Publications

The CCT/TRiC chaperonin is found in the cytosol of all eukaryotic cells and assists protein folding in an ATP-dependent manner. The heterozygous double mutation T400P and R516H in subunit CCT2 is known to cause Leber congenital amaurosis (LCA), a hereditary congenital retinopathy. This double mutation also renders the function of subunit CCT2, when it is outside of the CCT/TRiC complex, to be defective in promoting autophagy. Here, we show using steady-state and transient kinetic analysis that the corresponding double mutation in subunit CCT2 from Saccharomyces cerevisiae reduces the off-rate of ADP during ATP hydrolysis by CCT/TRiC. We also report that the ATPase activity of CCT/TRiC is stimulated by a non-folded substrate. Our results suggest that the closed state of CCT/TRiC is stabilized by the double mutation owing to the slower off-rate of ADP, thereby impeding the exit of CCT2 from the complex that is required for its function in autophagy.

The chaperonin GroEL is a multi-subunit molecular machine that assists in protein folding in the E. coli cytosol. Past studies have shown that GroEL undergoes large allosteric conformational changes during its reaction cycle. However, a measurement of subunit dynamics and their relation to the allosteric cycle of GroEL has been missing. Here, we report single-molecule FRET measurements that directly probe the conformational transitions of one subunit within GroEL and its single-ring variant under equilibrium conditions. We find that four microstates span the conformational manifold of the protein and interconvert on the submillisecond time scale. A unique set of relative populations of these microstates, termed a macrostate, is obtained by varying solution conditions, e.g., adding different nucleotides or the co-chaperone GroES. Strikingly, ATP titration studies demonstrate that the partition between the apo and ATP-liganded conformational macrostates traces a sigmoidal response with a Hill coefficient similar to that obtained in bulk experiments of ATP hydrolysis, confirming the essential role of the observed dynamics in the function of GroEL.

The NtrC family of AAA+ proteins are bacterial transcriptional regulators that control σ54-dependent RNA polymerase transcription under certain stressful conditions. MopR, which is a member of this family, is responsive to phenol and stimulates its degradation. Biochemical studies to understand the role of ATP and phenol in oligomerization and allosteric regulation, which are described here, show that MopR undergoes concentration-dependent oligomerization in which dimers assemble into functional hexamers. The oligomerization occurs in a nucleation-dependent manner with a tetrameric intermediate. Additionally, phenol binding is shown to be responsible for shifting MopRs equilibrium from a repressed state (high affinity toward ATP) to a functionally active, derepressed state with low-affinity for ATP. Based on these findings, we propose a model for allosteric regulation of MopR. IMPORTANCE The NtrC family of bacterial transcriptional regulators are enzymes with a modular architecture that harbor a signal sensing domain followed by a AAA+ domain. MopR, a NtrC family member, responds to phenol and activates phenol adaptation pathways that are transcribed by σ54-dependent RNA polymerases. Our results show that for efficient ATP hydrolysis, MopR assembles as functional hexamers and that this activity of MopR is regulated by its effector (phenol), ATP, and protein concentration. Our findings, and the kinetic methods we employ, should be useful in dissecting the allosteric mechanisms of other AAA+ proteins, in general, and NtrC family members in particular.

Understanding proteinligand interactions in a cellular context is an important goal in molecular biology and biochemistry, and particularly for drug development. Investigators must demonstrate that drugs penetrate cells and specifically bind their targets. Towards that end, we present a native mass spectrometry (MS)-based method for analyzing drug uptake and target engagement in eukaryotic cells. This method is based on our previously introduced direct-MS method for rapid analysis of proteins directly from crude samples. Here, direct-MS enables label-free studies of proteindrug binding in human cells and is used to determine binding affinities of lead compounds in crude samples. We anticipate that this method will enable the application of native MS to a range of problems where cellular context is important, including proteinprotein interactions, drug uptake and binding, and characterization of therapeutic proteins.

A fundamental problem that has hindered the use of the classic Monod-Wyman-Changuex (MWC) allosteric model since its introduction is that it has been difficult to determine the values of its parameters in a reliable manner because they are correlated with each other and sensitive to the data-fitting method. Consequently, experimental data are often fitted to the Hill equation, which provides a measure of cooperativity but no insights into its origin. In this work, we derived a general relationship between the value of the Hill coefficient and the parameters of the MWC model. It is shown that this relationship can be used to select the best estimate of the true combination of the MWC parameter values from all the possible ones found to fit the data. Here, this approach was applied to fits to the MWC model of curves of the fraction of GroEL molecules in the high-affinity (R) state for ATP as a function of ATP concentration. Such curves were collected at different temperatures, thereby providing insight into the hydrophobic effect associated with the ATP-promoted allosteric switch of GroEL. More generally, the relationship derived here should facilitate future thermodynamic analysis of other MWC-type allosteric systems.

The chaperonin GroEL and its co-chaperonin GroES form both GroEL-GroES bullet-shaped and GroEL-GroES(2) football-shaped complexes. The residence time of protein substrates in the cavities of these complexes is about 10 and 1 s, respectively. There has been much controversy regarding which of these complexes is the main functional form. Here, we show using computational analysis that GroEL protein substrates have a bimodal distribution of folding times, which matches these residence times, thereby suggesting that both bullet-shaped and football-shaped complexes are functional. More generally, co-existing complexes with different stoichiometries are not mutually exclusive with respect to having a functional role and can complement each other.

Advances in native mass spectrometry and single-molecule techniques have made it possible in recent years to determine the values of successive ligand binding constants for large multi-subunit proteins. Given these values, it is possible to distinguish between different allosteric mechanisms and, thus, obtain insights into how various bio-molecular machines work. Here, we describe for ring-shaped homo-oligomers, in particular, how the relationship between the values of successive ligand binding constants is diagnostic for concerted, sequential and probabilistic allosteric mechanisms.This article is part of a discussion meeting issue 'Allostery and molecular machines'.

The strength and specificity of protein complex formation is crucial for most life processes and is determined by interactions between residues in the binding partners. Double-mutant cycle analysis provides a strategy for studying the energetic coupling between amino acids at the interfaces of such complexes. Here we show that these pairwise interaction energies can be determined from a single high-resolution native mass spectrum by measuring the intensities of the complexes formed by the two wild-type proteins, the complex of each wild-type protein with a mutant protein, and the complex of the two mutant proteins. This native mass spectrometry approach, which obviates the need for error-prone measurements of binding constants, can provide information regarding multiple interactions in a single spectrum much like nuclear Overhauser effects (NOEs) in nuclear magnetic resonance. Importantly, our results show that specific inter-protein contacts in solution are maintained in the gas phase.

The ability to query enzyme molecules individually is transforming our view of catalytic mechanisms. Quiescin sulfhydryl oxidase (QSOX) is a multidomain catalyst of disulfide-bond formation that relays electrons from substrate cysteines through two redox-active sites to molecular oxygen. The chemical steps in electron transfer have been delineated, but the conformational changes accompanying these steps are poorly characterized. Here we use single-molecule Förster resonance energy transfer (smFRET) to probe QSOX conformation in resting and cycling enzyme populations. We report the discovery of unanticipated roles for conformational changes in QSOX beyond mediating electron transfer between redox-active sites. In particular, a state of the enzyme not previously postulated or experimentally detected is shown to gate, via a conformational transition, the entrance into a sub-cycle within an expanded QSOX kinetic scheme. By tightly constraining mechanistic models, smFRET data can reveal the coupling between conformational and chemical transitions in complex enzymatic cycles.

The Engrailed Homeodomain (EnHD) transcription factor of Drosophila melanogaster was fused to the enhanced green fluorescent protein (eGFP) either at its C- or N-terminus via three- or ten-residue flexible linkers. Here, we show that EnHD undergoes destabilization upon fusing it to eGFP regardless of the linker length used and whether the tethering is to its N- or C-terminus. The destabilization is reflected in melting points that are lower by up to 9°C. Thermodynamic analysis and coarse-grained molecular dynamic simulations indicate that this destabilization is due to eGFP-promoted entropic stabilization of the denatured state ensemble of EnHD. Our results provide, therefore, an example for destabilizing interdomain allostery. They are also important given the widespread use of eGFP tagging in cell biology, as they indicate that such tagging can cause unintended protein destabilization and concomitant effects.

Computational analysis of proteomes in all kingdoms of life reveals a strong tendency for N-terminal domains in two-domain proteins to have shorter sequences than their neighboring C-terminal domains. Given that folding rates are affected by chain length, we asked whether the tendency for N-terminal domains to be shorter than their neighboring C-terminal domains reflects selection for faster-folding N-terminal domains. Calculations of absolute contact order, another predictor of folding rate, provide additional evidence that N-terminal domains tend to fold faster than their neighboring C-terminal domains. A possible explanation for this bias, which is more pronounced in prokaryotes than in eukaryotes, is that faster folding of N-terminal domains reduces the risk for protein aggregation during folding by preventing formation of nonnative interdomain interactions. This explanation is supported by our finding that two-domain proteins with a shorter N-terminal domain are much more abundant than those with a shorter C-terminal domain.

Enzymatic inhibition by product molecules is an important and widespread phenomenon. We describe an approach to study product inhibition at the single-molecule level. Individual HRP molecules are trapped within surface-tethered lipid vesicles, and their reaction with a fluorogenic substrate is probed. While the substrate readily penetrates into the vesicles, the charged product (resorufin) gets trapped and accumulates inside the vesicles. Surprisingly, individual enzyme molecules are found to stall when a few tens of product molecules accumulate. Bulk enzymology experiments verify that the enzyme is noncompetitively inhibited by resorufin. The initial reaction velocity of individual enzyme molecules and the number of product molecules required for their complete inhibition are broadly distributed and dynamically disordered. The two seemingly unrelated parameters, however, are found to be substantially correlated with each other in each enzyme molecule and over long times. These results suggest that, as a way to counter disorder, enzymes have evolved the means to correlate fluctuations at structurally distinct functional sites.

The eukaryotic cytoplasmic chaperonin-containing TCP-1 (CCT) is a complex formed by two back-to-back stacked hetero-octameric rings that assists the folding of actins, tubulins, and other proteins in an ATP-dependent manner. Here, we tested the significance of the hetero-oligomeric nature of CCT in its function by introducing, in each of the eight subunits in turn, an identical mutation at a position that is conserved in all the subunits and is involved in ATP hydrolysis, in order to establish the extent of 'individuality' of the various subunits. Our results show that these identical mutations lead to dramatically different phenotypes. For example, Saccharomyces cerevisiae yeast cells with the mutation in subunit CCT2 display heat sensitivity and cold sensitivity for growth, have an excess of actin patches, and are the only strain here generated that is pseudo-diploid. By contrast, cells with the mutation in subunit CCT7 are the only ones to accumulate juxtanuclear protein aggregates that may reflect an impaired stress response in this strain. System-level analysis of the strains using RNA microarrays reveals connections between CCT and several cellular networks, including ribosome biogenesis and TOR2, that help to explain the phenotypic variability observed.

The 60-kDa heat shock protein (mHsp60) is a vital cellular complex that mediates the folding of many of the mitochondrial proteins. Its function is executed in cooperation with the cochaperonin, mHsp10, and requires ATP. Recently, the discovery of a new mHsp60-associated neurodegenerative disorder, Mit-CHAP-60 disease, has been reported. The disease is caused by a point mutation at position 3 (D3G) of the mature mitochondrial Hsp60 protein, which renders it unable to complement the deletion of the homologous bacterial protein in Escherichia coli (Magen, D., Georgopoulos, C., Bross, P., Ang, D., Segev, Y., Goldsher, D., Nemirovski, A., Shahar, E., Ravid, S., Luder, A., Heno, B., Gershoni-Baruch, R., Skorecki, K., and Mandel, H. (2008) Am. J. Hum. Genet. 83, 30-42). The molecular basis of the MitCHAP-60 disease is still unknown. In this study, we present an in vitro structural and functional analysis of the purified wild-type human mHsp60 and the MitCHAP-60 mutant. We show that the D3G mutation leads to destabilization of the mHsp60 oligomer and causes its disassembly at low protein concentrations. We also show that the mutant protein has impaired protein folding and ATPase activities. An additional mutant that lacks the first three amino acids (N-del), including Asp-3, is similarly impaired in refolding activity. Surprisingly, however, this mutant exhibits profound stabilization of its oligomeric structure. These results suggest that the D3G mutation leads to entropic destabilization of the mHsp60 oligomer, which severely impairs its chaperone function, thereby causing the disease.

The chaperonin GroEL assists protein folding by undergoing ATP-induced conformational changes that are concerted within each of its two back-to-back stacked rings. Here we examined whether concerted allosteric switching gives rise to all-or-none release and folding of domains in a chimeric fluorescent protein substrate, CyPet-YPet. Using this substrate, it was possible to determine the folding yield of each domain from its intrinsic fluorescence and that of the entire chimera by measuring Förster resonance energy transfer between the two domains. Hence, it was possible to determine whether release of one domain is accompanied by release of the other domain (concerted mechanism), or whether their release is not coupled. Our results show that the chimera's release tends to be concerted when folding is assisted by a wild-type GroEL variant, but not when assisted by the F44W/D155A mutant that undergoes a sequential allosteric switch. A connection between the allosteric mechanism of this molecular machine and its biological function in assisting folding is thus established.

Motivation: The folding of many proteins in vivo and in vitro is assisted by molecular chaperones. A well-characterized molecular chaperone system is the chaperonin GroEL/GroES from Escherichia coli which has a homolog found in the eukaryotic cytosol called CCT. All chaperonins have a ring structure with a cavity in which the substrate protein folds. An interesting difference between prokaryotic and eukaryotic chaperonins is in the nature of the ATP-mediated conformational changes that their ring structures undergo during their reaction cycle. Prokaryotic chaperonins are known to exhibit a highly cooperative concerted change of their cavity surface while in eukaryotic chaperonins the change is sequential. Approximately 70% of proteins in eukaryotic cells are multi-domain whereas in prokaryotes single-domain proteins are more common. Thus, it was suggested that the different modes of action of prokaryotic and eukaryotic chaperonins can be explained by the need of eukaryotic chaperonins to facilitate folding of multi-domain proteins. Results: Using a 2D square lattice model, we generated two large populations of single-domain and double-domain substrate proteins. Chaperonins were modeled as static structures with a cavity wall with which the substrate protein interacts. We simulated both concerted and sequential changes of the cavity surfaces and demonstrated that folding of single-domain proteins benefits from concerted but not sequential changes whereas double-domain proteins benefit also from sequential changes. Thus, our results support the suggestion that the different modes of allosteric switching of prokaryotic and eukaryotic chaperonin rings have functional implications as it enables eukaryotic chaperonins to better assist multi-domain protein folding.

Direct or indirect inter-residue interactions in proteins are often reflected by mutations at one site that compensate for mutations at another site. Various bioinformatic methods have been developed for detecting such correlated mutations in order to obtain information about intra- and inter-protein interactions. Here, we show by carrying out a correlated mutation analysis for non-interacting proteins that the signal due to inter-residue interactions is of similar magnitude to the 'noise' that arises from other evolutionary processes related to common ancestry. A new method for detecting correlated mutations is presented that reduces this evolutionary noise by taking into account evolutionary distances in the protein family. It is shown that this method yields better signal-to-noise ratios and, therefore, can much better resolve, for example, correlated mutations that reflect true inter-residue interactions.

An interesting example of an allosteric protein is the chaperonin GroEL. It undergoes adenosine 5-triphosphate-induced conformational changes that are reflected in binding of adenosine 5-triphosphate with positive cooperativity within rings and negative cooperativity between rings. Herein, correlated mutations in chaperonins are analyzed to unravel routes of allosteric communication in GroEL and in its complex with its co-chaperonin GroES. It is shown that analysis of correlated mutations in the chaperonin family can provide information about pathways of allosteric communication within GroEL and between GroEL and GroES. The results are discussed in the context of available structural, genetic, and biochemical data concerning short- and long-range interactions in the GroE system.

GroEL is an allosteric protein that facilitates protein folding in an ATP-dependent manner. Herein, the relationship between cooperative ATP binding by GroEL and the kinetics of GroE-assisted folding of two substrates with different GroES dependence, mouse dihydrofolate reductase (mDHFR) and mitochondrial malate dehydrogenase, is examined by using cooperativity mutants of GroEL. Strong intra-ring positive cooperativity in ATP binding by GroEL decreases the rate of GroEL-assisted mDHFR folding owing to a slow rate of the ATP-induced transition from the protein-acceptor state to the protein- release state. Inter-ring negative cooperativity in ATP binding by GroEL is found to affect the kinetic partitioning of mDHFR, but not of mitochondrial malate dehydrogenase, between folding in solution and folding in the cavity underneath GroES. Our results show that protein folding by this 'two-stroke motor' is coupled to cooperative ATP binding.

GroEL with an intrinsic fluorescent probe was generated by introducing the mutation Phe44 --> Trp. Different concentrations of ATP were rapidly mixed with GroEL containing this mutation, and the time-resolved change in fluorescence emission, upon excitation at 280 nm, was followed. Three kinetic phases were observed: a fast phase with a large amplitude and two slower phases with small amplitudes. The phases were assigned by (i) determining their dependence on ATP concentration; (ii) measuring their sensitivity to the mutation Arg197 --> Ala, which decreases cooperativity in ATP binding; and (iii) by carrying out mixing experiments of GroEL also with ADP, ATP gamma S, and ATP without K+. The apparent rate constant corresponding to the fast phase displays a bi-sigmoidal dependence on ATP concentration with Hill coefficients that are strikingly similar to those determined in steady-state experiments. This phase, which reflects ATP-induced conformational changes, is sensitive to the mutation Arg197 --> Ala in a manner that parallels steady-state experiments. The rate of conformational change in the presence of ATP is > 100 sec(-1), which is fast relative to most protein folding rates, whereas in the absence of ATP it is similar to 0.7 s(-1). The second phase reflects the transition from an ATP-bound state of GroEL to an ADP-bound state. The third phase, with the smallest amplitude, reflects release of residual contaminants. The results in this study are found to be consistent with the nested model for cooperativity in ATP binding by GroEL [Yifrach, O., and Horovitz, A. (1995) Biochemistry 34, 5303-5308].

Curves of initial rates of ATP hydrolysis by GroEL as a function of ATP concentration, in the presence of fixed concentrations of GroES, were found to deviate from sigmoidal kinetics. Instead of the lag phase typical of sigmoidal curves, a linear phase is observed at low ATP concentrations. Consequently, a good fit of the data to the Hill equation could not be achieved. Such curves could be simulated using a linear combination of Hill equations, thus indicating that more than one allosteric transition is taking place in the ATP concentration range studied. The data were fitted to a fractional saturation equation for ATP binding to GroEL based on a partition function that includes both GroES and ATP-liganded states of GroEL. Using this equation, it was possible to estimate in a reliable manner the value of the allosteric constant, L'₂, for the transition of the ring distal to GroES in the GroEL-GroES complex from the low (T)- to the high (R)-affinity state for ATP. The value of L'₂ is found to be 4 x 10^-5 whereas the value of the allosteric constant, L₂, for the transition of the second ring of GroEL from the T to R state is 2 x 10^-9 [Yifrach, O., and Horovitz, A. (1995) Biochemistry 34, 5303-5308]. Comparison of these values shows that GroES promotes the T to R transition of the ring distal to GroES in the GroEL- GroES complex. Owing to the relatively low affinity of the R conformation for nonfolded proteins, this transition will lead to release of protein substrates from trans ternary complexes of GroEL, GroES, and protein substrate. The role of this release mechanism may be to assist the folding of relatively large proteins that cannot form cis ternary complexes and/or to facilitate degradation of damaged proteins which cannot fold.

A protein engineering approach for detecting and measuring local conformational changes that accompany allosteric transitions in proteins is described. Using this approach, we can identify interactions that are made or broken during allosteric transitions. The method is applied to probe for changes in pairwise interactions in the chaperonin GroEL during its ATP- induced allosteric transitions. Two pairwise interactions are investigated: one between subunits (Asp-41 with Thr-522) and the other within subunits (Glu-409 with Arg-501). We find that the intraring intersubunit interaction between Asp-41 and Thr-522 changes little during the allosteric transitions of GroEL, indicating that the hydrogen bond between these residues is maintained. In contrast, the intrasubunit salt bridge between Glu-409 and Arg-501 becomes significantly weaker during the ATP-induced allosteric transitions of GroEL. Our results are consistent with the electron microscopy observations of an ATP-induced hinge movement of the apical domains relative to the equatorial domains.

The crystal structures of the chaperonin GroEL Arg13 → Gly; Ala126 → Val double mutant, without and in complex with ATPγS, have been determined at atomic resolution. Here, we show that the double mutation Arg13 → Gly; Ala126 → Val disrupts negative co-operativity between GroEL rings, with respect to ATP, but has little effect on the positive co-operativity within each ring. Our results help to explain why the double mutation facilitated the crystallization of GroEL and why breaking of dyad symmetry between rings is not observed in crystal structures of this mutant. Our results may also help to explain why the observed structural differences between the GroEL double mutant and its ATPγS-bound form are small.

Co-operativity in ATP hydrolysis by GroEL can be described by a model in which each ring of GroEL is in equilibrium between a low (T) and high (R) affinity state for ATP. According to this model, the GroEL double-ring is in equilibrium between three states: TT, TR and RR. In order to find out which states bind non-folded proteins, we measured the co-operativity in ATP hydrolysis by GroEL in the absence and presence of non-folded α-lactalbumin, under equilibrium conditions between GroEL and the non-folded protein. The non-folded protein is found to bind preferentially the T state of GroEL rings and to stimulate the ATPase activity of GroEL by (1) a direct effect on GroEL rings in the T state and (2) a shift in the equilibrium from the RR state toward the more active TR state. The coupling between co-operativity in ATP hydrolysis by GroEL and protein substrate binding and release by this molecular chaperone is shown.

A search for co-ordinated amino acid changes in the hsp60 family of chaperonins suggested that cysteine residues at positions 137 and 518 in the Escherichia coli chaperonin GroEL may interact with each other. In order to determine whether this interaction indeed exists we constructed a double-mutant cycle comprising wild-type GroEL, the single mutants Cys137→Ser and Cys518→Ser and the corresponding double mutant. The effects of the two mutations on the function of GroEL, in assisting the refolding of a non-folded protein substrate (rhodanese), are shown to be non-additive. It is also shown that ADP by itself specifically destabilities the Cys518→Ser mutant GroEL particle with this effect being suppressed in the double mutant. The observed pattern of co-ordinated mutations in the hsp60 family of chaperonins is thus shown to reflect a real interaction, though most likely indirect, between Cys137 and Cys518 in GroEL. Our study demonstrates that patterns of co-ordinated mutations combined with double-mutant cycle analysis can provide structural informantion on interactions in a protein without an available three-dimensional structure at atomic resolution.