† These authors contributed equally to this work. *For correspondence. E-mail: chajung@kiost.ac.kr; Tel.: +82-31-400-6297; Fax: +82-31-406-6297 § Supplemental material for this article may be found at http://www.springerlink.com/content/120956. Young Jun An 1† , Jung-Hyun Na 1† , Myung-Il Kim 1† , and Sun-Shin Cha 1,2,3 * 1 Marine Biotechnology Research Center, Korea Institute of Ocean Science and Technology, Ansan 15627, Republic of Korea 2 Department of Marine Biotechnology, Korea University of Science and Technology, Daejeon 34113, Republic of Korea 3 Department of Convergence Study on the Ocean Science and Technology, Ocean Science and Technology School, Korea Maritime and Ocean University, Pusan 49112, Republic of Korea (Received Aug 20, 2015 / Revised Sep 14, 2015 / Accepted Sep 14, 2015) Journal of Microbiology (2015) Vol. 53, No. 10, pp. 711–717 Copyright  2015, The Microbiological Society of Korea DOI 10.1007/s12275-015-5417-5 Structural basis for the ATP-independent proteolytic activity of LonB proteases and reclassification of their AAA+ modules § Lon proteases degrade defective or denature proteins as well as some folded proteins for the control of cellular protein quality. There are two types of Lon proteases, LonA and LonB. Each consists of two functional components: a pro- tease component and an ATPase associated with various cellular activities (AAA+ module). Here, we report the 2.03 Å-resolution crystal structure of the isolated AAA+ module (iAAA+ module) of LonB from Thermococcus onnurineus NA1 (TonLonB). The iAAA+ module, having no bound nucleotide, adopts a conformation virtually identical to the ADP-bound conformation of AAA+ modules in the hexa- meric structure of TonLonB; this provides insights into the ATP-independent proteolytic activity observed in a LonB protease. Structural comparison of AAA+ modules between LonA and LonB revealed that the AAA+ modules of Lon proteases are separated into two distinct clades depending on their structural features. The AAA+ module of LonB be- longs to the ‘H2 & Ins1 insert clade (HINS clade)’ defined for the first time in this study, while the AAA+ module of LonA is a member of the HCLR clade. Keywords: AAA+ proteins, PS-1 insert, H2 insert, Ins1, Lon proteases, Thermococcus onnurineus NA1, ATP-indepen- dent proteolytic activity Introduction ATP-dependent proteases play important roles in cellular protein quality control and homeostasis as well as regulation of many cellular processes by selectively degrading regulatory proteins (Goldberg and St John, 1976; Hershko and Ciechan- over, 1992; Gottesman, 2003; Sauer et al., 2004; McClellan et al., 2005; Talarico et al., 2005). In prokaryotic cells and in the organelles of higher eukaryotes, energy-dependent proteolysis is accomplished by oligomeric ATP-dependent proteases, such as Lon, FtsH, ClpAP, ClpXP, and HslUV (Gottesman, 1996). Each consists of two functional parts: a protease component, assembled into barrel-shaped com- plexes with an internal chamber in which the proteolytic sites are sequestered, and an oligomeric ring-shaped ATPase as- sociated with various cellular activities (AAA+ module). Substrate proteins are recognized by the AAA+ ring, either directly or via adaptor proteins. ATP binding/hydrolysis cycles drive conformational changes of the AAA+ ring that create mechanical forces that denature substrates and trans- locate them into the proteolytic sites (Sauer and Baker, 2011). Lon and FtsH contrast with ClpAP, ClpXP, and HslUV, which are assembled from an independently expressed AAA+ component and a protease component, since AAA+ and protease components of Lon and FtsH exist in a single polypeptide. Lon is the first identified ATP-dependent protease whose orthologues are found in all kingdoms of life including hu- man being. Emerging evidence suggests that Lon proteases are divided into two subgroups, LonA and LonB. LonA, found in all bacteria and in eukaryotic cell organelles, and LonB, found only in Archea, are different in their domain compositions. LonA is composed of an N-terminal domain (LAN), an AAA+ domain, and a protease domain, whereas LonB consists of a protease domain and an AAA+ domain containing a membrane-spanning region that anchors the protein in the cytoplasmic surface of the membrane (Rota- nova et al., 2006). Here, we report the crystal structure of the isolated AAA+ module (iAAA+ module) of LonB from Thermococcus onnurineus NA1 (TonLonB) with the mem- brane-spanning region deleted. The limited proteolysis of the hexameric TonLonB by trypsin gave rise to the iAAA+ module. Structural comparison of the iAAA+ module with AAA+ modules in the hexameric TonLonB structure (Cha et al., 2010) suggests that mechanical forces could be gen- erated even in the absence of ATP. In addition, the structure of the iAAA+ module discriminated from the AAA+ module of a LonA protease reveals that the AAA+ modules of Lon proteases are separated into two distinct clades depending on their structural features. Materials and Methods Expression, purification, and crystallization TonLonB was expressed and purified as previously described