Caseinolytic protease L (ClpL) is a member of the heat shock protein (Hsp) 100 family, which is found mostly in Gram‐positive bacteria. Here, ClpL, a major HSP in Streptococcus pneumoniae (pneumococcus), was biochemically characterized in vitro. Recombinant ClpL shows nucleotide hydrolase, refolding, holdase and disaggregation activity using either Mg2+ or Mn2+ and does not require the DnaK system for chaperone activity. ClpL exhibits two features distinct from other HSP100 family proteins: (a) Mn2+ enhances hydrolase activity, as well as chaperone activity; and (b) NTPase activity. ClpL forms a hexamer in the presence of ADP, ATP and ATP‐γ‐S. Mutational analysis using double‐mutant proteins mutated at the two Walker A motifs (K127A/T128A and K458A/T459A) revealed that both nucleotide‐binding domains are involved in chaperone activity, ATP hydrolase activity and hexamerization. Overall, pneumococcal ClpL is a unique Mn2+‐dependent Hsp100 family member that has chaperone activity without other co‐chaperones.

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