure 1(B). Each subunit of TARBP1 consists of a catalytic core domain of six parallel β-strands (β3-β2-β1-β5-β4-β6) packed between two layers of α-helices (α1 and α8 on one side, α5 and α7 on the other side). The catalytic core is a typical Rossmann fold. However, at the C-terminus, an unusual knot is formed by threading the β6 strand through the β4–β5 loop [Fig. 1(C)]. The binding site for cofactor is located at the knotted C-terminus, as defined by the bound AdoHcy. TARBP1 forms a homodimer, with the two monomers in almost perpendicular mode [Fig. 1(A)]. It dimerizes mainly through a number of interface contacts between helices α2, α8, and loop β6–α8 of each monomer. Dimerization buries 20.4% of the total surface area of each monomer, resulting in a large buried interface of 2948.4 Å2, with 1474.2 Å2 contributed from each monomer. The dimerization interface involves one of the loop (β6–α8) participating the formation of cofactor binding site. The loop from one monomer is stabilized by the interactions with the other monomer. The involvement of active site residues in the dimerization interface indicates the critical role of dimerization in activity of the enzyme. Our crystal structure of TARBP1 was determined with the bound cofactor AdoHcy. The cofactor binds to the protein in a bent conformation in a pocket of two loops of the knotted region, with the homocysteine moiety positioned folding back towards the adenosine moiety [Fig. 2(A)]. This bent conformation is different from the extended conformation of AdoMet/AdoHcy found in most of the AdoMet-dependent methyltransferases. However, in all the available structures of SPOUT family members, the co-factor is bound in the same conformation as TARBP1-AdoHcy complex.31 The binding pocket involves the loops between β4–α6, β5–α7, and β6–α8 [Fig. 2(B)]. The AdoHcy-binding interactions [Fig. 2(C)] are mostly between the adenosine moiety and the protein backbone. An extensive set of van der Waals contacts were established between the adenine moiety and residues V1543, Q1545, V1584, E1585, and S1600. In addition, the adenine ring forms hydrogen bond with L1595 (amide nitrogen), I1586 (amide nitrogen and backbone carbonyl) and Q1588. The ribose ring forms hydrogen bond with G1566 (amide nitrogen) and E1544. It also makes van der Waals contacts with residues V1543, G1571, and V160. The homocysteine moiety is held by the interaction with R156, N130, E131, and S1594. The sulfur atom (also likely the reactive methyl group in AdoMet) of AdoHcy is pointing to an adjacent concavity where the RNA substrates most likely to bind. The groove itself and surrounding surface are positively charged, which provides an ideal environment for nucleotide binding. Similar putative substrate binding site was also observed in other SPOUT family proteins.32 The active site of TARBP1 methyltransferase domain. (A) Stereo view (cross-eyed) of the bend conformation of AdoHcy. The structure is shown in carton representation. The loops involved in cofactor binding are colored in red. AdoHcy is shown in stick-and ball representation and the 2mFo − DFc map of the compound is shown in mesh (contoured to 1.0 a). (B) Stereo view (cross-eyed) of the cofactor binding site. All the residues interacting with AdoHcy are labeled and shown in stick-and-ball representation, with carbon atoms colored in green cyan. (C) Electro static potential surface of TARBP1 dimer. The surface is colored in red for negative charge, blue for positive charge and white for neutral. The close-up view of the positively charged grove where the RNA substrates could bind is shown on the right. We have compared the structure of TARBP1 methyltransferase domain against a non-redundant set of structures. DALI33 search revealed a set of closely related structures to TARBP1 (see Fig. 3). All these proteins are from the SPOUT superfamily and harbor the conserved motifs I-III of SPOUT superfamily.35 The fold of the core domain of all the structures, and the mode of the cofactor binding are similar to that of other previously reported SPOUT family proteins.31 The most similar structure is the SpoU Methyltransferase AviRb from Streptomyces viridochromogenes (PDB code: 1 × 7O,36 Z-score 20.1). The core domain of the two structures can be superimposed with a RMSD of 1.9 Å for all Cα atoms. The RMSD value of the superimposition of methyltransferase domain of TARBP1 and the TrmH tRNA-modifying enzyme from Aquifex aeolicus (PDB code: 1ZJR,32), a putative RNA methyltransferase of the TrmH family from Porphyromonas gingivalis (PDB code: 2I6D) and the YibK methyltransferase from Haemophilus influenzae HI0766 (PDB code:1J85,37) are 1.7, 1.7, and 2.3 Å, respectively (Z-scores from DALI search are 19.3, 19.0, 17.1, respectively). As the first structure of SPOUT family member from eukaryotes, this conserved structural fold of TARBP1 methyltransferase domain provides important evidence on the molecular evolution aspect of SPOUT family. Our results support the recently finding that all the members of this superfamily have a common ancestor.38 Multiple sequence alignment of catalytic domain of selected members of SPOUT family. Identical, very similar, similar residues are colored in red, green and blue respectively in alignment. Secondary structure elements of human TARBP1 are shown above the sequences and labeled: the helices are shown as cylinders, the strands are shown as arrow bars. The residues interacting with AdoHcy are labeled with orange triangles. The conserved motifs I-III of SPOUT family are underlined and labeled. The alignment was generated using Clustal W34 assisted with hand fittings. The sequences of catalytic core domains shown are: human TARBP1, TrmH tRNA-modifying enzyme from Aquifex aeolicus (O67577), SpoU Methyltransferase AviRb from Streptomyces viridochromogenes (AAG32066), putative RNA methyltransferase of the TrmH family from Porphyromonas gingivalis (AAQ67107) and the YibK methyltransferase from Haemophilus influenzae HI0766 (P44868). The HIV is still one of the gravest threats to public health now a day. Currently, the antiviral drugs for treatment of HIV infection are restricted to inhibitors of reverse transcriptase and the viral proteases. Despite of the efficient reducing of the level of viral replication, the effects of these drugs are short-term, due to the emergence of resistant strains and the unaffected latently infected strains. The Tat–TAR interaction is an attractive target for antiviral drug development, since Tat is involved in multiple stages of viral infection. As the cellular cofactor for Tat–TAR interaction, TARBP1 could be a target for interruption of the interaction. Our structure of TARBP1 can provide important structural information to facilitate the development of new antiviral drugs. We thank Peter Loppnau for excellent technical support in cloning. The Structural Genomics Consortium is a registered charity that receives funds from the Canadian Institutes for Health Research, the Canadian Foundation for Innovation, Genome Canada through the Ontario Genomics Institute, GlaxoSmithKline, Karolinska Institutet, the Knut and Alice Wallenberg Foundation, the Ontario Innovation Trust, the Ontario Ministry for Research and Innovation, Merck & Co., Inc., the Novartis Research Foundation, the Swedish Agency for Innovation Systems, the Swedish Foundation for Strategic Research and the Wellcome Trust.

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