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

MecA targets the competence transcription factor ComK to ClpC. As a consequence, this factor is degraded by the ClpC/ClpP protease. ClpC is a member of 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-terminus. The effects of these mutations were studied in vitro and in vivo. Deletion of the C-terminal domain resulted in a decreased binding affinity for MecA, a decreased ATPase activity in response to MecA addition and decreased degradative activity in vitro. In vivo, this deletion resulted in a failure to degrade ComK and in a decrease in thermal resistance for growth. Mutation 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 phenotype with respect to competence. Mutation of the C-terminal Walker A motif (K551Q) caused essentially the reverse phenotype both in vivo and in vitro. Although binding to MecA was only moderately impaired with 2 mM ATP, this 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 defect 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. Although the evidence suggests that the functions of the two ATP-binding domains overlap, it appears that the N-terminal nucleotide-binding domain of ClpC is particularly concerned with MecA-related functions, whereas the C-terminal domain plays a more general role in the activities of ClpC.

Specific interaction of CCR5 amino-terminal domain peptides containing sulfotyrosines with HIV-1 envelope glycoprotein gp120.

The HIV-1 envelope glycoprotein gp120 interacts consecutively with CD4 and the CCR5 coreceptor to mediate the entry of certain HIV-1 strains into target cells. Acidic residues and sulfotyrosines in the amino-terminal domain (Nt) of CCR5 are crucial for viral fusion and entry. We tested the binding of a panel of CCR5 Nt peptides to different soluble gp120/CD4 complexes and anti-CCR5 mAbs. The tyrosine residues in the peptides were sulfated, phosphorylated, or unmodified. None of the gp120/CD4 complexes associated with peptides containing unmodified or phosphorylated tyrosines. The gp120/CD4 complexes containing envelope glycoproteins from isolates that use CCR5 as a coreceptor associated with Nt peptides containing sulfotyrosines but not with peptides containing sulfotyrosines in scrambled Nt sequences. Finally, only peptides containing sulfotyrosines inhibited the entry of an R5 isolate. Our data show that proper posttranslational modification of the CCR5 Nt is required for gp120 binding and viral entry. More importantly, the Nt domain determines the specificity of the interaction between CCR5 and gp120s from isolates that use this coreceptor.

The N- and C-terminal domains of MecA recognize different partners in the competence molecular switch.

ComK is a transcription factor required for the expression of competence genes in Bacillus subtilis. Binding to MecA targets ComK for degradation by the ClpCP protease. MecA therefore acts as an adapter protein recruiting a regulatory protein for proteolysis. However, when ComS is synthesized, ComK is released from binding by MecA and thereby protected from degradation. MecA binds to three protein partners during these processes: ComK, ClpC and ComS. Using limited proteolysis, we have defined N- and C-terminal structural domains of MecA and evaluated the interactions of these domains with the protein partners of MecA. Using surface plasmon resonance, we have determined that the N-terminal domain of MecA interacts with ComK and ComS and the C-terminal domain with ClpC. MecA is shown to exist as a dimer with dimerization sites on both the N- and C-terminal domains. The C-terminal domain stimulates the ATPase activity of ClpC and is degraded by the ClpCP protease, while the N-terminal domain is inactive in both of these assays. In vivo data were consistent with these findings, as comG-lacZ expression was decreased in a strain overproducing the N-terminal domain, indicating reduced ComK activity. We propose a model in which binding of ClpC to the C-terminal domain of MecA induces a conformational change enabling the N-terminal domain to bind ComK with enhanced affinity. MecA is widespread among Gram-positive organisms and may act generally as an adapter protein, targeting proteins for regulated degradation.