Broad yet high substrate specificity: the challenge of AAA+ proteins Axel Mogk, * David Dougan, Jimena Weibezahn, Christian Schlieker, Kursad Turgay, and Bernd Bukau Zentrum fu ¨ r Molekulare Biologie Heidelberg, Universit€ at Heidelberg, Im Neuenheimer Feld 282, Heidelberg D-69120, Germany Received 31 August 2003, and in revised form 9 October 2003 Abstract AAA+ proteins remodel target substrates in an ATP-dependent manner, an activity that is of central importance for a plethora of cellular processes. While sharing a similar hexameric structure AAA+ proteins must exhibit differences in substrate recognition to fulfil their diverse biological functions. Here we describe strategies of AAA+ proteins to ensure substrate specificity. AAA domains can directly mediate substrate recognition, however, in general extra domains, added to the core AAA domain, control substrate interaction. Such extra domains may either directly recognize substrates or serve as a platform for adaptor proteins, which transfer bound substrates to their AAA+ partner proteins. The positioning of adaptor proteins in substrate recognition can enable them to control the activity of their partner proteins by coupling AAA+ protein activation to substrate availability. Ó 2003 Elsevier Inc. All rights reserved. Keywords: AAA+ superfamily; Adaptor protein; Chaperone; Protease; Substrate specificity 1. Introduction In all organisms, many vital cellular processes in- cluding membrane fusion, cell cycle regulation, orga- nelle biogenesis, as well as protein repair and degradation are controlled by members of a single protein superfamily, the AAA+ superfamily (Neuwald et al., 1999). The AAA+ superfamily comprises both the Hsp100/Clp family (Schirmer et al., 1996) and the more extensive ATPases associated with a variety of cellular activities (AAA) family (Beyer, 1997). AAA+ proteins share a common ATPase domain with a conserved se- quence of 230–250 amino acid residues referred to as the AAA domain. Each AAA domain consists of a core ATPase domain, containing the classic Walker A and B motifs, and a C-terminal a-helical domain. The struc- tural basis of this superfamily was confirmed by deter- mining the structure of various AAA+ proteins (Bochtler et al., 2000; Guo et al., 2002b; Lenzen et al., 1998; Li and Sha, 2002; Yu et al., 1998; Zhang et al., 2000). The superfamily can be further divided into two distinct classes, based on the number of AAA domains present in the protein (Schirmer et al., 1996). Class I proteins (e.g., ClpA, ClpB, and ClpC) contain two AAA domains, referred to as AAA-1 and AAA-2, separated by a linker sequence of variable length (Fig. 1). In contrast, class II proteins (e.g., ClpX and HslU(ClpY)) contain only one AAA domain (homologous to AAA-2). Here we shall concentrate primarily on prokaryotic AAA+ proteins, involved in protein quality control. AAA+ proteins play a prominent role in the refolding or removal of misfolded and aggregated proteins. ClpB is rescuing proteins from an aggregated state in coopera- tion with the DnaK chaperone system (Glover and Lindquist, 1998; Goloubinoff et al., 1999; Mogk et al., 1999). A number of AAA+ proteins (e.g., ClpA, ClpX, and HslU) associate with proteolytic components (ClpP, HslV) to form ATP-dependent proteases. Some AAA+ proteins (e.g., Lon) also contain a peptidase domain within a single polypeptide (Fig. 1). Proteases of the Hsp100/Clp protein family are the best understood proteolytic systems. ClpAP, ClpXP, and HslUV form barrel-like structures with two heptameric/hexameric rings of ClpP/HslV flanked on either side by hexameric rings of ClpX, ClpA or HslU (Beuron et al., 1998; Sousa et al., 2000). The active sites of ClpP and HslV are * Corresponding author. Fax: +49-6221-545894. E-mail address: a.mogk@zmbh.uni-heidelberg.de (A. Mogk). 1047-8477/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2003.10.009 Journal of Structural Biology 146 (2004) 90–98 Journal of Structural Biology www.elsevier.com/locate/yjsbi