Title Structures of restriction endonuclease HindIII in complex with its cognate DNA and divalent cations Author(s) Watanabe, Nobuhisa; Takasaki, Yozo; Sato, Chika; Ando, Shoji; Tanaka, Isao Acta Crystallographica. Section D, Biological Crystallography, 65(12), 1326-1333 Citation https://doi.org/10.1107/S0907444909041134 Issue Date 2009-12 Doc URL http://hdl.handle.net/2115/39986 Type article File Information watanabe_ActaCristD65.pdf Instructions for use Hokkaido University Collection of Scholarly and Academic Papers : HUSCAP electronic reprint Acta Crystallographica Section D Biological Crystallography ISSN 0907-4449 Editors: E. N. Baker and Z. Dauter Structures of restriction endonuclease HindIII in complex with its cognate DNA and divalent cations Nobuhisa Watanabe, Yozo Takasaki, Chika Sato, Shoji Ando and Isao Tanaka Acta Cryst. (2009). D65, 1326–1333 Copyright c International Union of Crystallography Author(s) of this paper may load this reprint on their own web site or institutional repository provided that this cover page is retained. Republication of this article or its storage in electronic databases other than as specified above is not permitted without prior permission in writing from the IUCr. For further information see http://journals.iucr.org/services/authorrights.html Acta Crystallographica Section D: Biological Crystallography welcomes the submission of papers covering any aspect of structural biology, with a particular emphasis on the struc- tures of biological macromolecules and the methods used to determine them. Reports on new protein structures are particularly encouraged, as are structure–function papers that could include crystallographic binding studies, or structural analysis of mutants or other modified forms of a known protein structure. The key criterion is that such papers should present new insights into biology, chemistry or structure. Papers on crystallo- graphic methods should be oriented towards biological crystallography, and may include new approaches to any aspect of structure determination or analysis. Crystallography Journals Online is available from journals.iucr.org Acta Cryst. (2009). D65, 1326–1333 Watanabe et al. · Restriction endonuclease Hind¡taxon¿III¡/taxon¿ research papers Acta Crystallographica Section D Hin Biological Structures of restriction endonuclease dIII in Crystallography complex with its cognate DNA and divalent cations ISSN 0907-4449 Nobuhisa Watanabe,a,b,c* The three-dimensional crystal structures of HindIII bound Received 13 July 2009 Yozo Takasaki,d Chika Sato,c to its cognate DNA with and without divalent cations were Accepted 8 October 2009 Shoji Andod and Isao Tanakac solved at 2.17 and 2.00 A˚ resolution, respectively. HindIII forms a dimer. The structures showed that HindIII belongs to PDB References: HindIII– the EcoRI-like ( -class) subfamily of type II restriction endo- cognate DNA complex, 2e52, a Department of Biotechnology and Biomaterial nucleases. The cognate DNA-complex structures revealed the r2e52sf; metal-ion bound, Chemistry, Graduate School of Engineering, 3a4k, r3a4ksf. Nagoya University, Japan, bSynchrotron specific DNA-recognition mechanism of HindIII by which it 2+ Radiation Research Center, Nagoya University, recognizes the palindromic sequence A/AGCTT. In the Mg Japan, cDivision of Molecular Life Science, ion-soaked structure the DNA was cleaved and two ions were Faculty of Advanced Life Science, Hokkaido bound at each active site, corresponding to the two-metal-ion d University, Japan, and Faculty of Medicine, mechanism. Saga University, Japan Correspondence e-mail: [email protected] 1. Introduction Restriction endonucleases are produced in bacteria, archaea and Chlorella viruses. These enzymes hydrolyze foreign DNA and thus allow microorganisms to avoid attack by phages etc. Based on subunit composition, cofactor requirements, DNA- recognition specificity and mode of action, restriction endo- nucleases have been classified into four types (Roberts et al., 2003). Type II restriction endonucleases recognize and digest specific palindromic sequences of double-stranded DNA. About 4000 type II restriction endonucleases have been discovered to date. The specificity of the type II restriction endonucleases has made them indispensable tools in bio- technology. However, the tertiary structures of only about 30 type II restriction endonucleases are available at present in REBASE (Roberts et al., 2007). Most of the type II restriction endonucleases have a common sequence motif PD-(D/E)xK (Stahl et al., 1998). Pingoud et al. (2005) showed that they contain a core comprised of a five-stranded mixed -sheet flanked by -helices and that the second and third -strands form a scaffold for the catalytic residues of PD-(D/E)xK. Typically, type II restriction enzymes utilize Mg2+ ion as a divalent cation. Recent reviews have focused on their struc- ture and function, the role of metal ions and their catalytic mechanisms (Pingoud & Jeltsch, 2001; Pingoud et al., 2005). The type II restriction endonuclease HindIII produced in Haemophilus influenzae Rd digests DNA at the palindromic sequence A/AGCTT. HindIII is comprised of 300 amino acids and has a molecular weight of 34 950 Da. The amino-acid sequences of this nuclease and the corresponding methyl- transferase were deduced from their genes (Nwankwo et al., 1994). We have previously reported protein-engineering # 2009 International Union of Crystallography studies on HindIII (Tang et al., 2000). One mutant, D108L, Printed in Singapore – all rights reserved showed a complete loss of enzyme activity, suggesting that 1326 doi:10.1107/S0907444909041134 Acta Cryst. (2009). D65, 1326–1333 electronic reprint research papers Table 1 further studies of substrate recognition Crystallographic data-collection and refinement statistics. and enzyme catalysis, we have deter- Iodine derivative Native Mg2+-soaked mined the HindIII crystal structure in its cognate DNA complex with and Beamline BL-5A NW-12A BL-6A Wavelength (A˚ ) 1.5 1.0 1.0 without metal ions. Space group P21 P21 P21 Unit-cell parameters (A˚ , ) a = 83.9, b = 130.9, a = 83.1, b = 131.5, a = 83.5, b = 132.2, c = 93.6, = 110.2 c = 93.7, = 111.2 c = 94.1, = 111.0 Resolution (A˚ ) 2.77 (2.87–2.77) 2.00 (2.07–2.00) 2.17 (2.25–2.17) 2. Materials and method Unique reflections 48465 130036 100664 Redundancy 3.6 7.6 1.9 2.1. Protein purification and Completeness (%) 95.3 (67.4) 99.9 (99.5) 99.8 (98.0) crystallization Rmerge (%) 9.2 (37.4) 5.6 (14.3) 5.4 (40.4) I/(I) 7.6 (1.6) 23.2 (12.5) 24.7 (3.5) Wild-type HindIII was expressed in a Mosaicity ( ) 1.00 0.25 0.49 His-tagged form (with ten His residues Refinement resolution (A˚ ) 2.00 2.17 Rwork/Rfree (5% of reflections) 17.7/21.7 17.5/22.1 at the N-terminus). The HindIII protein No. of waters 968 598 was purified by a modification of the ˚ R.m.s.d. bond lengths (A) 0.015 0.02 method reported previously (Tang et al., R.m.s.d. bond angles () 1.50 1.84 Average B factor (A˚ 2) 23.8 36.1 2000). The HindIII-RM gene inserted into the pET16b vector was expressed in Escherichia coli BL21 (DE3) pLysS. The bacteria were grown in 3 l LB medium containing 50 mgmlÀ1 ampicillin, harvested and sonicated to obtain crude extract. The extract in binding buffer (5 mM imidazole, 0.5 M NaCl and 20 mM Tris–HCl pH 8.0) was applied onto a column packed with His-Bind Resin (Novagen, San Diego, California, USA) in 50 mM NiSO4. The column was washed with binding buffer and then with wash buffer (60 mM imidazole, 0.5 M NaCl and 20 mM Tris–HCl pH 8.0). The protein was eluted by adding 0.05 M EDTA, 0.1 M EDTA, 0.1 M EDTA + 0.1 M imidazole, 0.1 M EDTA + 0.2 M imidazole, 0.1 M EDTA + 0.5 M imidazole and then 1.0 M imidazole to the column in a stepwise manner. HindIII protein eluted in the 0.1 M and 0.2 M imidazole fractions. The purified protein thus obtained was dialyzed against a buffer containing 20 mM Tris–HCl pH 8.0 and 0.2 M NaCl overnight. Protease Xa was added to the sample to remove the His tag by treatment at 285 K for 14 h. The sample was then added to the His-Bind resin column and the flowthrough fraction was collected and concentrated to 10 mg mlÀ1 with Amicon Ultra (Millipore, Billerica, Massa- chusetts). The weight ratio of protease Xa to HindIII was less than 0.1% and therefore no trace was observed in the concentrated sample. DNA oligonucleotides (50-GCCAAGCTTGGC-30) were synthesized and purified by HPLC (Hokkaido System Science, Sapporo, Japan). The oligonucleotides for use in crystal- lization trials were dissolved in Milli-Q reverse-osmose puri- fied water. The oligonucleotide solution was heated to 368 K for 10 min, followed by slow cooling to room temperature. Protein–DNA complexes were prepared by mixing annealed Figure 1 oligonucleotides with HindIII and incubating at 277 K over- Overall structure of the HindIII monomer. (a) HindIII monomer with night. The final protein–DNA complex solution contained labelled helices and -strands. (b) Topology diagram of HindIII. -Sheets HindIII protein at a concentration of 6.0 mg mlÀ1; the DNA are shown in red and helices are shown in cyan. duplex was present in a 1.5-fold molar excess relative to the protein dimer. Asp108 is located in the active site of the enzyme. We also Crystals of the HindIII–DNA complex were grown by the found that the mutant E86K showed higher activity than the hanging-drop vapour-diffusion method over a reservoir of 12– wild-type enzyme (Tang et al., 1999). However, the tertiary 16%(v/v) PEG 3350, 80–120 mM ammonium acetate. Drops structure of HindIII has not been determined. To facilitate were formed by mixing 1 ml HindIII–DNA complex solution Acta Cryst.