Abstract
ClpL is a member of the HSP100 family of AAA+ chaperones that is widely present in Gram-positive but surprisingly absent in Gram-negative bacteria. ClpL is involved in various cellular processes, including stress tolerance response, long-term survival, virulence, and antibiotic resistance. ClpL is poorly characterized, and its molecular mechanisms of chaperone activity are largely unclear. Here, we biochemically characterized the ClpL protein from Streptococcus mutans, a dental pathogen, to understand its biological functions. ClpL harbors five domains: N-domain, two nucleotide binding domains (NBD-1 and NBD-2), M-domain, and C-domain. NBD-1 and NBD-2 contain distinct Walker A and B motifs for ATP binding and hydrolysis, respectively. We found that ClpL predominantly exists as a trimer in solution; however, upon ATP binding, it rapidly forms a hexameric structure. To study structure-function activity, we constructed several substitution and deletion mutants. We found that mutations in the Walker A and B motifs interfered with ATP hydrolysis and oligomerization. Similarly, deletions of N-, M-, and C-domains abolished both ATPase activity and oligomerization. Because we previously found that ClpL acts as a chaperone, we analyzed the chaperone activity. Surprisingly, we found that the NBD-2 mutants did not display any chaperone activity, indicating that ATP binding and hydrolysis by NBD-2 are essential for the chaperone. However, NBD-1 mutants showed chaperone activities, but the activities were variable depending on the nature of the mutations. Our results indicate that unlike other HSP100 family chaperones, ClpL is a novel chaperone that does not require any additional secondary chaperones for its activity.