Roles of the two ClpC ATP binding sites in the regulation of competence and the stress response

K. Turgay et al., Roles of the two ClpC ATP binding sites in the regulation of competence and the stress response, MOL MICROB, 42(3), 2001, pp. 717-727
Citations number
Categorie Soggetti
Journal title
ISSN journal
0950-382X → ACNP
Year of publication
717 - 727
SICI code
MecA targets the competence transcription factor ComK to ClpC. As a consequ ence, this factor is degraded by the ClpC/ClpP protease. ClpC is a member o f the Clp/HSP100 family of ATPases and possesses two ATP binding sites. We have individually modified the Walker A motifs of these two sites and have also deleted a putative substrate recognition domain of ClpC at the C-termi nus. The effects of these mutations were studied in vitro and in vivo. Dele tion of the C-terminal domain resulted in a decreased binding affinity for MecA, a decreased ATPase activity in response to MecA addition and decrease d degradative activity in vitro. In vivo, this deletion resulted in a failu re to degrade ComK and in a decrease in thermal resistance for growth. Muta tion of the N-terminal Walker A box (K214Q) caused a drastically decreased ATPase activity in vitro, but did not interfere with MecA binding. in vivo, this mutation had no effect on thermal resistance, but had a clpC null phe notype with respect to competence. Mutation of the C-terminal Walker A moti f (K551Q) caused essentially the reverse phenotype both in vivo and in vitr o. Although binding to MecA was only moderately impaired with 2 mM ATP, thi s mutant protein displayed no response to 0.2 mM ATP, unlike the wild-type ClpC and the K214Q mutant protein. The ATPase activity of the K551Q mutant protein, induced by the addition of MecA plus ComS, was decreased about 10- fold but was not eliminated. In vivo, the K551Q mutation showed a partial d efect with respect to competence and a profound loss of thermal resistance. Sporulation was reduced drastically by the K551Q and less so by the K214Q mutation, but remained unaffected by deletion of the C-terminal domain. Alt hough the evidence suggests that the functions of the two ATP-binding domai ns overlap, it appears that the N-terminal nucleotide-binding domain of Clp C is particularly concerned with MecA-related functions, whereas the C-term inal domain plays a more general role in the activities of ClpC.