Although EcoR124 is one of the better-studied Type I restriction-modification enzymes, it still presents many challenges to detailed analyses because of its structural and functional complexity and missing structural information. In all available structures of its motor subunit HsdR, responsible for DNA translocation and cleavage, a large part of the HsdR C terminus remains unresolved. The crystal structure of the C terminus of HsdR, obtained with a crystallization chaperone in the form of pHluorin fusion and refined to 2.45 Å, revealed that this part of the protein forms an independent domain with its own hydrophobic core and displays a unique α-helical fold. The full-length HsdR model, based on the WT structure and the C-terminal domain determined here, disclosed a proposed DNA-binding groove lined by positively charged residues. In vivo and in vitro assays with a C-terminal deletion mutant of HsdR supported the idea that this domain is involved in complex assembly and DNA binding. Conserved residues identified through sequence analysis of the C-terminal domain may play a key role in protein-protein and protein-DNA interactions. We conclude that the motor subunit of EcoR124 comprises five structural and functional domains, with the fifth, the C-terminal domain, revealing a unique fold characterized by four conserved motifs in the IC subfamily of Type I restriction-modification systems. In summary, the structural and biochemical results reported here support a model in which the C-terminal domain of the motor subunit HsdR of the endonuclease EcoR124 is involved in complex assembly and DNA binding.

From the Center for Nanobiology and Structural Biology Institute of Microbiology Academy of Sciences of the Czech Republic Zamek 136 373 33 Nove Hrady Czech Republic

the College of Biomedical Sciences Larkin University Miami Florida 33169

the Department of Structural Cell Biology Molecular Mechanisms of DNA Repair Max Planck Institute of Biochemistry Am Klopferspitz 18 82152 Martinsried Germany

the Faculty of Sciences University of South Bohemia in Ceske Budejovice Branišovská 1760 370 05 České Budějovice Czech Republic

the Institute of Biochemistry University of Lübeck Ratzeburger Allee 160 Lübeck Germany and

the Institute of Microbiology Academy of Sciences of the Czech Republic Vídeňská 1083 142 20 Praha 4 Czech Republic

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Roberts R. J., Vincze T., Posfai J., and Macelis D. (2015) REBASE: a database for DNA restriction and modification: enzymes, genes and genomes. Nucleic Acids Res. 43, D298–D299 10.1093/nar/gku1046 PubMed DOI PMC

Murray N. E. (2002) Immigration control of DNA in bacteria: self versus non-self. Microbiology 148, 3–20 10.1099/00221287-148-1-3 PubMed DOI

Oliveira P. H., Touchon M., and Rocha E. P. (2014) The interplay of restriction-modification systems with mobile genetic elements and their prokaryotic hosts. Nucleic Acids Res. 42, 10618–10631 10.1093/nar/gku734 PubMed DOI PMC

Manso A. S., Chai M. H., Atack J. M., Furi L., De Ste Croix M., Haigh R., Trappetti C., Ogunniyi A. D., Shewell L. K., Boitano M., Clark T. A., Korlach J., Blades M., Mirkes E., Gorban A. N., et al. (2014) A random six-phase switch regulates pneumococcal virulence via global epigenetic changes. Nat. Commun. 5, 5055 10.1038/ncomms6055 PubMed DOI PMC

Furuta Y., Namba-Fukuyo H., Shibata T. F., Nishiyama T., Shigenobu S., Suzuki Y., Sugano S., Hasebe M., and Kobayashi I. (2014) Methylome diversification through changes in DNA methyltransferase sequence specificity. PLoS Genet. 10(4), e1004272 10.1371/journal.pgen.1004272 PubMed DOI PMC

Budroni S., Siena E., Dunning Hotopp J. C., Seib K. L., Serruto D., Nofroni C., Comanducci M., Riley D. R., Daugherty S. C., Angiuoli S. V., Covacci A., Pizza M., Rappuoli R., Moxon E. R., Tettelin H., and Medini D. (2011) Neisseria meningitidis is structured in clades associated with restriction modification systems that modulate homologous recombination. Proc. Natl. Acad. Sci. U.S.A. 108, 4494–4499 10.1073/pnas.1019751108 PubMed DOI PMC

Erwin A. L., Sandstedt S. A., Bonthuis P. J., Geelhood J. L., Nelson K. L., Unrath W. C., Diggle M. A., Theodore M. J., Pleatman C. R., Mothershed E. A., Sacchi C. T., Mayer L. W., Gilsdorf J. R., and Smith A. L. (2008) Analysis of genetic relatedness of Haemophilus influenzae isolates by multilocus sequence typing. J. Bacteriol. 190, 1473–1483 10.1128/JB.01207-07 PubMed DOI PMC

Loenen W. A., Dryden D. T., Raleigh E. A., Wilson G. G., and Murray N. E. (2014) Highlights of the DNA cutters: a short history of the restriction enzymes. Nucleic Acids Res. 42, 3–19 10.1093/nar/gkt990 PubMed DOI PMC

Roberts R. J., Belfort M., Bestor T., Bhagwat A. S., Bickle T. A., Bitinaite J., Blumenthal R. M., Degtyarev S. K. h., Dryden D. T., Dybvig K., Firman K., Gromova E. S., Gumport R. I., Halford S. E., Hattman S., et al. (2003) A nomenclature for restriction enzymes, DNA methyltransferases, homing endonucleases and their genes. Nucleic Acids Res. 31, 1805–1812 10.1093/nar/gkg274 PubMed DOI PMC

Youell J., and Firman K. (2008) EcoR124I: from plasmid-encoded restriction-modification system to nanodevice. Microbiol. Mol. Biol. Rev. 72, 365–377, table of contents 10.1128/MMBR.00043-07 PubMed DOI PMC

Suri B., Shepherd J. C., and Bickle T. A. (1984) The EcoA restriction and modification system of Escherichia coli 15T-: enzyme structure and DNA recognition sequence. EMBO J. 3, 575–579 PubMed PMC

Sommer R., and Schaller H. (1979) Nucleotide sequence of the recognition site of the B-specific restriction modification system in E. coli. Mol. Gen. Genet. 168, 331–335 PubMed

Bertani G., and Weigle J. J. (1953) Host controlled variation in bacterial viruses. J. Bacteriol. 65, 113–121 PubMed PMC

Horiuchi K., and Zinder N. D. (1972) Cleavage of bacteriophage fl DNA by the restriction enzyme of Escherichia coli B. Proc. Natl. Acad. Sci. U.S.A. 69, 3220–3224 10.1073/pnas.69.11.3220 PubMed DOI PMC

Kennaway C. K., Obarska-Kosinska A., White J. H., Tuszynska I., Cooper L. P., Bujnicki J. M., Trinick J., and Dryden D. T. (2009) The structure of M. EcoKI Type I DNA methyltransferase with a DNA mimic antirestriction protein. Nucleic Acids Res. 37, 762–770 10.1093/nar/gkn988 PubMed DOI PMC

Liu Y. P., Tang Q., Zhang J. Z., Tian L. F., Gao P., and Yan X. X. (2017) Structural basis underlying complex assembly and conformational transition of the type I R-M system. Proc. Natl. Acad. Sci. U.S.A. 114, 11151–11156 10.1073/pnas.1711754114 PubMed DOI PMC

Park S. Y., Lee H. J., Song J. M., Sun J., Hwang H. J., Nishi K., and Kim J. S. (2012) Structural characterization of a modification subunit of a putative type I restriction enzyme from Vibrio vulnificus YJ016. Acta Crystallogr. D 68, 1570–1577 10.1107/S0907444912038826 PubMed DOI

Kim J. S., DeGiovanni A., Jancarik J., Adams P. D., Yokota H., Kim R., and Kim S. H. (2005) Crystal structure of DNA sequence specificity subunit of a type I restriction-modification enzyme and its functional implications. Proc. Natl. Acad. Sci. U.S.A. 102, 3248–3253 10.1073/pnas.0409851102 PubMed DOI PMC

Calisto B. M., Pich O. Q., Piñol J., Fita I., Querol E., and Carpena X. (2005) Crystal structure of a putative type I restriction-modification S subunit from Mycoplasma genitalium. J. Mol. Biol. 351, 749–762 10.1016/j.jmb.2005.06.050 PubMed DOI

Gao P., Tang Q., An X., Yan X., and Liang D. (2011) Structure of HsdS subunit from Thermoanaerobacter tengcongensis sheds lights on mechanism of dynamic opening and closing of type I methyltransferase. PLoS ONE 6, e17346–e17346 10.1371/journal.pone.0017346 PubMed DOI PMC

Lapkouski M., Panjikar S., Janscak P., Smatanova I. K., Carey J., Ettrich R., and Csefalvay E. (2009) Structure of the motor subunit of type I restriction-modification complex EcoR124I. Nat. Struct. Mol. Biol. 16, 94–95 10.1038/nsmb.1523 PubMed DOI

Uyen N. T., Park S. Y., Choi J. W., Lee H. J., Nishi K., and Kim J. S. (2009) The fragment structure of a putative HsdR subunit of a type I restriction enzyme from Vibrio vulnificus YJ016: implications for DNA restriction and translocation activity. Nucleic Acids Res. 37, 6960–6969 10.1093/nar/gkp603 PubMed DOI PMC

Csefalvay E., Lapkouski M., Guzanova A., Csefalvay L., Baikova T., Shevelev I., Bialevich V., Shamayeva K., Janscak P., Kuta Smatanova I., Panjikar S., Carey J., Weiserova M., and Ettrich R. (2015) Functional coupling of duplex translocation to DNA cleavage in a type I restriction enzyme. PLoS ONE 10, e0128700 10.1371/journal.pone.0128700 PubMed DOI PMC

Davies G. P., Martin I., Sturrock S. S., Cronshaw A., Murray N. E., and Dryden D. T. (1999) On the structure and operation of type I DNA restriction enzymes. J. Mol. Biol. 290, 565–579 10.1006/jmbi.1999.2908 PubMed DOI

Obarska-Kosinska A., Taylor J. E., Callow P., Orlowski J., Bujnicki J. M., and Kneale G. G. (2008) HsdR subunit of the type I restriction-modification enzyme EcoR124I: biophysical characterisation and structural modelling. J. Mol. Biol. 376, 438–452 10.1016/j.jmb.2007.11.024 PubMed DOI PMC

Kennaway C. K., Taylor J. E., Song C. F., Potrzebowski W., Nicholson W., White J. H., Swiderska A., Obarska-Kosinska A., Callow P., Cooper L. P., Roberts G. A., Artero J. B., Bujnicki J. M., Trinick J., Kneale G. G., and Dryden D. T. (2012) Structure and operation of the DNA-translocating type I DNA restriction enzymes. Genes Dev. 26, 92–104 10.1101/gad.179085.111 PubMed DOI PMC

Piekarowicz A., Kłyz A., Kwiatek A., and Stein D. C. (2001) Analysis of type I restriction modification systems in the Neisseriaceae: genetic organization and properties of the gene products. Mol. Microbiol. 41, 1199–1210 PubMed

Grinkevich P., Iermak I., Luedtke N. A., Mesters J. R., Ettrich R., and Ludwig J. (2016) pHluorin-assisted expression, purification, crystallization and X-ray diffraction data analysis of the C-terminal domain of the HsdR subunit of the Escherichia coli type I restriction-modification system EcoR124I. Acta Crystallogr. F Struct. Biol. Commun. 72, 672–676 10.1107/S2053230X16011626 PubMed DOI PMC

Kobe B., Ve T., and Williams S. J. (2015) Fusion-protein-assisted protein crystallization. Acta Crystallogr F Struct. Biol. Commun. 71, 861–869 10.1107/S2053230X15011061 PubMed DOI PMC

Frishman D., and Argos P. (1995) Knowledge-based protein secondary structure assignment. Proteins 23, 566–579 10.1002/prot.340230412 PubMed DOI

Holm L., and Laakso L. M. (2016) Dali server update. Nucleic Acids Res. 44, W351–W355 10.1093/nar/gkw357 PubMed DOI PMC

Konagurthu A. S., Whisstock J. C., Stuckey P. J., and Lesk A. M. (2006) MUSTANG: a multiple structural alignment algorithm. Proteins 64, 559–574 10.1002/prot.20921 PubMed DOI

Hubacek J., and Glover S. W. (1970) Complementation analysis of temperature-sensitive host specificity mutations in Escherichia coli. J. Mol. Biol. 50, 111–127 10.1016/0022-2836(70)90108-7 PubMed DOI

Janscak P., Abadjieva A., and Firman K. (1996) The type I restriction endonuclease R. EcoR124I: over-production and biochemical properties. J. Mol. Biol. 257, 977–991 10.1006/jmbi.1996.0217 PubMed DOI

Sisáková E., Weiserová M., Dekker C., Seidel R., and Szczelkun M. D. (2008) The interrelationship of helicase and nuclease domains during DNA translocation by the molecular motor EcoR124I. J. Mol. Biol. 384, 1273–1286 10.1016/j.jmb.2008.10.017 PubMed DOI PMC

Miesenböck G., De Angelis D. A., and Rothman J. E. (1998) Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins. Nature 394, 192–195 10.1038/28190 PubMed DOI

McPherson A. (1982) Preparation and Analysis of Protein Crystals, pp. 19–49, Wiley, New York

Cianci M., Bourenkov G., Pompidor G., Karpics I., Kallio J., Bento I., Roessle M., Cipriani F., Fiedler S., and Schneider T. R. (2017) P13, the EMBL macromolecular crystallography beamline at the low-emittance PETRA III ring for high- and low-energy phasing with variable beam focusing. J. Synchrotron Radiat. 24, 323–332 10.1107/S1600577516016465 PubMed DOI PMC

Kabsch W. (2010) XDS. XDS. Acta Crystallogr. D 66, 125–132 10.1107/S0907444909047337 PubMed DOI PMC

Winn M. D., Ballard C. C., Cowtan K. D., Dodson E. J., Emsley P., Evans P. R., Keegan R. M., Krissinel E. B., Leslie A. G., McCoy A., McNicholas S. J., Murshudov G. N., Pannu N. S., Potterton E. A., Powell H. R., et al. (2011) Overview of the CCP4 suite and current developments. Acta Crystallogr. D 67, 235–242 PubMed PMC

Matthews B. W. (1968) Solvent content of protein crystals. J. Mol. Biol. 33, 491–497 10.1016/0022-2836(68)90205-2 PubMed DOI

Vagin A., and Teplyakov A. (1997) MOLREP: an automated program for molecular replacement. J. Appl. Cryst. 30, 1022–1025 10.1107/S0021889897006766 DOI

van Thor J. J., Georgiev G. Y., Towrie M., and Sage J. T. (2005) Ultrafast and low barrier motions in the photoreactions of the green fluorescent protein. J. Biol. Chem. 280, 33652–33659 10.1074/jbc.M505473200 PubMed DOI

Cowtan K. (2008) Fitting molecular fragments into electron density. Acta Crystallogr. D 64, 83–89 10.1107/S0907444907033938 PubMed DOI PMC

Murshudov G. N., Skubák P., Lebedev A. A., Pannu N. S., Steiner R. A., Nicholls R. A., Winn M. D., Long F., and Vagin A. A. (2011) REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D 67, 355–367 10.1107/S0907444911001314 PubMed DOI PMC

Emsley P., Lohkamp B., Scott W. G., and Cowtan K. (2010) Features and development of Coot. Acta Crystallogr. D 66, 486–501 10.1107/S0907444910007493 PubMed DOI PMC

McCoy A. J., Grosse-Kunstleve R. W., Adams P. D., Winn M. D., Storoni L. C., and Read R. J. (2007) Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 10.1107/S0021889807021206 PubMed DOI PMC

Afonine P. V., Grosse-Kunstleve R. W., Echols N., Headd J. J., Moriarty N. W., Mustyakimov M., Terwilliger T. C., Urzhumtsev A., Zwart P. H., and Adams P. D. (2012) Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367 10.1107/S0907444912001308 PubMed DOI PMC

Krieger E., Koraimann G., and Vriend G. (2002) Increasing the precision of comparative models with YASARA NOVA: a self-parameterizing force field. Proteins 47, 393–402 10.1002/prot.10104 PubMed DOI

Sinha D., Bialevich V., Shamayeva K., Guzanova A., Sisakova A., Csefalvay E., Reha D., Krejci L., Carey J., Weiserova M., and Ettrich R. (2018) A residue of motif III positions the helicase domains of motor subunit HsdR in restriction-modification enzyme EcoR124I. J. Mol. Model. 24, 176 10.1007/s00894-018-3722-8 PubMed DOI

Qi D., and Scholthof K. B. (2008) A one-step PCR-based method for rapid and efficient site-directed fragment deletion, insertion, and substitution mutagenesis. J. Virol. Methods 149, 85–90 10.1016/j.jviromet.2008.01.002 PubMed DOI

Taylor I., Patel J., Firman K., and Kneale G. (1992) Purification and biochemical characterisation of the EcoR124 type I modification methylase. Nucleic Acids Res. 20, 179–186 10.1093/nar/20.2.179 PubMed DOI PMC

Yanisch-Perron C., Vieira J., and Messing J. (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC19 vectors. Gene 33, 103–119 10.1016/0378-1119(85)90120-9 PubMed DOI

Patel J., Taylor I., Dutta C. F., Kneale G., and Firman K. (1992) High-level expression of the cloned genes encoding the subunits of and intact DNA methyltransferase, M. EcoR124. Gene 112, 21–27 10.1016/0378-1119(92)90298-4 PubMed DOI

Jacob F., and Wollman E. L. (1955) Etude génétique d'un bactériophage tempéré d'Escherichia coli: III: effet du rayonnement ultraviolet sur la recombinaison génétique. Ann. Inst. Pasteur 88, 724–749 PubMed

R Development Core Team (2017) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria.

Pagès H., Aboyoun P., Gentleman R., and DebRoy S. (2017) Biostrings: Efficient Manipulation of Biological Strings, Version 2.48.0

Sievers F., Wilm A., Dineen D., Gibson T. J., Karplus K., Li W., Lopez R., McWilliam H., Remmert M., Söding J., Thompson J. D., and Higgins D. G. (2011) Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 PubMed PMC

Dereeper A., Audic S., Claverie J. M., and Blanc G. (2010) BLAST-EXPLORER helps you building datasets for phylogenetic analysis. BMC Evol. Biol. 10, 8 10.1186/1471-2148-10-8 PubMed DOI PMC

Guindon S., Dufayard J. F., Lefort V., Anisimova M., Hordijk W., and Gascuel O. (2010) New algorithms and methods to estimate maximum-likelihood phylogenies: assessing the performance of PhyML 3.0. Syst. Biol. 59, 307–321 10.1093/sysbio/syq010 PubMed DOI

Dryden D. T., Murray N. E., and Rao D. N. (2001) Nucleoside triphosphate-dependent restriction enzymes. Nucleic Acids Res. 29, 3728–3741 10.1093/nar/29.18.3728 PubMed DOI PMC

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PDB
2y7c, 2y7h, 5YBB, 2AR0, 3KHK, 2LKD, 3UFB, 1YF2, 1YDX, 3OKG, 2W00, 3H1T, 4BE7, 4B4B, 4BEC, 4XJX, 5JN3, 6H2J, 5W1H, 2DOB, 1W7S, 5J3N