- Email: [email protected]
P60 domain
FEBS Letters 584 (2010) 4222–4226
journal homepage: www.FEBSLetters.org
Solution structure of the N-terminal catalytic domain of human H-REV107 – A novel circular permutated NlpC/P60 domain Xiaobai Ren a,b, Jian Lin a,b,⇑, Changwen Jin a,b,c, Bin Xia a,b,c,⇑⇑ a
Beijing Nuclear Magnetic Resonance Center, Peking University, Beijing 100871, China College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China c School of Life Sciences, Peking University, Beijing 100871, China b
a r t i c l e
i n f o
Article history: Received 12 August 2010 Revised 3 September 2010 Accepted 4 September 2010 Available online 17 September 2010 Edited by Stuart Ferguson Keywords: H-REV107 Tumor suppressor Phospholipase Lecithin retinol acyltransferase NMR
a b s t r a c t H-REV107 is a Ca2+-independent phospholipase A1/2, and it is also a pro-apoptosis protein belonging to the novel class II tumor suppressor family, H-REV107-like family. Here we report the solution structure of the N-terminal catalytic domain of human H-REV107, which has a similar architecture to classical NlpC/P60 domains, even though their fold topologies are different due to circular permutation in the primary sequence. The phospholipase active site possesses a structurally conserved Cys–His–His catalytic triad as found in NlpC/P60 peptidases, indicating H-REV107 should adopt a similar catalytic mechanism towards phospholipid substrates to that of NlpC/P60 peptidases towards peptides. As H-REV107 is highly similar to lecithin retinol acyltransferase, our study also provides structural insight to this essential enzyme in retinol metabolism. Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction H-REV107 (also known as H-REV107-1, H-REV107-3 and HRSL3) is a representative of a novel class II tumor suppressor family, designated as H-REV107-like family [1]. The H-REV107 gene is ubiquitously expressed in most normal tissues, but obviously downregulated in many kinds of tumor cells like carcinoma of ovary, cervix, and kidney etc. [2,3]. Overexpression of H-REV107like proteins efficiently inhibits the growth of tumor cells and induces apoptosis [4–7]. H-REV107-like proteins were found to be homologous to lecithin retinol acyltransferase (LRAT), an enzyme that catalyzes the transfer of the sn-1 acyl group of phosphatidylcholine (PC) to alltrans-retinol and forming a retinyl ester [8,9]. Recently, it was demonstrated that H-REV107 and other members of H-REV107like family, TIG3 and HRASLS2, possess Ca2+-independent phospholipase A1/2 activity towards a variety of phospholipid substrates in vivo and in vitro, indicating their involvement in phospholipid metabolism in cells [10–13].
Based on primary sequence analysis, it was proposed that LRAT family proteins, including H-REV107, show a circular permutation with respect to classical members of NlpC/P60 superfamily, which mostly comprises prokaryotic peptidases [14]. H-REV107 contains a conserved proline-rich motif at the N terminus and a putative Cterminal transmembrane domain which was found to be crucial for cellular localization, but not necessary for the enzyme activity [4,12]. Here we report the solution structure of the catalytic N-terminal domain of H-REV107 (H-REV107N) without the putative C-terminal transmembrane domain, the first structure of the LRAT family, which provides structural insight for the enzyme activities. 2. Materials and methods 2.1. Sample preparation The plasmid construction, protein expression and purification, and NMR sample preparation were described previously [15]. 2.2. NMR spectroscopy
⇑ Corresponding author at: Beijing Nuclear Magnetic Resonance Center, Peking University, Beijing 100871, China. ⇑⇑ Corresponding author at: Beijing Nuclear Magnetic Resonance Center, Peking University, Beijing 100871, China. E-mail addresses: [email protected] (J. Lin), [email protected] (B. Xia).
The backbone sequential and side-chain assignments of HREV107N were reported previously [15]. The three-dimensional 15 N-edited, 13C-edited aliphatic and 13C-edited aromatic NOESYHSQC spectra (mixing time 100 ms) were collected on Bruker
0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.09.015
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Table 1 Restraints and structural statistics. Restraints Intra-residue distance restraints Sequential distance restraints Medium range distance restraints Long range distance restraints Total unambiguous distance restraints Total ambiguous distance restraints Hydrogen bond restraints Dihedral angle restraints (u and w)
1317 655 315 542 2832 654 26 74
Restraints violations Distance (>0.3 Å) Dihedral angle (>5°)
0 0
AMBER energy (kcal/mol) Mean AMBER energy NOE distance restraints violation energy Torsion angle restraints violation energy
6192.14 ± 14.00 21.44 ± 2.57 1.00 ± 0.24
RMSD from mean structure (Å) Backbone heavy atoms in secondary structures All heavy atoms in secondary structures
0.33 ± 0.06 0.83 ± 0.11
Ramachandran statistics (%) Residues in most favored regions Residues in additional allowed regions Residues in generously allowed regions Residues in disallowed regions
81.1 17.9 0.4 0.7
Avance 800 MHz spectrometer at 298 K. Hydrogen–deuterium exchange experiments were performed on Bruker Avance 600 MHz spectrometer at 298 K. The chemical shifts were referenced to internal 2,2-dimethyl-2-silapentanesulfonic acid (DSS) [16]. All NMR spectra were processed using NMRPipe [17] and analyzed using NMRView [18]. 2.3. Structure calculations Distance restraints for structure calculation were generated by using the three-dimensional 15N-edited, 13C-edited aliphatic and
C-edited aromatic NOESY-HSQC spectra. Dihedral angle restraints were determined from backbone chemical shifts using TALOS [19]. Hydrogen bond restraints were obtained based on data from the hydrogen–deuterium exchange experiments. The tautomeric states of all four histidine residues were determined from 2D 1H to 15N HMQC spectrum (Supplementary Fig. 1) [20]. The structure calculations were performed using CYANA [21]. A total of 200 structures were calculated using CYANA, and 100 structures with the lowest target function values were selected. Then the structure refinement was carried out using the generalized Born (GB) solvent model in AMBER [22–23]. Finally, the 20 lowest energy structures were selected for representation. The final structures were analyzed using PROCHECK_NMR [24] and MOLMOL [25]. 3. Results 3.1. Solution structure of H-REV107 N-terminal domain The N-terminal phospholipase domain of H-REV107 (residues 1–125, H-REV107N) was expressed in Escherichia coli as a water soluble and well folded protein. The solution structure of HREV107N was determined with high quality with 3486 inter-proton NOE-derived distance constraints together with 74 dihedral angle and 26 hydrogen bond constraints. The constraints and structural statistics are summarized in Table 1. The 20 representative structures of H-REV107N have been deposited under PDB ID: 2KYT. The structure of H-REV107N comprises a six-stranded anti-parallel b-sheet (b1, residues 13–18; b2, residues 21–29; b3, residues 32–37; b4, residues 58–64; b5, residues 73–76; b6, residues 103– 104) and three a helices (a1, residues 65–68; a2, residues 89– 99; a3, residues 110–122) with a b1–b2–b3–b4–a1–b5–a2–b6– a3 sequential connectivity (Fig. 1). The short helix a1 is on one side of the b-sheet, while helices a2 and a3 are on the other side. The
Fig. 1. Solution structure of H-REV107 N. (A and C) Superimposition of the backbone trace of the 20 representative structures of H-REV107 N with a 65° rotation. (B and D) Ribbon diagrams of the mean structure with the secondary structural elements labeled oriented as (A and C), respectively.
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Fig. 2. Structural comparison of H-REV107 N and the NlpC/P60 domain of Spr (PDB: 2K1G). A, primary sequence alignment of H-REV107 N and the NlpC/P60 domain of Spr from E. coli performed by ClustalX [33] and pictured by ESPript 2.2 (http://espript.ibcp.fr/ESPript/ESPript/). The secondary structural elements are labeled, and the individual secondary elements in the two proteins are labeled in different colors (H-REV107 in red, and Spr in blue). The residue numbers are respectively labeled on different side of the sequences. The three residues of the Cys-His-His catalytic triad are indicated by blue triangles. B, structural comparison of H-REV107 N (pink) and the NlpC/P60 domain of Spr (PDB: 2K1G) (pale blue) in ribbon diagram (27). The two swapped a-helices are highlighted in red for H-REV107 N and in blue for Spr. C and D, topologies of H-REV107 N and 2K1G with the sequence number labeled. The colors of the two swapped a-helices and the five b-strands are corresponding to those in B.
N-terminal tail (residues 1–12) and the long loop (residues 38–57) connecting strand b3 and b4 are very flexible. 3.2. Structural comparison of H-REV107N and classical NlpC/P60 domain As H-REV107 was proposed to be a circular permutation with the catalytic domain of classical NlpC/P60 family proteins, we compared the structure of H-REV107N with that of the NlpC/P60 peptidase domain of E. coli lipoprotein Spr (PDB: 2K1G) [14,26]. Indeed, the overall architectures of the two structures are quite similar, even though their topologies are different (Fig. 2). Superimposing the five b-strands b1–b5 in H-REV107N with the corresponding b-strands in Spr reveals a backbone heavy atom root mean square deviation (RMSD) of 1.8 Å for the five b-strands, while the backbone heavy atom RMSD of helices a2 and a3 in H-REV107 and the corresponding a-helices in Spr is 4.0 Å (Fig. 2B). The major difference between the two structures is that helices a2 and a3 in H-REV107N are located at the C-terminus of the polypeptides, but the two structurally corresponding a-helices in Spr are at the Nterminus of the NlpC/P60 peptidase domain, which is typical of circular permutation (Fig. 2A). The structure of H-REV107N provides a direct evidence for the existence of circular permutation in NlpC/ P60 domain proteins.
tural comparison also reveals that residues C113, H23 and H35 of H-REV107 are located at almost identical positions as residues C68, H119 and H131 that form a Cys–His–His catalytic triad in the NlpC/P60 peptidase domain of Spr [26]. Side-chains of these corresponding residues also point in similar directions, respectively (Fig. 3). The two histidine residues in the catalytic triad are in the neutral Ne2–H tautomeric state, the same as in the structure
4. Discussion In this paper, we report the solution structure of the N-terminal phospholipase domain of H-REV107, the first structure of the novel H-REV107-like tumor suppressor family, which provides the structural evidence for the circular permutation of NlpC/P60 domain proteins. In previous reports, residues H23 and C113 of H-REV107 were found to be essential for its phospholipase activity [11–13]. Struc-
Fig. 3. The catalytic triads of H-REV107 N and the NlpC/P60 domain of Spr (PDB: 2K1G). The side chain heavy atoms of the Cys-His-His catalytic triad residues in each structure are shown in different colors (H-REV107 in red and 2K1G in blue). The distances between C113 Sc and H23 Nd1 atoms and between H23 Ne2 and H35 Nd1 atoms from the mean structure of H-REV107N are indicated in red, and the corresponding distances (C68 Sc to H119 Nd1, and H119 Ne2 to H131 Nd1) from the mean structure of 2K1G are indicated in blue. The conserved arginine residue of HREV107 near the catalytic triad is shown in red.
X. Ren et al. / FEBS Letters 584 (2010) 4222–4226
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Fig. 4. Sequence alignments of the catalytic domains of H-REV107 and LRAT family proteins HRASL2, TIG3 and LRAT generated by ClustalX [33] and pictured by ESPript 2.2 (http://espript.ibcp.fr/ESPript/ESPript/). The secondary structural elements of H-REV107N are labeled. The residue numbers of H-REV107 are labeled on the top. The three residues of Cys-His-His catalytic triad and the conserved arginine residues are indicated by blue triangles.
of Spr. Thus, even though the two enzymes have different substrates, H-REV107 should adopt a similar catalytic mechanism as that of the NlpC/P60 peptidase domain of Spr, in which the deprotonation of the residue C113 thiol by the basic side-chain of residue H23 generates a nucleophile anionic sulfur to attack the carbonyl group of the substrate, and the third polar residue H35 assists to properly orient the side-chain of H23 [14,26]. H-REV107-like family proteins also belong to the lecithin retinol acyltransferase (LRAT) protein family, which are essential enzymes in the all-trans-retinol metabolism. Except for the predicted N- and C-terminal transmembrane domain of LRAT [27], the catalytic domains of H-REV107 and LRAT are highly homologous (identity 26%) [28,29] (Fig. 4). The structure of HREV107N is also the first structure of LRAT family, which may provide structural insight for understanding the mechanism of LRAT. It was reported that C161 and H60 are essential for the activity of LRAT and formed catalytic dyad through the sequence alignments and mutagenesis [28]. However, in the thiol enzymes, a third polar residue that is also important for the catalysis is still not clearly identified even though extensive mutagenesis studies have been carried out [28–30]. Based on the primary sequence alignments and the structure of H-REV107N (Fig. 4), we propose that the conserved residues C161, H60, and H72 of LRAT should form a Cys– His–His catalytic triad as that in H-REV107, and H72 should correspond to H35 of H-REV107 and play the role to position the sidechain of H60. It has been reported that the H72Q mutant of LRAT is a little more active than wild-type LRAT [30], suggesting that glutamine can play a similar role as histidine at this position [26,31]. In addition, the positively charged residue R18 of HREV107 is just located near the catalytic triad and it is highly conserved in the LRAT family, which may play a role in stabilizing the phosphate group of the phospholipid substrates as the arginine residue in cytosolic phospholipase A2 does [32] (Fig. 3). In summary, we have solved the solution structure of the N-terminal catalytic domain of H-REV107, which is the first structure of H-REV107-like family, and also the first structure of circular permutated NlpC/P60 domains. According to the structural analysis, the phospholipase active site of H-REV107 consists a Cys–His–His catalytic triad, indicating H-REV107 should adopt a similar catalytic mechanism as NlpC/P60 peptidases, which also provides structural insights for the lecithin retinol acyltransferase. Acknowledgements All NMR experiments were carried out at Beijing NMR center. This research was supported by Grant 2009CB521703 (to BX) from
the 973 Program of China, and Grants 30125009 (to BX) and 30800172 (to JL) from the National Science Foundation of China. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.febslet.2010.09.015. References [1] Hughes, P.J. and Stanway, G. (2000) The 2A proteins of three diverse picornaviruses are related to each other and to the H-rev107 family of proteins involved in the control of cell proliferation. J. Gen. Virol. 81, 201–207. [2] Husmann, K., Sers, C., Fietze, E., Mincheva, A., Lichter, P. and Schafer, R. (1998) Transcriptional and translational downregulation of H-REV107, a class II tumour suppressor gene located on human chromosome 11q11–12. Oncogene 17, 1305–1312. [3] Roder, K., Latasa, M.J. and Sul, H.S. (2002) Silencing of the mouse H-rev107 gene encoding a class II tumor suppressor by CpG methylation. J. Biol. Chem. 277, 30543–30550. [4] Sers, C., Emmenegger, U., Husmann, K., Bucher, K., Andres, A.C. and Schafer, R. (1997) Growth-inhibitory activity and downregulation of the class II tumorsuppressor gene H-rev107 in tumor cell lines and experimental tumors. J. Cell Biol. 136, 935–944. [5] Sers, C. et al. (2002) The class II tumour suppressor gene H-REV107–1 is a target of interferon-regulatory factor-1 and is involved in IFNgamma-induced cell death in human ovarian carcinoma cells. Oncogene 21, 2829–2839. [6] Nazarenko, I., Schafer, R. and Sers, C. (2007) Mechanisms of the HRSL3 tumor suppressor function in ovarian carcinoma cells. J. Cell Sci. 120, 1393–1404. [7] Tsai, F.M., Shyu, R.Y. and Jiang, S.Y. (2007) RIG1 suppresses Ras activation and induces cellular apoptosis at the golgi apparatus. Cell. Signal. 19, 989–999. [8] MacDonald, P.N. and Ong, D.E. (1988) Evidence for a lecithin-retinol acyltransferase activity in the rat small intestine. J. Biol. Chem. 263, 12478– 12482. [9] Saari, J.C. and Bredberg, D.L. (1989) Lecithin:retinol acyltransferase in retinal pigment epithelial microsomes. J. Biol. Chem. 264, 8636–8640. [10] Uyama, T., Jin, X.H., Tsuboi, K., Tonai, T. and Ueda, N. (2009) Characterization of the human tumor suppressors TIG3 and HRASLS2 as phospholipidmetabolizing enzymes. Biochim. Biophys. Acta 1791, 1114–1124. [11] Uyama, T., Morishita, J., Jin, X.H., Okamoto, Y., Tsuboi, K. and Ueda, N. (2009) The tumor suppressor gene H-Rev107 functions as a novel Ca2+-independent cytosolic phospholipase A1/2 of the thiol hydrolase type. J. Lipid Res. 50, 685– 693. [12] Han, B.G. et al. (2010) Expression, purification and biochemical characterization of the N-terminal regions of human TIG3 and HRASLS3 proteins. Protein Expr. Purif. 71, 103–107. [13] Duncan, R.E., Sarkadi-Nagy, E., Jaworski, K., Ahmadian, M. and Sul, H.S. (2008) Identification and functional characterization of adipose-specific phospholipase A2 (AdPLA). J. Biol. Chem. 283, 25428–25436. [14] Anantharaman, V. and Aravind, L. (2003) Evolutionary history, structural features and biochemical diversity of the NlpC/P60 superfamily of enzymes. Genome Biol. 4, R11. [15] Ren, X., Lin, J., Jin, C. and Xia, B. (2010). 1H, 13C and 15N resonance assignments of human H-REV107 N-terminal domain. Biomol. NMR Assign. (in press). [16] Markley, J.L., Bax, A., Arata, Y., Hilbers, C.W., Kaptein, R., Sykes, B.D., Wright, P.E. and Wuthrich, K. (1998) Recommendations for the presentation of NMR
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X. Ren et al. / FEBS Letters 584 (2010) 4222–4226 structures of proteins and nucleic acids. IUPAC-IUBMB-IUPAB Inter-Union Task Group on the Standardization of Data Bases of Protein and Nucleic Acid Structures Determined by NMR Spectroscopy. J. Biomol. NMR 12, 1–23. Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J. and Bax, A. (1995) NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR 6, 277–293. Johnson, B.A. and Blevins, R.A. (1994) NMRView: a computer program for the visualization and analysis of NMR data. J. Biomol. NMR 4, 603–614. Cornilescu, G., Delaglio, F. and Bax, A. (1999) Protein backbone angle restraints from searching a database for chemical shift and sequence homology. J. Biomol. NMR 13, 289–302. Pelton, J.G., Torchia, D.A., Meadow, N.D. and Roseman, S. (1993) Tautomeric states of the active-site histidines of phosphorylated and unphosphorylated IIIGlc, a signal-transducing protein from Escherichia coli, using twodimensional heteronuclear NMR techniques. Protein Sci. 2, 543–558. Güntert, P., Mumenthaler, C. and Wüthrich, K. (1997) Torsion angle dynamics for NMR structure calculation with the new program DYANA. J. Mol. Biol. 273, 283–298. Pearlman, D.A.C., David, A., Caldwell, James W., Ross, Wilson S., Cheatham, Thomas E., Debolt, Steve, Ferguson, David, Seibel, George and Kollman, Peter (1995) AMBER, a package of computer programs for applying molecular mechanics, normal mode analysis, molecular dynamics and free energy calculations to simulate the structural and energetic properties of molecules. Comput. Phys. Commun. 91, 1–41. Xia, B., Tsui, V., Case, D.A., Dyson, H.J. and Wright, P.E. (2002) Comparison of protein solution structures refined by molecular dynamics simulation in vacuum, with a generalized Born model, and with explicit water. J. Biomol. NMR 22, 317–331.
[24] Laskowski, R.A., Rullmannn, J.A., MacArthur, M.W., Kaptein, R. and Thornton, J.M. (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J. Biomol. NMR 8, 477–486. [25] Koradi, R., Billeter, M. and Wüthrich, K. (1996) MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 14, 29–32. [26] Aramini, J.M. et al. (2008) Solution NMR structure of the NlpC/P60 domain of lipoprotein Spr from Escherichia coli: structural evidence for a novel cysteine peptidase catalytic triad. Biochemistry 47, 9715–9717. [27] Moise, A.R., Golczak, M., Imanishi, Y. and Palczewski, K. (2007) Topology and membrane association of lecithin: retinol acyltransferase. J. Biol. Chem. 282, 2081–2090. [28] Jahng, W.J., Xue, L. and Rando, R.R. (2003) Lecithin retinol acyltransferase is a founder member of a novel family of enzymes. Biochemistry 42, 12805–12812. [29] Xue, L. and Rando, R.R. (2004) Roles of cysteine 161 and tyrosine 154 in the lecithin-retinol acyltransferase mechanism. Biochemistry 43, 6120–6126. [30] Mondal, M.S., Ruiz, A., Hu, J., Bok, D. and Rando, R.R. (2001) Two histidine residues are essential for catalysis by lecithin retinol acyl transferase. FEBS Lett. 489, 14–18. [31] Li, J., Derewenda, U., Dauter, Z., Smith, S. and Derewenda, Z.S. (2000) Crystal structure of the Escherichia coli thioesterase II, a homolog of the human Nef binding enzyme. Nat. Struct. Biol. 7, 555–559. [32] Dessen, A., Tang, J., Schmidt, H., Stahl, M., Clark, J.D., Seehra, J. and Somers, W.S. (1999) Crystal structure of human cytosolic phospholipase A2 reveals a novel topology and catalytic mechanism. Cell 97, 349–360. [33] Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. and Higgins, D.G. (1997) The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Res. 25, 4876–4882.
journal homepage: www.FEBSLetters.org
Solution structure of the N-terminal catalytic domain of human H-REV107 – A novel circular permutated NlpC/P60 domain Xiaobai Ren a,b, Jian Lin a,b,⇑, Changwen Jin a,b,c, Bin Xia a,b,c,⇑⇑ a
Beijing Nuclear Magnetic Resonance Center, Peking University, Beijing 100871, China College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China c School of Life Sciences, Peking University, Beijing 100871, China b
a r t i c l e
i n f o
Article history: Received 12 August 2010 Revised 3 September 2010 Accepted 4 September 2010 Available online 17 September 2010 Edited by Stuart Ferguson Keywords: H-REV107 Tumor suppressor Phospholipase Lecithin retinol acyltransferase NMR
a b s t r a c t H-REV107 is a Ca2+-independent phospholipase A1/2, and it is also a pro-apoptosis protein belonging to the novel class II tumor suppressor family, H-REV107-like family. Here we report the solution structure of the N-terminal catalytic domain of human H-REV107, which has a similar architecture to classical NlpC/P60 domains, even though their fold topologies are different due to circular permutation in the primary sequence. The phospholipase active site possesses a structurally conserved Cys–His–His catalytic triad as found in NlpC/P60 peptidases, indicating H-REV107 should adopt a similar catalytic mechanism towards phospholipid substrates to that of NlpC/P60 peptidases towards peptides. As H-REV107 is highly similar to lecithin retinol acyltransferase, our study also provides structural insight to this essential enzyme in retinol metabolism. Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.
1. Introduction H-REV107 (also known as H-REV107-1, H-REV107-3 and HRSL3) is a representative of a novel class II tumor suppressor family, designated as H-REV107-like family [1]. The H-REV107 gene is ubiquitously expressed in most normal tissues, but obviously downregulated in many kinds of tumor cells like carcinoma of ovary, cervix, and kidney etc. [2,3]. Overexpression of H-REV107like proteins efficiently inhibits the growth of tumor cells and induces apoptosis [4–7]. H-REV107-like proteins were found to be homologous to lecithin retinol acyltransferase (LRAT), an enzyme that catalyzes the transfer of the sn-1 acyl group of phosphatidylcholine (PC) to alltrans-retinol and forming a retinyl ester [8,9]. Recently, it was demonstrated that H-REV107 and other members of H-REV107like family, TIG3 and HRASLS2, possess Ca2+-independent phospholipase A1/2 activity towards a variety of phospholipid substrates in vivo and in vitro, indicating their involvement in phospholipid metabolism in cells [10–13].
Based on primary sequence analysis, it was proposed that LRAT family proteins, including H-REV107, show a circular permutation with respect to classical members of NlpC/P60 superfamily, which mostly comprises prokaryotic peptidases [14]. H-REV107 contains a conserved proline-rich motif at the N terminus and a putative Cterminal transmembrane domain which was found to be crucial for cellular localization, but not necessary for the enzyme activity [4,12]. Here we report the solution structure of the catalytic N-terminal domain of H-REV107 (H-REV107N) without the putative C-terminal transmembrane domain, the first structure of the LRAT family, which provides structural insight for the enzyme activities. 2. Materials and methods 2.1. Sample preparation The plasmid construction, protein expression and purification, and NMR sample preparation were described previously [15]. 2.2. NMR spectroscopy
⇑ Corresponding author at: Beijing Nuclear Magnetic Resonance Center, Peking University, Beijing 100871, China. ⇑⇑ Corresponding author at: Beijing Nuclear Magnetic Resonance Center, Peking University, Beijing 100871, China. E-mail addresses: [email protected] (J. Lin), [email protected] (B. Xia).
The backbone sequential and side-chain assignments of HREV107N were reported previously [15]. The three-dimensional 15 N-edited, 13C-edited aliphatic and 13C-edited aromatic NOESYHSQC spectra (mixing time 100 ms) were collected on Bruker
0014-5793/$36.00 Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2010.09.015
X. Ren et al. / FEBS Letters 584 (2010) 4222–4226
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Table 1 Restraints and structural statistics. Restraints Intra-residue distance restraints Sequential distance restraints Medium range distance restraints Long range distance restraints Total unambiguous distance restraints Total ambiguous distance restraints Hydrogen bond restraints Dihedral angle restraints (u and w)
1317 655 315 542 2832 654 26 74
Restraints violations Distance (>0.3 Å) Dihedral angle (>5°)
0 0
AMBER energy (kcal/mol) Mean AMBER energy NOE distance restraints violation energy Torsion angle restraints violation energy
6192.14 ± 14.00 21.44 ± 2.57 1.00 ± 0.24
RMSD from mean structure (Å) Backbone heavy atoms in secondary structures All heavy atoms in secondary structures
0.33 ± 0.06 0.83 ± 0.11
Ramachandran statistics (%) Residues in most favored regions Residues in additional allowed regions Residues in generously allowed regions Residues in disallowed regions
81.1 17.9 0.4 0.7
Avance 800 MHz spectrometer at 298 K. Hydrogen–deuterium exchange experiments were performed on Bruker Avance 600 MHz spectrometer at 298 K. The chemical shifts were referenced to internal 2,2-dimethyl-2-silapentanesulfonic acid (DSS) [16]. All NMR spectra were processed using NMRPipe [17] and analyzed using NMRView [18]. 2.3. Structure calculations Distance restraints for structure calculation were generated by using the three-dimensional 15N-edited, 13C-edited aliphatic and
C-edited aromatic NOESY-HSQC spectra. Dihedral angle restraints were determined from backbone chemical shifts using TALOS [19]. Hydrogen bond restraints were obtained based on data from the hydrogen–deuterium exchange experiments. The tautomeric states of all four histidine residues were determined from 2D 1H to 15N HMQC spectrum (Supplementary Fig. 1) [20]. The structure calculations were performed using CYANA [21]. A total of 200 structures were calculated using CYANA, and 100 structures with the lowest target function values were selected. Then the structure refinement was carried out using the generalized Born (GB) solvent model in AMBER [22–23]. Finally, the 20 lowest energy structures were selected for representation. The final structures were analyzed using PROCHECK_NMR [24] and MOLMOL [25]. 3. Results 3.1. Solution structure of H-REV107 N-terminal domain The N-terminal phospholipase domain of H-REV107 (residues 1–125, H-REV107N) was expressed in Escherichia coli as a water soluble and well folded protein. The solution structure of HREV107N was determined with high quality with 3486 inter-proton NOE-derived distance constraints together with 74 dihedral angle and 26 hydrogen bond constraints. The constraints and structural statistics are summarized in Table 1. The 20 representative structures of H-REV107N have been deposited under PDB ID: 2KYT. The structure of H-REV107N comprises a six-stranded anti-parallel b-sheet (b1, residues 13–18; b2, residues 21–29; b3, residues 32–37; b4, residues 58–64; b5, residues 73–76; b6, residues 103– 104) and three a helices (a1, residues 65–68; a2, residues 89– 99; a3, residues 110–122) with a b1–b2–b3–b4–a1–b5–a2–b6– a3 sequential connectivity (Fig. 1). The short helix a1 is on one side of the b-sheet, while helices a2 and a3 are on the other side. The
Fig. 1. Solution structure of H-REV107 N. (A and C) Superimposition of the backbone trace of the 20 representative structures of H-REV107 N with a 65° rotation. (B and D) Ribbon diagrams of the mean structure with the secondary structural elements labeled oriented as (A and C), respectively.
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Fig. 2. Structural comparison of H-REV107 N and the NlpC/P60 domain of Spr (PDB: 2K1G). A, primary sequence alignment of H-REV107 N and the NlpC/P60 domain of Spr from E. coli performed by ClustalX [33] and pictured by ESPript 2.2 (http://espript.ibcp.fr/ESPript/ESPript/). The secondary structural elements are labeled, and the individual secondary elements in the two proteins are labeled in different colors (H-REV107 in red, and Spr in blue). The residue numbers are respectively labeled on different side of the sequences. The three residues of the Cys-His-His catalytic triad are indicated by blue triangles. B, structural comparison of H-REV107 N (pink) and the NlpC/P60 domain of Spr (PDB: 2K1G) (pale blue) in ribbon diagram (27). The two swapped a-helices are highlighted in red for H-REV107 N and in blue for Spr. C and D, topologies of H-REV107 N and 2K1G with the sequence number labeled. The colors of the two swapped a-helices and the five b-strands are corresponding to those in B.
N-terminal tail (residues 1–12) and the long loop (residues 38–57) connecting strand b3 and b4 are very flexible. 3.2. Structural comparison of H-REV107N and classical NlpC/P60 domain As H-REV107 was proposed to be a circular permutation with the catalytic domain of classical NlpC/P60 family proteins, we compared the structure of H-REV107N with that of the NlpC/P60 peptidase domain of E. coli lipoprotein Spr (PDB: 2K1G) [14,26]. Indeed, the overall architectures of the two structures are quite similar, even though their topologies are different (Fig. 2). Superimposing the five b-strands b1–b5 in H-REV107N with the corresponding b-strands in Spr reveals a backbone heavy atom root mean square deviation (RMSD) of 1.8 Å for the five b-strands, while the backbone heavy atom RMSD of helices a2 and a3 in H-REV107 and the corresponding a-helices in Spr is 4.0 Å (Fig. 2B). The major difference between the two structures is that helices a2 and a3 in H-REV107N are located at the C-terminus of the polypeptides, but the two structurally corresponding a-helices in Spr are at the Nterminus of the NlpC/P60 peptidase domain, which is typical of circular permutation (Fig. 2A). The structure of H-REV107N provides a direct evidence for the existence of circular permutation in NlpC/ P60 domain proteins.
tural comparison also reveals that residues C113, H23 and H35 of H-REV107 are located at almost identical positions as residues C68, H119 and H131 that form a Cys–His–His catalytic triad in the NlpC/P60 peptidase domain of Spr [26]. Side-chains of these corresponding residues also point in similar directions, respectively (Fig. 3). The two histidine residues in the catalytic triad are in the neutral Ne2–H tautomeric state, the same as in the structure
4. Discussion In this paper, we report the solution structure of the N-terminal phospholipase domain of H-REV107, the first structure of the novel H-REV107-like tumor suppressor family, which provides the structural evidence for the circular permutation of NlpC/P60 domain proteins. In previous reports, residues H23 and C113 of H-REV107 were found to be essential for its phospholipase activity [11–13]. Struc-
Fig. 3. The catalytic triads of H-REV107 N and the NlpC/P60 domain of Spr (PDB: 2K1G). The side chain heavy atoms of the Cys-His-His catalytic triad residues in each structure are shown in different colors (H-REV107 in red and 2K1G in blue). The distances between C113 Sc and H23 Nd1 atoms and between H23 Ne2 and H35 Nd1 atoms from the mean structure of H-REV107N are indicated in red, and the corresponding distances (C68 Sc to H119 Nd1, and H119 Ne2 to H131 Nd1) from the mean structure of 2K1G are indicated in blue. The conserved arginine residue of HREV107 near the catalytic triad is shown in red.
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Fig. 4. Sequence alignments of the catalytic domains of H-REV107 and LRAT family proteins HRASL2, TIG3 and LRAT generated by ClustalX [33] and pictured by ESPript 2.2 (http://espript.ibcp.fr/ESPript/ESPript/). The secondary structural elements of H-REV107N are labeled. The residue numbers of H-REV107 are labeled on the top. The three residues of Cys-His-His catalytic triad and the conserved arginine residues are indicated by blue triangles.
of Spr. Thus, even though the two enzymes have different substrates, H-REV107 should adopt a similar catalytic mechanism as that of the NlpC/P60 peptidase domain of Spr, in which the deprotonation of the residue C113 thiol by the basic side-chain of residue H23 generates a nucleophile anionic sulfur to attack the carbonyl group of the substrate, and the third polar residue H35 assists to properly orient the side-chain of H23 [14,26]. H-REV107-like family proteins also belong to the lecithin retinol acyltransferase (LRAT) protein family, which are essential enzymes in the all-trans-retinol metabolism. Except for the predicted N- and C-terminal transmembrane domain of LRAT [27], the catalytic domains of H-REV107 and LRAT are highly homologous (identity 26%) [28,29] (Fig. 4). The structure of HREV107N is also the first structure of LRAT family, which may provide structural insight for understanding the mechanism of LRAT. It was reported that C161 and H60 are essential for the activity of LRAT and formed catalytic dyad through the sequence alignments and mutagenesis [28]. However, in the thiol enzymes, a third polar residue that is also important for the catalysis is still not clearly identified even though extensive mutagenesis studies have been carried out [28–30]. Based on the primary sequence alignments and the structure of H-REV107N (Fig. 4), we propose that the conserved residues C161, H60, and H72 of LRAT should form a Cys– His–His catalytic triad as that in H-REV107, and H72 should correspond to H35 of H-REV107 and play the role to position the sidechain of H60. It has been reported that the H72Q mutant of LRAT is a little more active than wild-type LRAT [30], suggesting that glutamine can play a similar role as histidine at this position [26,31]. In addition, the positively charged residue R18 of HREV107 is just located near the catalytic triad and it is highly conserved in the LRAT family, which may play a role in stabilizing the phosphate group of the phospholipid substrates as the arginine residue in cytosolic phospholipase A2 does [32] (Fig. 3). In summary, we have solved the solution structure of the N-terminal catalytic domain of H-REV107, which is the first structure of H-REV107-like family, and also the first structure of circular permutated NlpC/P60 domains. According to the structural analysis, the phospholipase active site of H-REV107 consists a Cys–His–His catalytic triad, indicating H-REV107 should adopt a similar catalytic mechanism as NlpC/P60 peptidases, which also provides structural insights for the lecithin retinol acyltransferase. Acknowledgements All NMR experiments were carried out at Beijing NMR center. This research was supported by Grant 2009CB521703 (to BX) from
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