Seryl phosphorylation modulates chaperonin function of Mycobacterial chaperonin, Cpn60.1.

The GroEL chaperonin forms a 14‑subunit double ring that encapsulates client proteins for folding with the aid of the cochaperonin GroES. Mycobacteria encode two GroEL homologs: Cpn60.2 (which is essential) and Cpn60.1 (which is non-essential but which has been proposed to have roles in pathogenesis). Cpn60.1 homologues exhibited impaired oligomerization and thereby failed to exhibit chaperonin activity. We previously showed that phosphorylation of MtCpn60.1 S393 correlates with oligomeric assembly. Here, we show that canonical oligomerization residues are replaced by serines in Cpn60.1 homologues, enabling a phosphorylation‑switch. We generated phospho-mimetic variants at these positions and assessed their ability to functionally replace Escherichia coli GroEL. Using functional complementation and serial-depletion assays, we demonstrate that although the wild-type MtCpn60.1 failed, a double phospho-mimetic variant (S393D + S75D) exhibited chaperonin activity in groEL conditional mutant strains but did not functionally replace GroEL in a groEL knock out strain. Further, native PAGE analysis of the S75D/S393D double phospho-mimetic variant was consistent with higher-order oligomerization, while mass spectrometry of MtCpn60.1 confirmed S75 phosphorylation and identified additional phosphoserine-containing peptides. Homology modeling suggests that phosphorylation may alter a residue-pair interaction involved in oligomerization. Together, these findings support a model in which phosphorylation contributes to higher-order oligomerization and partial chaperonin activity of Cpn60.1.