Crystal Structures of the CBS and DRTGG Domains of the Regulatory Region of Clostridium perfringens Pyrophosphatase Complexed with the Inhibitor, AMP, and Activator, Diadenosine Tetraphosphate H. Tuominen 1 , A. Salminen 1 , E. Oksanen 2 , J. Jämsen 1 , O. Heikkilä 1 , L. Lehtiö 3 , N. N. Magretova 4 , A. Goldman 2 , A. A. Baykov 4 ⁎ and R. Lahti 1 ⁎ 1 Department of Biochemistry and Food Chemistry, University of Turku, Vatselankatu 2, FI-20014 Turku, Finland 2 Institute of Biotechnology, University of Helsinki, Biocenter 3, P.O. Box 65, FI-00014 Helsinki, Finland 3 Pharmaceutical Sciences, Department of Biosciences, Åbo Akademi University, BioCity, FI-20520 Turku, Finland 4 A. N. Belozersky Institute of Physico-Chemical Biology, Moscow State University, Moscow 119899, Russia Received 22 December 2009; received in revised form 10 March 2010; accepted 10 March 2010 Available online 19 March 2010 Nucleotide-binding cystathionine β-synthase (CBS) domains serve as regulatory units in numerous proteins distributed in all kingdoms of life. However, the underlying regulatory mechanisms remain to be estab- lished. Recently, we described a subfamily of CBS domain-containing pyrophosphatases (PPases) within family II PPases. Here, we express a novel CBS-PPase from Clostridium perfringens (CPE2055) and show that the enzyme is inhibited by AMP and activated by a novel effector, diadenosine 5′,5-P1,P4-tetraphosphate (AP 4 A). The structures of the AMP and AP 4 A complexes of the regulatory region of C. perfringens PPase (cpCBS), comprising a pair of CBS domains interlinked by a DRTGG domain, were determined at 2.3 Å resolution using X-ray crystallography. The structures obtained are the first structures of a DRTGG domain as part of a larger protein structure. The AMP complex contains two AMP molecules per cpCBS dimer, each bound to a single monomer, whereas in the activator-bound complex, one AP 4 A molecule bridges two monomers. In the nucleotide-bound structures, activator binding induces significant opening of the CBS domain interface, compared with the inhibitor complex. These results provide structural insight into the mechanism of CBS-PPase regulation by nucleotides. © 2010 Elsevier Ltd. All rights reserved. Edited by M. Guss Keywords: CBS domain; DRTGG domain; diadenosine tetraphosphate; AMP; inorganic pyrophosphatase *Corresponding authors. E-mail addresses: baykov@genebee.msu.su; reijo.lahti@utu.fi. Present address: O. Heikkilä, Department of Virology, University of Turku, Kiinanmyllynkatu 13, FI-20520 Turku, Finland. Abbreviations used: AP 4 A, diadenosine 5′,5-P1,P4-tetraphosphate; CBS, cystathionine β-synthase; cpCBS-PPase, CBS domain-containing PPase from Clostridium perfringens; cpCBS, regulatory region of cpCBS-PPase (residues 66–306); cpCBS-His 8 , regulatory region of cpCBS-PPase (residues 66–306) with eight His residues at the C-terminus; His 6 -th-cpCBS, regulatory region of cpCBS-PPase (residues 66–306) with six His residues and a thrombin-cleavable site at the N-terminus; PPase, inorganic pyrophosphatase; th-cpCBS, thrombin-treated His 6 -th-cpCBS with the His 6 tag removed; PDB, Protein Data Bank; PEG, polyethylene glycol; SAD, single-wavelength anomalous dispersion. doi:10.1016/j.jmb.2010.03.019 J. Mol. Biol. (2010) 398, 400–413 Available online at www.sciencedirect.com 0022-2836/$ - see front matter © 2010 Elsevier Ltd. All rights reserved.