Figure 1.

The conserved intersubunit residue A328 controls ClpB activity. (A) Hexameric model of AAA-1 ring of E. coli ClpB. Structure of the hexameric AAA-1 ring of E. coli ClpB and M-domains (red). The hexameric model is based on the crystal structure of Thermus thermophilus ClpB (pdb number 1qvr1) and was generated as described in Diemand and Lupas (2006). The enlarged section shows the catalytic site and bound AMPPNP. AAA-1 subunits are in beige and gray. The positions of Walker A and B motifs, the trans-acting arginine fingers and the analyzed mutational sites (A328, K476) are indicated. AMPPNP is shown in black. A sequence alignment of the analyzed subunit interface of ClpB homologs (Hsp101, Hsp104, Hsp78) and ClpA is provided. A328 is highlighted in red, arginine fingers in purple (TT, Thermus thermophilus; EC, Escherichia coli; AT, Arabidopsis thaliana; SC, Saccharomyces cerevisiae). (B) E. coli ΔclpB cells expressing the indicated plasmid-encoded clpB alleles under control of an IPTG-regulatable promoter were grown overnight at 30°C. Various dilutions (10⁰–10⁻⁷) were spotted on LB plates containing the indicated IPTG concentrations and incubated at 30, 37, or 42°C for 24 h.