Crystal Structure of a Putative Type I Restriction–Modification S Subunit from Mycoplasma genitalium Ba ´ rbara M. Calisto 1 , Oscar Q. Pich 2 , Jaume Pin ˜ ol 2 , Ignacio Fita 1 Enrique Querol 2 * and Xavier Carpena 1 * 1 Institut de Biologia Molecular de Barcelona (IBMB-CSIC) Parc Cientı ´fic de Barcelona Josep-Samitier 1-5, 08028 Barcelona, Spain 2 Institut de Biotecnologia i Biomedicina and Departament de Bioquı ´mica i Biologia Molecular, Universitat Auto `noma de Barcelona, 08193 Bellaterra, Barcelona, Spain The crystal structure of the eubacteria Mycoplasma genitalium ORF MG438 polypeptide, determined by multiple anomalous dispersion and refined at 2.3 A ˚ resolution, reveals the organization of S subunits from the Type I restriction and modification system. The structure consists of two globular domains, with about 150 residues each, separated by a pair of 40 residue long antiparallel a-helices. The globular domains correspond to the variable target recognition domains (TRDs), as previously defined for S subunits on sequence analysis, while the two helices correspond to the central (CR1) and C-terminal (CR2) conserved regions, respectively. The structure of the MG438 subunit presents an overall cyclic topology with an intramolecular 2-fold axis that superimposes the N and the C-half parts, each half containing a globular domain and a conserved helix. TRDs are found to be structurally related with the small domain of the Type II N6-adenine DNA MTase TaqI. These relationships together with the structural peculiarities of MG438, in particular the presence of the intramolecular quasi-symmetry, allow the proposal of a model for S subunits recognition of their DNA targets in agreement with previous experimental results. In the crystal, two subunits of MG438 related by a crystallographic 2-fold axis present a large contact area mainly involving the symmetric interactions of a cluster of exposed hydrophobic residues. Comparison with the recently reported structure of an S subunit from the archaea Methanococcus jannaschii highlights the structural features preserved despite a sequence identity below 20%, but also reveals important differences in the globular domains and in their disposition with respect to the conserved regions. q 2005 Published by Elsevier Ltd. Keywords: Type I restriction and modification system; HsdS; crystal structure; Mycoplasma genitalium; DNA recognition *Corresponding authors Introduction Type I restriction and modification (R-M) systems are heterooligomeric complexes that catalyze both the restriction and the specific modification of DNA. 1 Type I R-M systems comprise three different subunits, HsdS (S), HsdM (M) and HsdR (R), coded by three closely linked genes that are named hsd as the acronym for host specificity of DNA. The three subunits assemble either into a large complex with stoichiometry R 2 M 2 S 1 , which presents both methyl- transferase (MTase) and restrictase (REase) activities, or into a smaller M 2 S 1 complex showing only MTase activity. The S subunit confers DNA sequence specificity to both the MTase and REase complexes recognizing an asymmetric bipartite nucleotide target. 2 The two components of the target, of 3–4 bp and 4–5 bp, respectively, are separated by a non- specific spacer of 6–8 bp. The restriction or modifi- cation activities of Type I R-M systems are determined by the methylation status of the target sequence. The REase enzyme cleaves unmodified target sequences while hemimethylated target sequences are the substrate for modification. 0022-2836/$ - see front matter q 2005 Published by Elsevier Ltd. Abbreviations used: R-M, restriction and modification; Mtase, methyltransferase; REase, restrictase; CR, con- served region; TRD, target recognition domain; ORF, open reading frame; MAD, multiwavelength anomalous diffraction; SeMet, selenomethionine. E-mail addresses of the corresponding authors: enric.querol@uab.es; xcvcri@ibmb.csic.es doi:10.1016/j.jmb.2005.06.050 J. Mol. Biol. (2005) 351, 749–762