Glu257 in GroEL is a sensor involved in coupling polypeptide substrate binding to stimulation of ATP hydrolysis

allostery ͉ chaperonin GroEL ͉ potassium ion ͉ timing mechanism M uch mechanistic and structural information related to the operation of the chaperonin nanomachine has accumulated (1-11). As with many other cellular machines, the chaperonin nanomachine has evolved to operate at variable speed in response to biological demand. This implies the presence of one or more timing devices or switches that govern the cycling of the machinery through the various functional and conformational states. In the presence of GroES, the two rings of GroEL operate 180°out of phase with one another in the manner of a two-stroke, reciprocating motor (1, 2). A complete chaperonin cycle consequently consists of two hemicycles and two reciprocal resting states (12).In the accompanying article (12), we used stopped-flow FRET experiments to monitor the association and dissociation of GroES to and from the asymmetric, resting-state complex comprising (GroEL cis -[ADP]-[GroES]/GroEL trans -ADP), where the square brackets indicate ligands that are not free to exchange with unbound ligand. We showed that the dominant timing mechanism is located on the trans ring. This timer is regulated by allosteric transitions in the trans ring that are both responsive to the nucleotides ADP and ATP, the potassium ion, and substrate protein (SP) and which are communicated to the cis ring. Here, our focus is on the hydrolysis of ATP. We show that the steady-state hydrolysis of ATP is largely governed by the trans ring timer and its response to the nucleotides ADP and ATP, K ϩ , and SP. We also show, paradoxically, that the hydrolysis of ATP in the newly formed cis ring is largely indifferent to the presence of encapsulated SP and to variations in [K ϩ ] that have profound effects on the rate of ATP hydrolysis in the steady state.

Section: Resultssupporting

confidence: 85%

“…2 A; see also ref. 22). A small burst corresponding to Ϸ0.5 mol of Pi per mole of GroEL 14 remained, followed by a linear rate of Pi production.…”

Section: Resultsmentioning

confidence: 94%

Setting the chaperonin timer: The effects of K ⁺ and substrate protein on ATP hydrolysis

Grason¹,

Gresham²,

Widjaja³

et al. 2008

“…1) and recruitment of another GroES on the trans ring (ring A) (10,11). It is also known that preincubating the acceptor-state asymmetric complex with ADP (∼30 μM) can reduce the size of the burst of Pi release almost to zero (11,33). Both phenomena can be attributed to the slow release of ADP from ring A that precedes and prevents ATP binding to the same ring.…”

Section: Significancementioning

confidence: 99%

Substrate protein switches GroE chaperonins from asymmetric to symmetric cycling by catalyzing nucleotide exchange

Ye¹,

Lorimer

2013

The complex kinetics of Pi and ADP release by the chaperonin GroEL/GroES is influenced by the presence of unfolded substrate protein (SP). Without SP, the kinetics of Pi release are described by four phases: a "lag," a "burst" of ATP hydrolysis by the nascent cis ring, a "delay" caused by ADP release from the nascent trans ring, and steady-state ATP hydrolysis. The release of Pi precedes the release of ADP. The rate-determining step of the asymmetric cycle is the release of ADP from the trans ring of the GroEL-GroES 1 "bullet" complex that is, consequently, the predominant species. In the asymmetric cycle, the two rings of GroEL function alternately, 180°out of phase. In the presence of SP, a change in the kinetic mechanism occurs. With SP present, the kinetics of ADP release are also described by four phases: a lag, a "surge" of ADP release attributable to SP-induced ADP/ATP exchange, and a "pause" during which symmetrical "football" particles are formed, followed by steady-state ATP hydrolysis. SP catalyzes ADP/ATP exchange on the trans ring. Now ADP release precedes the release of Pi, and the rate-determining step of the symmetric cycle becomes the hydrolysis of ATP by the symmetric GroEL-GroES 2 football complex that is, consequently, the predominant species. A FRET-based analysis confirms that asymmetric GroEL-GroES 1 bullets predominate in the absence of SP, whereas symmetric GroEL-GroES 2 footballs predominate in the presence of SP. This evidence suggests that symmetrical football particles are the folding functional form of the chaperonin machine in vivo.T he GroEL/GroES chaperonin machine is an indispensable, cellular device of exquisite complexity (1-3). Ultimately driven by the hydrolysis of ATP, it optimizes the folding of unfolded, client proteins under conditions where that thermodynamically favorable process does not occur (4). GroEL, the "engine," comprises two heptameric rings stacked back-to-back. Its subunits consist of equatorial, intermediate, and apical domains, which move in a concerted, rigid-body manner, swiveling on hinges located at the domain interfaces (5, 6). Each ring cycles through a progression of allosterically controlled conformational states in response to the binding of adenosine nucleotides and their associated metal ions, Mg 2+ and K + , the cochaperonin GroES, and, when present, the substrate protein (SP) (1-3). GroES functions like the lid on a cooking pot, transiently encapsulating a single molecule of SP, within the GroEL ring, the so-called Anfinsen cage (7). However, the SP does not remain encapsulated. Instead, with each chaperonin hemicycle, specifically in response to the binding of ATP to the opposite ring, first GroES and then SP is released, regardless of whether or not the SP has progressed to the folded state (8).Based mostly on studies of GroEL/GroES in the absence of SP, one of us (G.H.L.) likened its behavior to that of a twostroke motor (9). The two rings operate ∼180°out of phase with one another, hydrolyzing ATP alternately. The rate-determining step in ...

“…A slightly larger value is estimated from the data of Inobe et al (20), who measured k cat ∼ 0.09 sec −1 . Slow release of ADP has been inferred in the past based on bulk kinetic experiments (12,26).…”

Section: Frank Et Almentioning

confidence: 99%

Out-of-equilibrium conformational cycling of GroEL under saturating ATP concentrations

Frank

Goomanovsky

Davidi³

et al. 2010

Self Cite

The molecular chaperone GroEL exists in at least two allosteric states, T and R, that interconvert in an ATP-controlled manner. Thermodynamic analysis suggests that the T-state population becomes negligible with increasing ATP concentrations, in conflict with the requirement for conformational cycling, which is essential for the operation of molecular machines. To solve this conundrum, we performed fluorescence correlation spectroscopy on the single-ring version of GroEL, using a fluorescent switch recently built into its structure, which turns "on," i.e., increases its fluorescence dramatically, when ATP is added. A series of correlation functions was measured as a function of ATP concentration and analyzed using singular-value decomposition. The analysis assigned the signal to two states whose dynamics clearly differ. Surprisingly, even at ATP saturation, ∼50% of the molecules still populate the T state at any instance of time, indicating constant out-of-equilibrium cycling between T and R. Only upon addition of the cochaperonin GroES does the T-state population vanish. Our results suggest a model in which the T/R ratio is controlled by the rate of ADP release after hydrolysis, which can be determined accordingly.allostery | conformational dynamics | fluorescence correlation spectroscopy | molecular chaperones | chaperonins A TP-driven protein machines are abundant and contribute to multiple essential biological processes (1). Such protein machines undergo motor-like rotational motion (like F 1 -ATPase), carry cargos (like kinesin), or fold proteins (like GroEL, the subject of this paper). An important feature of all ATP-driven molecular machines is a functional cycle that involves sequential transitions between several conformational states (2). Understanding the dynamics of conformational cycling is, therefore, essential for the full elucidation of the mechanism of action of a protein machine.The Escherichia coli molecular chaperone GroEL is a machine that assists protein folding by undergoing a series of allosteric transitions that facilitate protein substrate binding and release (3,4). GroEL is made up of two homoheptameric rings, stacked back-to-back, with a cavity at each end (5), in which protein folding can take place. The allosteric transitions of GroEL are induced by ATP binding that occurs with positive cooperativity within rings and negative cooperativity between rings (6). It has been suggested that the intraring positive cooperativity is an outcome of a concerted switch between two conformations, Tand R, with low and high affinities for ATP, respectively. GroEL functions in conjunction with a heptameric ring-shaped cochaperonin, GroES. Binding of GroES to the so-called cis ring induces an additional conformational change that leads to the R' conformation and triggers dissociation of bound protein substrate into the cavity (7). The structures of the three relatively stable conformations, T, R, and R', have been determined using x-ray crystallography and electron microscopy (8, 9). It is not known ...