Structural and functional investigation of Φ-EL-chaperonin mediated protein folding
Chaperonins are ubiquitous, sequence related protein complexes that aid in the folding of nascent and misfolded polypeptides in an ATP driven pathway. Recently a GroEL-like, 860 kilo Dalton chaperonin protein complex was identified and isolated from the bacteriophage EL, a virus that infects the Gram-negative bacterium Pseudomonas aeruginosa. The bacteriophage EL contains 201 predicted open reading frames and is the only known phage that encodes for its own chaperonin known as &PHgr;-EL-chaperonin. To understand the importance of &PHgr;-EL-chaperonin in phage EL life cycle, the recombinant &PHgr;-EL-chaperonin protein was expressed in Escherichia coli (E. coli) purified to homogeneity and the structure of &PHgr;-EL-chaperonin was studied in ATP and ADP bound states using cryo-electron microscopy (cryo-EM). The &PHgr;-EL-chaperonin is a 61 KDa protein that self assembles into a tetradecamer composed of two back-to-back stacked identical rings in the presence of ATP. The 7Å cryo-EM reconstruction of ATP bound &PHgr;-EL-chaperonin provides insight into the architecture of the subunit interaction within and between the rings. The striking features include co-operative ATP binding between the rings and a novel subunit interaction across the inter-ring interface that accounted for its high structural stability in the ATP bound state. Furthermore, &PHgr;-EL-chaperonin does not require a co-chaperonin for hydrolyzing ATP, which was verified upon adding ADP to the protein sample and solving the structure using cryo-EM. The 9Å cryo-EM reconstruction of the ADP bound &PHgr;-EL-chaperonin revealed large conformational changes that occur as a result of ATP hydrolysis. Interestingly, both the chaperonin rings hydrolyze ATP simultaneously triggering conformational changes in both the rings and dissociates equatorially into two separate heptamers that vii then acquire a closed conformation. Particularly, the equatorial domains and apical domains extend away from the center of the ring and encase an enormous cavity inside the ring. The cavity presumably accommodates and mediates folding of large viral gene products that cannot be folded by the host chaperonin system. Additionally, dynamic light scattering (DLS) experiments were conducted to further investigate the mechanism of &PHgr;-EL-chaperonin. Interestingly, the experiments revealed the presence of a transient double ringed state of &PHgr;-EL-chaperonin that exists in the absence of nucleotide. The DLS data suggests that the transient double ringed state is formed by the back-to-back stacking of nucleotide free singe rings. Furthermore, this transient state of the chaperonin is required to expose the ATP binding pockets that were hidden in the &PHgr;-EL-chaperonin single rings. The ATP binding to this transient state regains the lost inter-ring contacts and forms the stable double ringed open structure. A bioinformatic analysis was carried out to further substantiate the novel findings in the &PHgr;-EL-chaperonin mechanism. The analysis strongly suggests that the &PHgr;-EL-chaperonin has evolved from Group I chaperonin with multiple insertions and deletions in its sequence that is accountable for this novel mechanism of protein folding. In addition, phylogenetic analysis unveiled that the &PHgr;-EL-chaperonin has evolutionarily diverged and is distantly related to Group I chaperonins. The distinctive features of &PHgr;-EL-chaperonin including co-chaperonin independent ATPase activity, ring-ring dissociation with respect to ATP hydrolysis strongly proposes a novel mechanism of &PHgr;-EL-chaperonin. Furthermore, the sequential and mechanistic differences between &PHgr;-EL-chaperonin and Group I chaperonin implies that the &PHgr;-EL-chaperonin should be classified into a new chaperonin group.
Molugu, Sudheer Kumar, "Structural and functional investigation of Φ-EL-chaperonin mediated protein folding" (2011). ETD Collection for University of Texas, El Paso. AAI3457756.