Hays S. Rye

Characterization of the Active Intermediate of a GroEL–GroES-Mediated Protein Folding Reaction

Recent studies of GroE-mediated protein folding indicate that substrate proteins are productively released from a cis ternary complex in which the nonnative substrate is sequestered within the GroEL channel underneath GroES. Here, we examine whether protein folding can occur in this space. Stopped-flow fluorescence anisotropy of a pyrene-rhodanese-GroEl complex indicates that addition of GroES and ATP (but not ADP) leads to a rapid change in substrate flexibility at GroEL. Strikingly, when GroES release is blocked by the use of either a nonhydrolyzable ATP analog or a single-ring GroEL mutant, substrates complete folding while remaining associated with chaperonin. We conclude that the cis ternary complex, in the presence of ATP, is the active state intermediate in the GroE-mediated folding reaction: folding is initiated in this state and for some substrates may be completed prior to the timed release of GroES triggered by ATP hydrolysis.

Distinct actions of cis and trans ATP within the double ring of the chaperonin GroEL

The chaperonin GroEL is a double-ring structure with a central cavity in each ring that provides an environment for the efficient folding of proteins when capped by the co-chaperone GroES in the presence of adenine nucleotides. Productive folding of the substrate rhodanese has been observed in cis ternary complexes, where GroES and polypeptide are bound to the same ring, formed with either ATP, ADP or non-hydrolysable ATP analogues,, suggesting that the specific requirement for ATP is confined to an action in the trans ring that evicts GroES and polypeptide from the cis side. We show here, however, that for the folding of malate dehydrogenase and Rubisco there is also an absolute requirement for ATP in the cis ring, as ADP and AMP-PNP are unable to promote folding. We investigated the specific roles of binding and hydrolysis of ATP in the cis and trans rings using mutant forms of GroEL that bind ATP but are defective in its hydrolysis. Binding of ATP and GroES in cis initiated productive folding inside a highly stable GroEL–ATP–GroES complex. To discharge GroES and polypeptide, ATP hydrolysis in the cis ring was required to form a GroEL–ADP–GroES complex with decreased stability, priming the cis complex for release by ATP binding (without hydrolysis) in the trans ring. These observations offer an explanation of why GroEL functions as a double-ring complex.

GroEL-GroES Cycling: ATP and Nonnative Polypeptide Direct Alternation of Folding-Active Rings

The double-ring chaperonin GroEL mediates protein folding in the central cavity of a ring bound by ATP and GroES, but it is unclear how GroEL cycles from one folding-active complex to the next. We observe that hydrolysis of ATP within the cis ring must occur before either nonnative polypeptide or GroES can bind to the trans ring, and this is associated with reorientation of the trans ring apical domains. Subsequently, formation of a new cis-ternary complex proceeds on the open trans ring with polypeptide binding first, which stimulates the ATP-dependent dissociation of the cis complex (by 20- to 50-fold), followed by GroES binding. These results indicate that, in the presence of nonnative protein, GroEL alternates its rings as folding-active cis complexes, expending only one round of seven ATPs per folding cycle.

GroEL-Mediated Protein Folding: Making the Impossible, Possible

ABSTRACT Protein folding is a spontaneous process that is essential for life, yet the concentrated and complex interior of a cell is an inherently hostile environment for the efficient folding of many proteins. Some proteins—constrained by sequence, topology, size, and function—simply cannot fold by themselves and are instead prone to misfolding and aggregation. This problem is so deeply entrenched that a specialized family of proteins, known as molecular chaperones, evolved to assist in protein folding. Here we examine one essential class of molecular chaperones, the large, oligomeric, and energy utilizing chaperonins or Hsp60s. The bacterial chaperonin GroEL, along with its co-chaperonin GroES, is probably the best-studied example of this family of protein-folding machine. In this review, we examine some of the general properties of proteins that do not fold well in the absence of GroEL and then consider how folding of these proteins is enhanced by GroEL and GroES. Recent experimental and theoretical studies suggest that chaperonins like GroEL and GroES employ a combination of protein isolation, unfolding, and conformational restriction to drive protein folding under conditions where it is otherwise not possible.

GroEL stimulates protein folding through forced unfolding

Many proteins cannot fold without the assistance of chaperonin machines like GroEL and GroES. The nature of this assistance, however, remains poorly understood. Here we demonstrate that unfolding of a substrate protein by GroEL enhances protein folding. We first show that capture of a protein on the open ring of a GroEL–ADP–GroES complex, GroELs physiological acceptor state for non-native proteins in vivo, leaves the substrate protein in an unexpectedly compact state. Subsequent binding of ATP to the same GroEL ring causes rapid, forced unfolding of the substrate protein. Notably, the fraction of the substrate protein that commits to the native state following GroES binding and protein release into the GroEL–GroES cavity is proportional to the extent of substrate-protein unfolding. Forced protein unfolding is thus a central component of the multilayered stimulatory mechanism used by GroEL to drive protein folding.

Visualizing Groel/Es in the Act of Encapsulating a Folding Protein

The GroEL/ES chaperonin system is required for the assisted folding of many proteins. How these substrate proteins are encapsulated within the GroEL-GroES cavity is poorly understood. Using symmetry-free, single-particle cryo-electron microscopy, we have characterized a chemically modified mutant of GroEL (EL43Py) that is trapped at a normally transient stage of substrate protein encapsulation. We show that the symmetric pattern of the GroEL subunits is broken as the GroEL cis-ring apical domains reorient to accommodate the simultaneous binding of GroES and an incompletely folded substrate protein (RuBisCO). The collapsed RuBisCO folding intermediate binds to the lower segment of two apical domains, as well as to the normally unstructured GroEL C-terminal tails. A comparative structural analysis suggests that the allosteric transitions leading to substrate protein release and folding involve concerted shifts of GroES and the GroEL apical domains and C-terminal tails.

Triggering Protein Folding within the GroEL-GroES Complex

The folding of many proteins depends on the assistance of chaperonins like GroEL and GroES and involves the enclosure of substrate proteins inside an internal cavity that is formed when GroES binds to GroEL in the presence of ATP. Precisely how assembly of the GroEL-GroES complex leads to substrate protein encapsulation and folding remains poorly understood. Here we use a chemically modified mutant of GroEL (EL43Py) to uncouple substrate protein encapsulation from release and folding. Although EL43Py correctly initiates a substrate protein encapsulation reaction, this mutant stalls in an intermediate allosteric state of the GroEL ring, which is essential for both GroES binding and the forced unfolding of the substrate protein. This intermediate conformation of the GroEL ring possesses simultaneously high affinity for both GroES and non-native substrate protein, thus preventing escape of the substrate protein while GroES binding and substrate protein compaction takes place. Strikingly, assembly of the folding-active GroEL-GroES complex appears to involve a strategic delay in ATP hydrolysis that is coupled to disassembly of the old, ADP-bound GroEL-GroES complex on the opposite ring.

Probing the conformation of the fibronectin III1-2 domain by fluorescence resonance energy transfer.

Fibronectin (FN) matrix is crucial for cell and tissue functions during embryonic development, wound healing, and oncogenesis. Assembly of FN matrix fibrils requires FN domains that mediate interactions with integrin receptors and with other FN molecules. In addition, regulation of FN matrix assembly depends on the first two FN type III modules, III1 and III2, which harbor FN-binding sites. We propose that interactions between these two modules sequester FN-binding sites in soluble FN and that these sites become exposed by FN conformational changes during assembly. To test the idea that III1–2 has a compact conformation, we constructed CIIIY, a conformational sensor of III1–2 based on fluorescent resonance energy transfer between cyan and yellow fluorescent proteins conjugated at its N and C termini. We demonstrate energy transfer in CIIIY and show that fluorescent resonance energy transfer was eliminated by proteolysis and by treatment with mild denaturants that disrupted intramolecular interactions between the two modules. We also show that mutations of key charged residues resulted in conformational changes that exposed binding sites for the N-terminal 70-kDa FN fragment. Collectively, these results support a conformation-dependent mechanism for the regulation of FN matrix assembly by III1–2.

The C-terminal Tails of the Bacterial Chaperonin GroEL Stimulate Protein Folding by Directly Altering the Conformation of a Substrate Protein

Background: Chaperonins like GroEL-GroES are required for the folding of many proteins. Results: The GroEL C termini alter substrate protein conformation and accelerate folding. Conclusion: Optimal protein folding requires the partial unfolding of misfolded states, a process that involves the GroEL C terminus. Significance: Chaperonins can actively facilitate protein folding by altering the conformations of folding intermediates. Many essential cellular proteins fold only with the assistance of chaperonin machines like the GroEL-GroES system of Escherichia coli. However, the mechanistic details of assisted protein folding by GroEL-GroES remain the subject of ongoing debate. We previously demonstrated that GroEL-GroES enhances the productive folding of a kinetically trapped substrate protein through unfolding, where both binding energy and the energy of ATP hydrolysis are used to disrupt the inhibitory misfolded states. Here, we show that the intrinsically disordered yet highly conserved C-terminal sequence of the GroEL subunits directly contributes to substrate protein unfolding. Interactions between the C terminus and the non-native substrate protein alter the binding position of the substrate protein on the GroEL apical surface. The C-terminal tails also impact the conformational state of the substrate protein during capture and encapsulation on the GroEL ring. Importantly, removal of the C termini results in slower overall folding, reducing the fraction of the substrate protein that commits quickly to a productive folding pathway and slowing several kinetically distinct folding transitions that occur inside the GroEL-GroES cavity. The conserved C-terminal tails of GroEL are thus important for protein folding from the beginning to the end of the chaperonin reaction cycle.

CONSTRUCTION OF SINGLE-RING AND TWO-RING HYBRID VERSIONS OF BACTERIAL CHAPERONIN GROEL

Publisher Summary The chapter presents a study related to the construction of single-ring and two-ring hybrid versions of bacterial chaperonin GroEL. A single-ring version of the bacterial chaperonin, GroEL, has been designed using information from the crystal structure of the native double-ring assembly. This molecule, designated SR1, has proved to be functional as a folding chamber, inside which proteins, such as rhodanese and green fluorescent protein (GFP), bound to the ring in nonnative form, can reach native form on addition of GroES and ATP. In particular, the substrate protein folds to the native state within the central channel underneath GroES and remains within this space because the absence of a second GroEL ring prevents the normal ATP mediated signaling that releases GroES. SR1 has been designed by examination of the model of unliganded GroEL at 2.8 A, derived from the orthorhombic crystal. This reveals a contact surface between the two rings that is composed of both electrostatic and hydrophobic interactions. Each subunit in the homotetradecameric complex forms contacts through the inferior aspect of its equatorial domain with that region of two neighboring subunits in the opposite ring. The essential double-ring nature of GroEL, with critical signaling occurring between its rings during the folding reaction, encouraged the study of double-ring complexes containing defects in only one ring.