A camel passes through the eye of a needle: protein unfolding activity of Clp ATPases

Living cells maintain an elaborate network of molecular processes that depend on the function of large protein complexes. The principles of assembly and mechanism of those “protein machines” that carry out so many essential cellular functions provide a challenging new frontier to be explored in the post-genomic era (Alberts, 1998). Research on Clp ATPases is an ongoing inquiry into the inner workings of machinery designed to maintain the quality of cellular proteins by performing the functions of molecular chaperones and energy-dependent proteases. Although many unanswered questions remain, major progress in understanding the mechanism of protein quality control by Clp ATPases has been achieved in recent years. The place of Clp ATPases within the AAA+ superfamily of ATPases associated with various cellular activities (Neuwald et al., 1999) suggests that analogous mechanisms might by applicable to a substantially broader range of biological processes. ClpA from Escherichia coli, the ATP-binding component of an energy-dependent “caseinolytic protease” was the first Clp ATPase discovered (Hwang et al., 1987; Katayama-Fujimura et al., 1987). It was followed by identification of a number of closely related ATPases: ClpB, ClpC, ClpE, ClpL, ClpV, ClpX, and HslU (ClpY) (reviewed in (Butler et al., 2006; Dougan et al., 2002a; Gottesman, 2003; Schlieker et al., 2005)). Four members of the family: ClpA, ClpB, ClpX, and HslU will be the focus of the present review, as these are the most thoroughly characterized Clp ATPases. Many Clp ATPases, with the notable exception of ClpB, form complexes with peptidase subunits (ClpP or HslV, see Fig. 1). The ClpAP, ClpXP, and HslUV proteases are similar in design to the eukaryotic 26S proteasome, with the ATPase subunits guarding the entrances to the proteolytic chambers (Kessel et al., 1995; Rohrwild et al., 1997). Clp proteases are found in all prokaryotes as well as in mitochondria and chloroplasts of eukaryotic cells (Gottesman, 2003). Fig. 1 Domain organization and function of selected Clp ATPases. AAA+ sequence modules are shown along with the N-terminal domains of ClpA and ClpB (N), the middle domain of ClpB (M), the Zn-binding domain of ClpX (Zn), and the intermediate domain of HslU (I). ... ClpB is different from ClpA, ClpX, and HslU because it does not associate with peptidase subunits (see Fig. 1) and is found not only in prokaryotes and in the eukaryotic organelles, but also in the cytosol of yeast and plants. Indeed, the function of ClpB is distinct from that of other Clp ATPases: ClpB is not involved in protein degradation (Woo et al., 1992), instead, it disaggregates and reactivates strongly aggregated proteins (Goloubinoff et al., 1999; Motohashi et al., 1999; Zolkiewski, 1999). The aggregation-reversing activity of ClpB requires cooperation with the Hsp70/Hsp40 chaperone machinery (bacterial DnaK/DnaJ). Presently, Clp ATPases are considered to be a subgroup of the AAA+ protein superfamily (Lupas and Martin, 2002; Neuwald et al., 1999). Thus, their protein degradation and disaggregation activities are just two of a wide variety of AAA+ functions that include membrane fusion and vesicular trafficking, cytoskeletal regulation and intracellular transport, organelle biogenesis, DNA replication and repair, and transcription regulation (Hanson and Whiteheart, 2005; Ogura and Wilkinson, 2001; Vale, 2000). The functional diversity of AAA+ ATPases is based on the common protein architecture: all AAA+ proteins contain conserved sequence modules (one or two, see Fig. 1) with ATP-binding motifs (Walker A and B, sensor-1 and -2) (reviewed in (Hanson and Whiteheart, 2005)). The AAA+ ATPases form ring- or cylinder-shaped oligomers (usually hexamers) with a narrow channel located at the center (see Fig. 2) and the nucleotide binding sites at the inter-subunit interfaces. In addition to the AAA+ modules, the ATPases contain other domains that often specify their cellular localization, substrate identity, and/or their interacting partners (Iyer et al., 2004). Many AAA+ ATPases, including ClpA and ClpX, interact with partner proteins that modulate their function (Dougan et al., 2002b). Fig. 2 Substrate threading mechanism of ClpA. (Left), ClpA contains two AAA+ modules (D1, D2) and forms a cylinder-shaped hexamer with a narrow central channel. The degradation tag (red) at the terminus of a protein substrate (blue) is inserted into the ClpA ...