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...

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The signal recognition particle (SRP) from Escherichia coli consists of 4.5S RNA and protein Ffh. It is essential for targeting ribosomes that are translating integral membrane proteins to the translocation pore in the plasma membrane. Independently of Ffh, 4.5S RNA also interacts with elongation factor G (EF-G) and the 30S ribosomal subunit. Here we use a cross-linking approach to probe the conformation of 4.5S RNA in SRP and in the complex with the 30S ribosomal subunit and to map the binding site. The UV-activatable cross-linker p-azidophenacyl bromide (AzP) was attached to positions 1, 21, and 54 of wildtype or modified 4.5S RNA. In SRP, cross-links to Ffh were formed from AzP in all three positions in 4.5S RNA, indicating a strongly bent conformation in which the 5 0 end (position 1) and the tetraloop region (including position 54) of the molecule are close to one another and to Ffh. In ribosomal complexes of 4.5S RNA, AzP in both positions 1 and 54 formed cross-links to the 30S ribosomal subunit, independently of the presence of Ffh. The major cross-linking target on the ribosome was protein S7; minor cross-links were formed to S2, S18, and S21. There were no cross-links from 4.5S RNA to the 50S subunit, where the primary binding site of SRP is located close to the peptide exit. The functional role of 4.5S RNA binding to the 30S subunit is unclear, as the RNA had no effect on translation or tRNA translocation on the ribosome.

ClpB cooperates with the DnaK chaperone system in the reactivation of protein from aggregates and is a member of the ATPases associated with a variety of cellular activities (AAA؉) protein family. The underlying disaggregation reaction is dependent on ATP hydrolysis at both AAA cassettes of ClpB but the role of each AAA cassette in the reaction cycle is largely unknown. Here we analyze the activity of the separately expressed and purified nucleotide binding domains of ClpB from Thermus thermophilus. The two fragments show different biochemical properties: the first construct is inactive in ATPase activity assays and binds nucleotides weakly, the second construct has a very high ATPase activity and interacts tightly with nucleotides. Both individual fragments have lost their chaperone function and are not able to form large oligomers. When combined in solution, however, the two fragments form a stable heterodimer with oligomerization capacities equivalent to wildtype ClpB. This non-covalent complex regains activity in reactivating protein aggregates in cooperation with the DnaK chaperone system. Upon complex formation the ATPase activity of fragment 2 is reduced to a level similar to wild-type ClpB. Hence functional ClpB can be reassembled from its isolated AAA cassettes showing that covalent linkage of these domains is not a prerequisite for the chaperone activity. The observation that the intrinsically high ATPase activity of AAA2 is suppressed by AAA1 allows a hypothetical assignment of their mechanistic function. Whereas the energy gained upon ATP hydrolysis at the AAA2 is likely to drive a conformational change of the structure of ClpB, AAA1 might function as a regulator of the chaperone cycle.ClpB co-operates with the DnaK chaperone system consisting of the principal component DnaK and the co-chaperones DnaJ and GrpE in the disaggregation and reactivation of protein aggregates. This chaperone network was first identified in Saccharomyces cerevisiae to be essential for the survival of yeast at elevated temperatures (1). The mechanism of this chaperone-mediated thermotolerance was elucidated and linked to the protein disaggregation activity of the ClpB-DnaK chaperone network (2). This reaction is strictly dependent on the presence and hydrolysis of ATP. In Escherichia coli 25% of the heat-labile proteins were found to interact with these chaperones under heat shock conditions, indicating a broad substrate specificity of the ClpB-DnaK system in vivo and a preference for large multidomain proteins as substrates (3).The chaperone activity of ClpB and the DnaK system was also studied in vitro using a variety of substrate proteins (2-6). Model substrates from different organisms, with different sizes and functions have been established leading to the characterization of chaperone function under a variety of experimental conditions (5-8).According to all determined crystal structures, the fold of AAA cassettes (ATPases associated with a variety of cellular activities) 4 is conserved (9, 10). AAA cassettes consist o...