proteins STRUCTURE O FUNCTION O BIOINFORMATICS STRUCTURE NOTE Crystal structure of Bacillus Subtilis CodW, a noncanonical HslV-like peptidase with an impaired catalytic apparatus Seong-Hwan Rho, 1 Hyun Ho Park, 1 Gil Bu Kang, 1 Young Jun Im, 1 Min Suk Kang, 2 Byung Kook Lim, 2 Ihn Sik Seong, 2 Jaehong Seol, 2 Chin Ha Chung, 2 Jimin Wang, 3 and Soo Hyun Eom 1 * 1 Department of Life Science, Cell Dynamics Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea 2 School of Biological Sciences, Seoul National University, Seoul 151-742, Korea 3 Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, Connecticut 06520 Key words: ATP-dependent protease; CodW; crystal structure; Ntn-hydrolase; autolysis. INTRODUCTION ATP-dependent proteases play vital roles in protein quality control and in regulating the levels of certain cellu- lar proteins. 1–3 Escherichia coli and other bacteria, includ- ing Bacillus subtilis, contain at least three types of multi- meric ATP-dependent proteases homologous to the eu- karyotic 26S proteasome: Lon, Clp and HslVU. 1,2,4,5 Of these, HslVU has been extensively studied as the simplest proteasome ancestor. 6–10 Like the 26S proteasome, HslVU is comprised of two multimeric components: the ATPase HslU, which belongs to the AAA superfamily of ATPases, 11 and the peptidase HslV, which shares common structural features with the catalytic b-type subunits of the 20S proteasome. Despite a sequence identity of only about 20%, they share the amino acids crucial for proteol- ysis and auto-cleavage [Fig. 1(A)]. Moreover, both use a threonine residue at the N-terminus as a catalytic nucleo- phile exposed by the processing of a methionine residue or a prosegment upon assembly, and are thus members of the N-terminal nucleophile (Ntn)-hydrolase family. 14–16 CodW is the proteolytic component of CodWX, which is highly homologous to HslVU 17 and has sequence identi- ties of 52 and 55% with E. coli and H. influenzae HslV , respectively. A previous electron microscopic analysis revealed that CodW and HslV have similar molecular architectures with strict sixfold symmetry and similar dimensions (11 nm in diameter and 10 nm in height). 18 In addition, both can form a hybrid protease capable of degrading SulA, suggesting CodWX and HslVU are close relatives. 15 Nonetheless, recent biochemical studies suggest that CodW is distinct from HslV in several respects. 15,18,19 First, although CodW is synthesized as a precursor with additional residues at its N-terminus, just like proteasomal b and other HslV proteases, unlike those others CodW is incapable of autolysis before the nucleo- phile Thr-6 upon assembly. 15 It may be, therefore, that CodW utilizes its N-terminal serine as a catalytic nucleo- phile. Second, CodWX is an alkaline protease that is maxi- mally active at pH 9.5, unlike HslUV which has a maximal activity at pH 8. Third, CodW by itself has no peptidase activity, even towards small peptides; it requires hydrolysis of ATP by CodX, which in turn leads to formation of a CodWX complex, for digestion of peptide and protein sub- strates. By contrast, uncomplexed HslV does show weak peptidase activity toward small peptides, and the binding of ATP is sufficient for the formation of HslVU, though ATP hydrolysis is required for the digestion of a protein substrate. This dissimilarity in the behaviors of CodW and Grant sponsor: Korea Science and Engineering Foundation (KOSEF); Grant num- bers: R01-2004-000-10936-0, R01-2007-000-10592-0 and Research Center for Bio- molecular Nanotechnology (GIST). Seong-Hwan Rho and Hyun Ho Park contributed equally to this work. *Correspondence to: Soo Hyun Eom, Department of Life Science, Gwangju Institute of Science and Technology, Gwangju 500-712, Korea. E-mail: eom@gist.ac.kr Received 8 June 2007; Revised 24 July 2007; Accepted 30 July 2007 Published online 2 November 2007 in Wiley InterScience (www.interscience.wiley. com). DOI: 10.1002/prot.21758 1020 PROTEINS V V C 2007 WILEY-LISS, INC.