Facilitated Oligomerization of Mycobacterial GroEL: Evidence for Phosphorylation-Mediated Oligomerization

Abstract

The distinctive feature of the GroES-GroEL chaperonin system in mediating protein folding lies in its ability to exist in a tetradecameric state, form a central cavity, and encapsulate the substrate via the GroES lid. However, recombinant GroELs of Mycobacterium tuberculosis are unable to act as effective molecular chaperones when expressed in Escherichia coli. We demonstrate here that the inability of M. tuberculosis GroEL1 to act as a functional chaperone in E. coli can be alleviated by facilitated oligomerization. The results of directed evolution involving random DNA shuffling of the genes encoding M. tuberculosis GroEL homologues followed by selection for functional entities suggested that the loss of chaperoning ability of the recombinant mycobacterial GroEL1 and GroEL2 in E. coli might be due to their inability to form canonical tetradecamers. This was confirmed by the results of domain-swapping experiments that generated M. tuberculosis-E. coli chimeras bearing mutually exchanged equatorial domains, which revealed that E. coli GroEL loses its chaperonin activity due to alteration of its oligomerization capabilities and vice versa for M. tuberculosis GroEL1. Furthermore, studying the oligomerization status of native GroEL1 from cell lysates of M. tuberculosis revealed that it exists in multiple oligomeric forms, including single-ring and double-ring variants. Immunochemical and mass spectrometric studies of the native M. tuberculosis GroEL1 revealed that the tetradecameric form is phosphorylated on serine-393, while the heptameric form is not, indicating that the switch between the singleand double-ring variants is mediated by phosphorylation.

Multiple chaperonins in bacteria—novel functions and non-canonical behaviors

“…While GroEL1 has been functionally characterized in these studies, the ability of GroEL2 in complementing E. coli GroEL was studied independently by two groups, which resulted in an understanding that GroEL2 can functionally replace E. coli GroEL in vivo and in vitro only upon abundance (Hu et al 2008;Kumar et al 2009;Fan et al 2012). Moreover, GroEL2 has been shown to localize at the cell wall (Hickey et al 2009) as a multimer and upon cleavage by a serine protease, Hip1, separates into the secretion-competent monomeric form.…”

“…Furthermore, evolutionary studies on Mtb groEL sequences have suggested rapid evolution of the groEL1 gene (Goyal et al 2006) and that the difference in the rates of evolution between GroEL1 and GroEL2 has been proposed to be due to differential interaction of these chaperonins with the host immune system (Hughes 1993). Although these studies have principally employed biochemical tools on purified proteins and convincingly demonstrated the inability of Mtb GroELs to act as chaperones, further genetic studies on these chaperonin genes have provided evidence towards the hypothesis that Mtb GroELs are inactive as chaperonins (Kumar et al 2009;Henderson et al 2010;Kumar and Mande 2011). Phenotypic investigations on the Mtb GroELs in different E. coli groEL mutant strains established that Mtb GroELs are ineffective in complementing E. coli GroEL (Kumar et al 2009).…”

“…Although these studies have principally employed biochemical tools on purified proteins and convincingly demonstrated the inability of Mtb GroELs to act as chaperones, further genetic studies on these chaperonin genes have provided evidence towards the hypothesis that Mtb GroELs are inactive as chaperonins (Kumar et al 2009;Henderson et al 2010;Kumar and Mande 2011). Phenotypic investigations on the Mtb GroELs in different E. coli groEL mutant strains established that Mtb GroELs are ineffective in complementing E. coli GroEL (Kumar et al 2009). Furthermore, directed evolution studies employing gene shuffling and domain swapping to derive functional GroELs from Mtb GroELs established that oligomerization is the prelude to the formation of an active GroEL chaperonin, and therefore, impaired oligomerization of Mtb GroELs results in their inactivity.…”

“…Furthermore, directed evolution studies employing gene shuffling and domain swapping to derive functional GroELs from Mtb GroELs established that oligomerization is the prelude to the formation of an active GroEL chaperonin, and therefore, impaired oligomerization of Mtb GroELs results in their inactivity. Furthermore, attempts to understand the cause of the inactivity led to the discovery that Mtb GroEL1 follows facilitated oligomerization to the tetradecameric state mediated by a phosphorylation switch, suggesting that determinants of oligomerization for Mtb GroEL1 are distinct from their E. coli counterpart (Kumar et al 2009). Such regulated oligomerization of GroEL has been postulated to help nutrient-deprived and slowgrowing Mtb to cope up with GroEL that, if perpetually active, would consume the ATP pools (Kumar and Mande 2011;Haslbeck et al 2005).…”

Comparative Biochemical Characterization of Two GroEL Homologs from the CyanobacteriumSynechococcus elongatusPCC 7942

“…However, the tetradecamer of one of the GroELs, Cpn60.1, has been detected in cell extracts. 32) The tetradecameric form is phosphorylated while the heptameric form is not, indicating phosphorylation-mediated regulation of the GroEL oligomeric states. The GroELs possess very weak ATPase activity.…”