The N Terminus of ClpB from Thermus thermophilus Is Not Essential for the Chaperone Activity

Abstract

ClpB fromClpB from Thermus thermophilus is a member of the AAA protein superfamily that is important for a variety of biological activities (1). Despite their different cellular functions AAA proteins (ATPases associated with a variety of cellular activities) employ a general mechanism. They mediate the assembly and disassembly of large protein complexes that are involved in processes like DNA replication, vesicle transport, or organelle biogenesis. The AAA protein superfamily comprises the Clp/ Hsp100 proteins with its members ClpA, ClpX, and ClpY on the one hand and ClpB on the other.ClpA, ClpX, and ClpY (HslU) interact with cellular peptidases to form ATP-dependent proteases. The role of these Clp proteins is the unfolding of substrates and the delivery of unfolded polypeptides to the protease subunit. In contrast ClpB from Escherichia coli and T. thermophilus and the Saccharomyces cerevisiae homologue Hsp104 do not appear to bind to cellular proteases. Instead they interact with the DnaK/ Hsp70 chaperone system to assist the disaggregation of protein aggregates (2-6).Clp proteins are further classified according to the number of nucleotide binding domains (NBD), 1 which are 220 -250 amino acids in length and show a high level of sequence homology. Class two proteins contain only one NBD whereas the class one members ClpA and ClpB contain two NBDs. In ClpB and Hsp104 ATP hydrolysis at both NBDs was shown to be necessary for the chaperone activity of the Clp proteins (7,8).Structural information on the class two protein HslU (ClpY) and the hexamerization domain (D2) of N-ethylmaleimide-sensitive fusion protein give insight into the domain architecture of AAA proteins (9 -11). These crystal structures show ringshaped oligomers composed of six monomers. The AAA modules of both proteins show a similar overall structure, consisting of the core NBD and a C-terminal, mostly helical, domain. The core NBD contains the Rossman fold including the phosphate binding loop of ATP-and GTP-binding proteins, which consists of five central ␤-sheets flanked by ␣-helices. In the case of HslU an additional helical domain is inserted in the NBD that is not present in other Clp proteins.In ClpB Eco the C-terminal domain of the second AAA cassette was shown to be important for oligomerization, ATPase activity, and chaperone function (12). Isolated C-domains of ClpA, ClpX, and ClpY, which associate with peptidases, were found to interact with different substrate proteins in vivo. Therefore the C-terminal domains were proposed to be sensor and substrate discrimination domains although the C-terminal domains show only little sequence homology between different Clp proteins (13).The class one Clp proteins have, in addition, an N-terminal domain that precedes the first NBD. The ClpA and ClpB mRNAs of E. coli contain internal translation initiation sites and are expressed in vivo as two gene products: the full-length proteins and shortened versions lacking the N-terminal domain (14,15). The N-terminal domains are supposed to consist of two...

ClpB N-terminal domain plays a regulatory role in protein disaggregation

“…ATPase activity assays were measured for ClpB WT (WT ClpB) and ClpB ΔN in the absence and presence of client proteins. Substrate proteins that showed CSPs upon addition of the isolated NTD in our NMR experiments also enhanced ATPase activity of ClpB by threefold to fivefold, similar to previously reported levels of activation by α-casein (17). These same substrates, however, were only able to activate ClpB ΔN by 1.1-to 1.8-fold, suggesting that the direct interaction of the ClpB NTD with the substrate is essential for the full allosteric activation of the ClpB ATPase domains (Fig.…”

“…Addition of substrate proteins has been widely reported to elevate ClpB ATPase activity (17), with some substrates such as α-casein stimulating ClpB in an NTD-dependent manner, while others inducing the same degree of activation for both ClpB and ClpB ΔN (ClpB hexamers lacking the NTD). We thus wanted to establish whether substrates that bind to monomeric NTD also displayed an NTD-dependent activation of the hexameric ClpB chaperone.…”

“…Its precise function remains unclear-it is not required for thermotolerance (17), yet it becomes important in vivo when Hsp70 activity is compromised (18,19). Although it was reported that the NTD is not required for disaggregation of many small aggregates, it is involved in the reactivation of several strongly aggregated proteins (17,18,20). Here, we use NMR to structurally characterize its interaction with substrate proteins and to elucidate its functional role in protein disaggregation.…”

Interplay between Heat Shock Proteins HSP101 and HSA32 Prolongs Heat Acclimation Memory Posttranscriptionally in Arabidopsis

“…S1). The NTD was shown to be dispensable for chaperone activity in Escherichia coli and Thermus thermophilus (Beinker et al, 2002;Mogk et al, 2003) and for thermotolerance in cyanobacteria and yeast (Clarke and Eriksson, 2000;Hung and Masison, 2006). However, work on E. coli ClpB showed that truncation of the NTD causes severe defects in molecular chaperone activity in vitro (Barnett et al, 2000;Li and Sha, 2003).…”

Trigonal DnaK-DnaJ Complex Versus Free DnaK and DnaJ

“…The stable K⅐J has been reported only for DnaK and DnaJ from T. thermophilus, but the chaperone function of K⅐J has been studied with a general interest because the current model for the mechanism of DnaK function assumes only transient but not stable interaction between DnaK and DnaJ (4,11,16,19,23). Related to this contention, the chaperone activity of uncomplexed TDnaK and TDnaJ (termed KϩJ) has been reported (14,23,24), but the difference from that of K⅐J is not clear. Here, we carefully compared chaperone activities of the K⅐J set (K⅐J plus TGrpE) and those of the KϩJ set (KϩJ plus TGrpE).…”