Chaperonins are ubiquitous and essential mediators of cellular protein folding that consist of 14–18 subunits arranged in two stacked rings1–4
. Each ring encompasses a central chamber that accommodates non-native polypeptides. All chaperonins share a similar subunit architecture, consisting of three distinct domains: an ATP-binding equatorial domain, a distal apical domain harboring the polypeptide-binding sites and an intermediate hinge domain. A key feature of chaperonins is their ability to close their chamber and encapsulate the bound substrate, thus providing a protected environment for protein folding. Opening and closing of the folding chamber is controlled by a conformational cycle driven by ATP binding and hydrolysis. Despite overall structural similarities, there are substantial differences between the eubacterial chaperonins, such as GroEL from E. coli2–5
, and the chaperonins from archaea and eukaryotes6,7
. Whereas bacterial, so-called group I chaperonins are homo-oligomeric, eukaryotic and archaeal group II chaperonins are generally hetero-oligomeric7
. The greatest difference between group I and group II chaperonins resides in their distinct strategies to mediate closure of their central folding chamber. Group I chaperonins use a ring-shaped cochaperone, GroES, as a detachable lid. In the presence of ATP, GroES binds GroEL and closes its central cavity5
. In contrast, group II chaperonins have a built-in lid formed by protrusions that extend from the tip of each apical domain8–10
. For the eukaryotic chaperonin TRiC (also called CCT), it has been shown that assembly of the iris-like lid is triggered by ATP hydrolysis9
. Despite intensive studies on the biochemistry and function of bacterial chaperonins, little is known of the mechanistic and biological significance underlying the unique structural features of eukaryotic and archaeal chaperonins.
The chaperonin folding cycle is crucially dependent on the synchronized action of individual subunits11–13
. Accordingly, chaperonins are highly allosteric protein machines11–13
. Subunits within each ring are coupled as a functional unit through positive cooperativity in ATP binding11,14–16
; this allows them to act in a concerted fashion to create the closed folding chamber. In addition, negative communication between the rings causes ATP binding to one ring to inhibit ATP binding to the adjacent ring14–18
. This feature is thought to ensure that only one ring is folding-active at a given time, allowing chaperonins to function as ‘two-stroke’ motors. This unique allosteric behavior is observed in both group I and group II chaperonins11,14,15,19,20
and is described as nested cooperativity, as the positive cooperative transition within each ring is nested into the overall negative cooperativity between them.
The basis of allosteric regulation in group I chaperonins has been extensively studied. Positive and negative cooperativity in GroEL are established independently of the GroES lid19,21
, although GroES profoundly influences the conformational changes of GroEL12,21–24
. In contrast, little is known about the molecular basis of allostery in eukaryotic and archaeal chaperonins. Their very different mechanisms of lid formation raise the question of how group II chaperonins regulate their conformational cycle. We here investigate the function of the built-in lid in the mammalian chaperonin TRiC and an archaeal chaperonin from Methanococcus maripaludis
. Our results show that the built-in lid is important in regulating the conformational cycle of group II chaperonins. We found that the apical protrusions have been incorporated into the allosteric network that communicates ATP-induced conformational changes between subunits. It thus appears that group I and group II chaperonins have evolved very different strategies for intersubunit coordination, despite the overall structural conservation of the chaperonin ring architecture. Our findings help explain how group II chaperonins act like two-stroke motors without the help of an extrinsic cofactor and show how conserved protein scaffolds can achieve allosteric regulation through widely divergent structural features.