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Department of Chemistry, York University, Toronto, Ontario M3J 1P3, CanadaCentre for Research in Mass Spectrometry, Department of Chemistry, York University, 4700 Keele Street, Toronto, Ontario M3J 1P3, Canada
Supported in part by Ministry of Business, Innovation and Employment Contract UOCX1208, New Zealand Royal Society Marsden Fund Contract UOC1013, and United States Army Research Laboratory and United States Army Research Office Contract/Grant W911NF-11-1-0481. To whom correspondence may be addressed: Biomolecular Interaction Centre, School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch, New Zealand. Tel.: 64-3-364-2987; Fax: 64-3-364-2590.
Biomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, Private Bag 4800, Christchurch 8041, New ZealandDepartment of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, 30 Flemington Road, Parkville, Victoria 3010, Australia
* The authors declare that they have no conflicts of interest with the contents of this article. 1 Supported by Maurice Wilkins Centre flexible research funding and a Journal of Cell Science travel fellowship. 4 The abbreviation used is:
Pyruvate kinase catalyzes the final step in glycolysis and is allosterically regulated to control flux through the pathway. Two models are proposed to explain how Escherichia coli pyruvate kinase type 1 is allosterically regulated: the “domain rotation model” suggests that both the domains within the monomer and the monomers within the tetramer reorient with respect to one another; the “rigid body reorientation model” proposes only a reorientation of the monomers within the tetramer causing rigidification of the active site. To test these hypotheses and elucidate the conformational and dynamic changes that drive allostery, we performed time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange studies followed by mutagenic analysis to test the activation mechanism. Global exchange experiments, supported by thermostability studies, demonstrate that fructose 1,6-bisphosphate binding to the allosteric domain causes a shift toward a globally more dynamic ensemble of conformations. Mapping deuterium exchange to peptides within the enzyme highlight site-specific regions with altered conformational dynamics, many of which increase in conformational flexibility. Based upon these and mutagenic studies, we propose an allosteric mechanism whereby the binding of fructose 1,6-bisphosphate destabilizes an α-helix that bridges the allosteric and active site domains within the monomeric unit. This destabilizes the β-strands within the (β/α)8-barrel domain and the linked active site loops that are responsible for substrate binding. Our data are consistent with the domain rotation model but inconsistent with the rigid body reorientation model given the increased flexibility at the interdomain interface, and we can for the first time explain how fructose 1,6-bisphosphate affects the active site.
). Allostery provides immediate enzymatic control of reaction flux in response to cellular signals (e.g. the feedback regulation of a branch point enzyme by the concentration of the pathway product). As such, allostery is critical for the modulation of cellular metabolism and is ubiquitous across all forms of life.
Pyruvate kinase (EC 22.214.171.124) catalyzes the final step in the central energy production pathway, glycolysis (
), and as such is a key enzyme in cellular metabolism. It mediates a phosphate transfer from phosphoenolpyruvate to ADP, producing pyruvate and ATP (Fig. 1A). Pyruvate, and to a lesser extent phosphoenolpyruvate, are common precursors in metabolism, including the synthesis of glucose, fatty acids, glycerol, and amino acids (
Not surprisingly, pyruvate kinase enzymes are almost always allosterically regulated. Moreover, cells often express multiple pyruvate kinase isozymes that are regulated by different effectors. The isozymes are usually homotropically activated by the substrate phosphoenolpyruvate to allow regulation of the glycolytic flux in response to elevated phosphoenolpyruvate levels (
). For example, Escherichia coli has two pyruvate kinase enzymes that are regulated by different allosteric activators. The type 1 pyruvate kinase, the focus of this work, is heterotropically activated by fructose 1,6-bisphosphate. The binding of fructose 1,6-bisphosphate has a K-type effect whereby it increases the affinity of the enzyme for the substrate, phosphoenolpyruvate (
Key to unraveling allosteric mechanisms are the structures of the enzyme with and without bound allosteric effectors. Atomic resolution x-ray structures of pyruvate kinase enzymes from mammalian, bacterial, and parasitic organisms have been reported (
). As is common in allosterically controlled enzymes, pyruvate kinase is oligomeric and is usually a homotetramer. Each monomer comprises three or four domains: A, B, C, and N-terminal domains (only present in mammalian enzymes) (
) (Fig. 1, B and C). The A-domain is a (β/α)8-barrel (TIM barrel) housing the active site at the top of the barrel. The B-domain, also known as the “lid domain,” is thought to close over the active site upon substrate binding. The active site is located in the cleft between the A- and B-domains, and this is where the essential mono- and divalent cations bind (
To unpick the molecular mechanism(s) by which pyruvate kinase achieves allosteric control upon fructose 1,6-bisphosphate binding, several groups have determined the three-dimensional crystal structures of various isozymes in both the allosteric activator-bound and -unbound states (
The fructose 1,6-bisphosphate-bound structures reveal that the allosteric activator binding site is located entirely within the C-domain and must transmit an allosteric signal over a 40-Å distance to the active site in the same subunit. The binding site is located in a pocket formed from an effector loop and the first two turns of the Cα5′-helix in Saccharomyces cerevisiae (
) suggest that the allosteric transition involves a rigid body rocking motion of the A- and C-domains that reorients these domains within the tetramer.
Based largely on structural studies, two models have been proposed by others to explain how E. coli pyruvate kinase type 1 is regulated by fructose 1,6-bisphosphate (Fig. 2) The first suggests that the domains within the monomer are reoriented along with changes in the orientation of the monomers within the tetramer (
) (Fig. 2A). The second competing model puts forward that the domains are rigid and that reorientation of the domains within the tetramer causes the active site to rigidify, presumably in a conformation that better binds the substrate phosphoenolpyruvate (
) to soak fructose 1,6-bisphosphate into E. coli pyruvate kinase type 1 crystals have caused them to crack. This indicates that fructose 1,6-bisphosphate binding causes conformational changes that affect crystal packing. Moreover, our attempts to co-crystallize E. coli pyruvate kinase type 1 with fructose 1,6-bisphosphate have not yielded crystals, despite using large and sparse matrix screens that can trial >1000 different conditions and successfully obtaining unliganded enzyme crystals in various conditions. One explanation for this is that the fructose 1,6-bisphosphate-bound protein is heterogeneous in some way.
To build a detailed picture of the conformational and dynamic changes that drive E. coli pyruvate kinase type 1 heterotopic allosteric activation by fructose 1,6-bisphosphate, we performed time-resolved electrospray ionization mass spectrometry coupled to a hydrogen-deuterium exchange study. The “new view” of allostery describes a dynamic process whereby a protein fluctuates within an ensemble of conformations (
). Although the concept of allostery has evolved considerably in recent years, allosteric mechanisms remain difficult to establish because defining changes in protein dynamics is challenging even for small proteins (
). Here, we present hydrogen-deuterium exchange data that suggest the protein samples a larger conformational ensemble upon fructose 1,6-bisphosphate binding. Mapping deuterium exchange to peptides within the protein reveals regions that show significantly altered conformational dynamics upon the binding of fructose 1,6-bisphosphate. Based on these dynamic data and further confirmation by mutagenic experiments, we are able to propose a new, detailed mechanism by which fructose 1,6-bisphosphate promotes substrate binding despite the allosteric binding site being 40 Å away from the active site.
Results and Discussion
Proteins exist as an ensemble of conformers, and conformational sampling allows proteins to fluctuate between different conformers (
), although in practice detecting high energy conformers is more challenging because of their small number in the population and transient nature. In the dynamic view of allostery, the binding of the allosteric modulator remodels the energy landscape and triggers a shift in the relative occupancy of states in the conformational ensemble (
); the conformation that represents the lowest energy state is changed, although many conformers may still be sampled. Importantly, this shift increases how often a given (activated or inhibited) state is sampled (
Fructose 1,6-Bisphosphate Binding Causes an Increase in Global Protein Flexibility
To gauge changes in global conformational dynamics, global hydrogen-deuterium exchange was measured as a time course (14 ms to 1.5 s) for E. coli pyruvate kinase type 1 with and without its allosteric activator, fructose 1,6-bisphosphate. The data were fitted to an exponential function, generating a kinetic plot of overall protein deuterium exchange (Fig. 3). Increases in deuterium exchange characteristically result from intermittent loss of hydrogen bonds within and between secondary structure elements due to changes in structure or conformational flexibility, leading to increased solvent access associated with destabilization of secondary structure and tertiary structure. By the same token, decreases in deuterium exchange typically result from stable hydrogen bond formation within secondary structure elements and decreased solvent accessibility.
The global hydrogen-deuterium exchange kinetic plot (Fig. 3) reveals that when fructose 1,6-bisphosphate is present the enzyme has a higher amplitude of deuterium exchange (∼Δ1966 Da), which is evident in the increased change in mass seen at 1–1.5 s. In the absence of fructose 1,6-bisphosphate, the amplitude is ∼Δ1884 Da. Moreover, we note that there is a difference in the burst phase (0–14 ms) between the differently treated proteins. This is consistent with a globally more dynamic protein that can exchange deuterium more readily. From this, we infer that the binding event causes a shift toward a more unstructured, heterogeneous protein.
Not only was there a change in the amount of deuterium taken up by the protein on the time-resolved electrospray ionization mass spectrometry time scale (milliseconds to seconds), but the rate of exchange was also augmented upon fructose 1,6-bisphosphate binding, corresponding to an increase from 1.9 ± 0.5 to 2.9 ± 0.8 s−1. This is also consistent with activator binding increasing the globally averaged dynamic conformational sampling the protein is undergoing over time: that is, fructose 1,6-bisphosphate increases not only the extent of conformational space being sampled but also how “fast” the protein explores conformational space (
To corroborate this global time-resolved electrospray ionization mass spectrometry experiment, we measured the thermal stability of the wild-type enzyme in the absence and presence (0.25 mm) of fructose 1,6-bisphosphate. We found that fructose 1,6-bisphosphate decreases the thermal stability by ∼5 °C from 54.3 ± 0.3 (S.D.) to 49.7 ± 0.3 °C. This had been previously observed for the E. coli pyruvate kinase type 1 enzyme (
). Taken together, these data suggest that fructose 1,6-bisphosphate binding to the allosteric domain causes a shift toward a globally more unstructured and dynamic ensemble of conformations. This may also explain our (and others' (
)) observation that treating crystals of E. coli pyruvate kinase type 1 with fructose 1,6-bisphosphate results in crystal cracking and the lack of conditions for co-crystallization.
Localized Changes in Deuterium Exchange Reveal Regions with Altered Conformational Flexibility
To provide detailed information on regions of the protein where conformational flexibility significantly changes upon fructose 1,6-bisphosphate binding, enzyme that was premixed with deuterium (59 ms to 1 s) was digested using pepsin, and we determined the amplitude of deuterium exchange on individual peptides over this time period. Digestion of pyruvate kinase type 1 was effective given the size of the protein, resulting in a 64% sequence coverage corresponding to 36 distinctive peptides and an average spatial resolution of 10 amino acid residues. Typical raw hydrogen-deuterium exchange mass spectrometry data of representative peptides (Fig. 4A) demonstrate the shift in the mass envelope caused by differences in deuterium exchange of unbound and bound pyruvate kinase peptides. Kinetic hydrogen-deuterium exchange profiles of the representative peptides were fit to a single exponential expression to illustrate a change in peptide deuterium exchange as a function of labeling time (Fig. 4B).
We then mapped the percentage of deuterium exchange for each peptide onto the pyruvate kinase structure to illustrate regions that have significant changes in local backbone dynamics (Fig. 5; the color spectrum indicates the level of exchange: blue to red, low to high exchange, respectively). Qualitatively, the protein in the absence of fructose 1,6-bisphosphate shows generally low exchange of deuterium (20–60%), commensurate with a stable, globular structure. In particular, the peptides close to the tetrameric interfaces (A/A′, peptide 291–294; and C/C′, peptide 452–467) show low exchange, which is consistent with our previous work demonstrating that pyruvate kinase type 1 is a very stable tetramer (KD4-1 < 10 nm) (
). Moreover, the peptides at the A/C interdomain interface (peptides 398–408 and 409–419; Fig. 5, circled) have relatively low deuterium exchange (47 and 56%) compared with the same peptide in the presence of fructose 1,6-bisphosphate (87 and 95%).
When mapping the degree of exchange in peptides derived from protein pretreated with fructose 1,6-bisphosphate and comparing this with the unbound peptides, it is evident that the pattern of exchange is different (Fig. 5, A and B). Fig. 6A shows only those peptides that exhibited a significant change in absolute deuterium exchange (>15% absolute difference) when comparing peptides derived from unbound enzyme with those from fructose 1,6-bisphosphate-bound enzyme. To permit straightforward interpretation of the data, the absolute changes are noted only as an increase or decrease in exchange (Fig. 6B). The percentage of change in deuterium exchange upon fructose 1,6-bisphosphate binding was then mapped to the structure to highlight regions of the protein that exhibited significant changes in backbone deuterium exchange (Fig. 6C). Clearly, there are regions that show significant changes, which we address in the sections below.
Dynamic Changes to the Allosteric Domain upon Fructose 1,6-Bisphosphate Binding
We first examined the allosteric binding domain to assess how fructose 1,6-bisphosphate binding affects its conformational flexibility. From Fig. 6, fructose 1,6-bisphosphate binding results in clear changes in deuterium exchange in the allosteric C-domain. Because a structure of E. coli pyruvate kinase type 1 with bound fructose 1,6-bisphosphate is not available, we have modeled fructose 1,6-bisphosphate binding to the allosteric binding domain using overlays with the S. cerevisiae pyruvate kinase structure (
) (Fig. 7, A and B). For S. cerevisiae pyruvate kinase, fructose 1,6-bisphosphate binding in the allosteric site is facilitated by residue Arg459, forming a strong electrostatic interaction with the 1′-phosphate group of fructose 1,6-bisphosphate. In addition, the 6′-phosphate makes a series of hydrogen bonds with side chains from the sequence Ser402-Thr-Ser-Gly-Thr-Thr407 (Thr378-Gln-Gly-Gly-Lys382 are the equivalent residues in E. coli pyruvate kinase type 1), and the sugar ring of the fructose 1,6-bisphosphate forms interactions with residues Gln483 and His491. Moreover, chemical modification of E. coli pyruvate kinase type 1 identifies Lys382 (equivalent to Thr406 in S. cerevisiae pyruvate kinase) as important for fructose 1,6-bisphosphate binding, providing some evidence that the allosteric binding site is located in a pocket similar to that of S. cerevisiae pyruvate kinase (
The binding site for fructose 1,6-bisphosphate in the allosteric domain shows key changes in conformational flexibility. Assuming fructose 1,6-bisphosphate binds in the same pocket and in a similar orientation, ligand binding requires a displacement of surface-accessible water molecules and a change in the hydrogen bonding network of the pocket, including residues of the Cα3′- and Cα4′-helices and the effector loop (Fig. 7C), so we expected to find changes to the backbone flexibility in these regions. We note first that the surface that binds fructose 1,6-bisphosphate has decreased exchange, consistent with fructose 1,6-bisphosphate binding decreasing the availability of amides and preventing exchange. In addition, there is a large increase in deuterium exchange for the peptide containing the effector loop, residues 452–467 (Fig. 6C). The effector loop is at the suspected activator binding site, and it shows a 38% increase in deuterium exchange. It has been implicated as the fructose 1,6-bisphosphate binding loop (
), and in the unbound state it is located very near the position of suspected fructose 1,6-bisphosphate binding. One interpretation of our result is that fructose 1,6-bisphosphate binding pushes the enzyme toward a conformational state that favors the effector loop positioned to the side. Fig. 7A shows the position of the effector loop when fructose 1,6-bisphosphate is bound in the S. cerevisiae structure, whereas Fig. 7B shows the effector loop position in the absence of fructose 1,6-bisphosphate (E. coli pyruvate kinase with fructose 1,6-bisphosphate modeled into the binding site from Protein Data Bank code 1A3W), illustrating the change in conformation for this loop that may occur.
The neighboring Cα3′-helix (residues 370–391) has a 17% decrease in deuterium exchange (Fig. 6, B and C). The decrease in dynamics for the Cα3′-helix may be due to hydrogen bonding with fructose 1,6-bisphosphate, decreasing the number of amide hydrogen atoms available for exchange. In the S. cerevisiae structure (Protein Data Bank code 1A3W), the 6′-phosphate of fructose 1,6-bisphosphate hydrogen bonds with Thr407 of the Cα3′-helix (
) supports the assumption that this helix also binds the allosteric activator in the E. coli enzyme. Hydrogen bond formation with fructose 1,6-bisphosphate may cause a reorientation of residue Gln379, weakening its interactions with residues Asn402 and Thr405 of the Cα4′-helix (Fig. 7D). The mutagenic study at Lys382 and the significant decrease in conformational flexibility we observed collectively support the concept that the Cα3′-helix is directly involved in interactions with fructose 1,6-bisphosphate and important for communicating the allosteric signal to the Cα4′-helix.
Significantly, fructose 1,6-bisphosphate binding also causes destabilization of the interdomain interface that links the allosteric domain (C-domain) and the (β/α)8-barrel (A-domain) within the monomer (Fig. 7). Our dynamic data, when mapped onto the structure, suggest that the interactions between the allosteric domain and the A-domain are largely mediated by the Cα4′-helix of the allosteric domain (circled in Figs. 5B and 7C) and the N-terminal loops of the (β/α)8-barrel. The binding of fructose 1,6-bisphosphate causes a 40% increase in deuterium exchange of an α-helix (Cα4′-helix) motif that forms the interface between the (β/α)8-barrel (A-domain) and the allosteric domain (C-domain). This strongly suggests that it may play an important part in the transition between inactive and active state populations. This result is inconsistent with the L. mexicana and T. cruzi pyruvate kinase crystal structures (
), which show a rigid body rocking of the A/C-core domains, and temperature factor analysis of these structures does not suggest that fructose 1,6-bisphosphate binding affects the mobility of the A/C interdomain interface. Our result is, however, consistent with the model put forward by Mattevi et al. (
) created variant enzymes to perturb salt bridges between the A- and C-domains. They created the substitutions K413Q, which is at the end of the Cα4′-helix, and R271L, which sits above the Cα3′-helix, resulting in severely altered functional behaviors compared with the wild-type enzyme. Lys413 is positioned on a peptide that has increased deuterium uptake in the presence of fructose 1,6-bisphosphate. Importantly, the K413Q amino acid substitution induced activation of the enzyme in the absence of fructose 1,6-bisphosphate, shown by a left shift of the phosphoenolpyruvate saturation curve (S0.5 for phosphoenolpyruvate binding decreased in K413Q compared with wild type) (
). Similar work in Bacillus stearothermophilus pyruvate kinase where the equivalent residue is substituted (W466Y) also demonstrated activation of the variant enzyme in the absence of the allosteric activator (
). In contrast, the R271L amino acid substitution resulted in a 5-fold increase in S0.5 for fructose 1,6-bisphosphate binding as well as a right shift of the phosphoenolpyruvate saturation curve in the absence of fructose 1,6-bisphosphate, suggesting that it does affect the Cα3′-helix, which binds fructose 1,5-bisphosphate. Thus, these two variants validate the significance of the A/C interdomain salt bridges for allosteric signal transfer between allosteric and catalytic domains, consistent with our hydrogen-deuterium exchange studies.
Next, we created four E. coli pyruvate kinase variants at the Cα4′-helix to probe the role of hydrogen bonds within the region that bridges the A- and C-domains on the allosteric mechanism. Thr405 interacts with the end of the Cα3′-helix, whereas Lys404 and Gln408 form hydrogen bonds with residues at the bottom of the A-domain, and Leu411 sits in a hydrophobic pocket at the N-terminal end of the (β/α)8-barrel. Kinetic analysis of these variants is presented in Table 1.
TABLE 1Kinetic parameters for variant enzymes with substitutions in the Cα4′-helix
The substitution of Thr405 to alanine results in a 5-fold increase in the S0.5 for fructose 1,6-bisphosphate from 0.006 ± 0.001 mm for the wild-type enzyme to 0.029 ± 0.009 mm for the T405A variant. This is consistent with the proposed interaction of the Cα4′- and Cα3′-helices whereby fructose 1,6-bisphosphate binding alters the hydrogen bonding pattern between Gln379 and the Cα4′-helix.
The Q408A variant, which bridges the A- and C-domains, shows a significant right shift of both ADP and phosphoenolpyruvate saturation curves in the presence of fructose 1,6-bisphosphate. In addition, the enzyme is catalytically compromised in the absence of fructose 1,6-bisphosphate (kcat = 95 ± 3 s−1 compared with wild-type kcat = 216 ± 4 s−1). Thus, the mechanism of allostery appears to be changed: for this variant, fructose 1,6-bisphosphate also affects the maximal rate (kcat = 95 ± 3 s−1 in the absence but 283 ± 30 s−1 in the presence of fructose 1,6-bisphosphate). Interestingly, Q408A has decreased thermostability (47.5 ± 0 °C), which is further destabilized 10 °C by fructose 1,6-bisphosphate (37.3 ± 0.3 °C). This variant, which bridges the Cα4′-helix with the A-domain via the main chains of Ala187 and Asp214 and the side chain of His216, suggests clear connections between the A- and C-domains and is consistent with a destabilizing role for the Cα4′-helix but suggests that the level of destabilization is carefully tuned for activation.
The substitution of Lys404 with alanine resulted in no change in function, suggesting that this residue plays either a minor or no role in the allosteric mechanism. The introduction of a negative charge to the hydrophobic (β/α)8-barrel in L411E resulted in an inactive enzyme and an extremely low unfolding temperature (Tm ∼ 23 °C), suggesting that amino acid substitutions at the A/C domain interface can destabilize the protein.
To summarize, hydrogen-deuterium exchange studies suggest that fructose 1,6-bisphosphate binding results in large changes in conformational flexibility of key elements within the allosteric domain. First, the effector loop, which in the unbound pyruvate kinase type 1 structure lies across the fructose 1,6-bisphosphate binding site, has increased conformational flexibility, consistent with its displacement upon binding. Second, the Cα3′-helix has decreased conformational flexibility, consistent with fructose 1,6-bisphosphate binding to this helix (Fig. 7B), as seen in the S. cerevisiae structure. Finally, the Cα4′-helix has increased conformational flexibility (Fig. 7D), which we propose is due to weakening interactions between the Cα3′- and Cα4′-helices as the Cα3′-helix binds fructose 1,6-bisphosphate (Fig. 7D). This is corroborated by mutagenic data. Substitution of Lys413 with alanine at the end of the Cα4′-helix breaks a salt bridge between the A- and C-domains and results in enzyme activation. R271L, which sits above the Cα3′-helix, affects both phosphoenolpyruvate and fructose 1,6-bisphosphate binding, consistent with a key role of the Cα3′-helix in the allosteric mechanism. Disruption of hydrogen bonds within the Cα4′-helix by substitutions at positions Thr405, which interacts with the end of the Cα3′-helix, and Gln408, which interacts with residues at the bottom of the A-domain, results in disruption of fructose 1,6-bisphosphate binding and altered catalysis. Substitution of Leu411 with glutamate suggests that changes at this interface can be destabilizing.
The Tetrameric A/A′ and C/C′ Interfaces Are Also Altered upon Fructose 1,6-Bisphosphate Binding
The tetrameric C/C′ interface that is formed by the allosteric domains shows significantly increased conformational flexibility upon fructose 1,6-bisphosphate binding, suggesting that the β-strand (Cβ5′-strand; residues 463–470) that forms this interface is destabilized. Fig. 8A shows the C/C′ interface interactions in the unbound E. coli pyruvate kinase type 1. These interactions include hydrogen bonds between residues Ala465 and Ala465′, Val467 and Asn463′, and Asn463 and Val467′. Fructose 1,6-bisphosphate binding causes a 38% increase in deuterium exchange in the peptide that includes this strand.
Although peptide coverage across the A/A′ interface is poor (7 of 26 residues in the fructose 1,6-bisphosphate-unbound monomer are within peptides that could be detected in both samples), several peptides show altered deuterium exchange (Fig. 8A). The peptide containing residues 291–294 has an increase in deuterium exchange (difference of +31%) and is less structured. In contrast, the peptide containing residues 255–258, which is close to the A/A′ interface, has decreased exchange (difference of −38%) and is, therefore, significantly more structured upon fructose 1,6-bisphosphate binding.
Our deuterium exchange data support an allosteric mechanism whereby fructose 1,6-bisphosphate binding destabilizes and decouples the subunits at the tetrameric interfaces to remove phosphoenolpyruvate cooperation between active sites (homotropic activation). This finding is corroborated by mutagenic studies from two groups, Lovell et al. (
) generated the E392A pyruvate kinase variant at the C/C interface, resulting in complete loss of homotropic activation by phosphoenolpyruvate, suggesting that in yeast the active sites communicate through this interface. Moreover, Lovell et al. (
) propose that the equivalent A/A interface residue of E. coli pyruvate kinase Gln279 in B. stearothermophilus pyruvate kinase is important in coupling phosphoenolpyruvate binding with cooperative and allosteric functions.
Overall, the peptides mapped to the tetrameric interfaces demonstrate increased conformational flexibility, indicating that the interfaces are destabilized upon fructose 1,6-bisphosphate binding. Interestingly, this result, together with the increased conformational sampling evidenced from the global hydrogen-deuterium exchange experiment above, contradicts the general hypothesis put forward by Morgan et al. (
) that “while the regulatory molecules and molecular mechanisms may vary between species, the underlying principle of tetrameric stabilization in response to effector binding is conserved.” That study used thermal unfolding to infer conformation stability, whereas the data from the hydrogen-deuterium exchange directly assess the degree of conformational flexibility both globally and within specific regions within the protein. In any event, together with the dramatic destabilization at the A/C interdomain interface Cα4′-helix (peptide 398–408), our results connect the domain and tetrameric interfaces and in particular their destabilization to the allosteric mechanism of E. coli pyruvate kinase type 1 enzyme (
Lastly, our data are consistent with the effect that fructose 1,6-bisphosphate has on phosphoenolpyruvate affinity. Fructose 1,6-bisphosphate binding blocks the homotropic allosteric mechanism by phosphoenolpyruvate: in the absence of fructose 1,6-bisphosphate, titration of phosphoenolpyruvate results in a sigmoidal response, a signature of cooperativity, whereas in the presence of fructose 1,6-bisphosphate a rectangular hyperbolic curve is observed, signaling a loss of cooperativity. Destabilization of the domain and tetrameric interfaces, such that the active site-containing A-domains are decoupled from one another, is consistent with the loss of phosphoenolpyruvate cooperation between active sites.
Signaling the Catalytic Site for Activation
Next, we traced changes in conformational flexibility associated with the active site (β/α)8-barrel domain to understand the mechanism by which fructose 1,6-bisphosphate binding affects catalysis.
Increased conformational flexibility of the β-strands within the (β/α)8-barrel accompanies destabilization of the Cα4′-helix, which sits at the interdomain interface between the allosteric and catalytic domains and interacts with the αβ-loops at the N terminus of the (β/α)8-barrel. Residue Gln408 of the key Cα4′-helix forms interactions with Asp187 of loop α3β3 and Asn214 of loop α4β4, whereas Lys404 of the Cα4′-helix interacts with Gly185 of loop α3β3 (Fig. 7E). Given the large change in conformational flexibility of the Cα4′-helix, unsurprisingly, the β-strands following the αβ-loops (β-strands 4 and 5, respectively) show an increase in deuterium exchange of 23 and 31%, respectively. Furthermore, β-strands 1, 2, and 3 also showed some destabilization (increase in deuterium exchange of 36, 22, and 27%, respectively), resulting in a largely unstable (β/α)8-barrel core. The destabilization of these β-strands suggests that the allosteric signal is communicated through the core of the (β/α)8-barrel to the active site.
Changes in deuterium exchange were also identified in the active site loops at the top of the (β/α)8-barrel (Fig. 9). Exchange in the peptide containing the β8α8 loop increased by 20%, and the peptide containing the neighboring β1α1 increased by 36%. Furthermore, β-strands 1, 2, 3, 4, and 5 in the core of the (β/α)8-barrel near the substrate binding sites were also destabilized. Specific substrate binding regions are modeled from L. mexicana pyruvate kinase crystal structures (Protein Data Bank code 3HQP). This reveals that the adenosine ring of the ATP (or ADP) is located within interaction distance of loops β1α1, β2α2, β3α3, and β8α8, whereas the oxalate molecule (an analogue of phosphoenolpyruvate/pyruvate) and catalytic ions are situated near β-strands 4 and 5 (Fig. 9). Thus, many of the catalytically important residues show increased conformational flexibility. A caveat to this analysis is that the peptides, in addition to residues belonging to β-strands 1, 3, and 4, also include residues that are with the βα-loops, which are likely to be dynamic. Unfortunately, the resolution limits of this experiment do not allow us to confirm whether the loop regions are experiencing high exchange, thus causing an averaged apparent high exchange of the peptide, or whether the entire peptide is experiencing high exchange.
Increased exchange in these specific active site loops in the E. coli pyruvate kinase type 1 enzyme implies that substrate binding is likely conserved across pyruvate kinase enzymes and that the binding of fructose 1,6-bisphosphate increases the flexibility of these active site loops (Fig. 9). Structural analysis comparing temperature factors of the L. mexicana structures in the presence and absence of fructose 1,6-bisphosphate identifies two active site loops (β7α7 and β8α8) and their preceding β-strands (7 and 8) in the core of the (β/α)8-barrel that have increased B-factors upon fructose 1,6-bisphosphate binding. Increased B-factors in these active site regions suggest that allosteric activation by fructose 1,6-bisphosphate binding increases the flexibility of active site loops for substrate and catalytic ion binding. The binding of the allosteric activator can redistribute protein conformational ensembles and alter the rates of interchange between these populations; this can manifest as a change in flexibility at the active site (
). The increased exchange can be explained by these loops having greater flexibility and therefore an increased range of conformational sampling, which would increase the chances of substrate binding for catalysis (
). Increased exchange in the active site at early labeling time points is consistent with the “intensification” model for catalysis-linked dynamics whereby the energy barrier between conformational substates is lowered upon activator binding (
The Flexible Lid Domain (B-domain) Is Not Allosterically Controlled by Fructose 1,6-Bisphosphate
The exchange profiles of peptides derived from the lid domain (B-domain) of pyruvate kinase type 1 are largely unchanged when the enzyme is pretreated with fructose 1,6-bisphosphate, suggesting that ligand binding in the allosteric site has only a minor effect upon the secondary structure of the lid domain. This result is consistent with previous studies that have shown that the movement of the flexible lid domain is not allosterically controlled (
); however, it is controlled by the presence, type, and position of substrates bound within the active site.
The results presented here provide an experimental characterization of site-specific dynamics for the allosteric activation of E. coli pyruvate kinase type 1, a central metabolic enzyme. The deuterium exchange data demonstrate that fructose 1,6-bisphosphate binding alters the conformational flexibility of specific regions, largely increasing the conformational sampling in the bound state. In addition, the data reveal altered dynamics in the allosteric binding region and also at the substrate binding site ∼40 Å away. The results are consistent with the view of allosteric activation that describes proteins as dynamic molecules existing as an ensemble of conformations (
Based on our interpretation of the observed dynamic changes during the allosteric transition to the active bound state, we propose that the following scheme results in allosteric activation of E. coli pyruvate kinase type 1 upon fructose 1,6-bisphosphate binding 1) The presence of fructose 1,6-bisphosphate destabilizes the effector loop and causes a structural rearrangement, displacing it to allow binding. 2) The fructose 1,6-bisphosphate then forms interactions with the Cα3′-helix, increasing the stability of the helix and weakening an interaction between the loop preceding Cα3′ and the Cα4′-helix. 3) Destabilization of the Cα4′-helix results in destabilization of the interdomain interface that links the allosteric domain (C-domain) and the (β/α)8-barrel (A-domain) within the monomer. 4) Altered interactions of the destabilized Cα4′-helix at the interface between the A- and C-domains and loops α3β3 and α4β4 result in destabilized β-strands following the αβ-loops, consequently producing a more conformationally flexible (β/α)8-barrel core. This destabilization is consistent with the β-strands being involved in allosteric signal transfer through the core of the (β/α)8-barrel to the active site. 5) The signal transfer through the core of the (β/α)8-barrel destabilizes the substrate binding loops at the active site. We propose that the active site loops then sample a greater number of conformations, some of which facilitate binding of the substrate and catalytic ions. 6) The destabilization of the tetrameric interfaces (A/A′ and C/C′) decouples the monomers attenuating the phosphoenolpyruvate homotropic allosteric signal between active sites, explaining why fructose 1,6-bisphosphate binding blocks the phosphoenolpyruvate allosteric mechanism.
In conclusion, we propose a new mechanism by which fructose 1,6-bisphosphate allosterically regulates pyruvate kinase. This work extends previous knowledge of the allosteric activation mechanism by explaining how fructose 1,6-bisphosphate promotes substrate binding and attenuating the phosphoenolpyruvate homotropic allosteric signal despite the allosteric binding site being 40 Å away from the active site.
K. A. D., R. C. J. D., and D. J. W. designed and coordinated the study and wrote the article with the help of S. A. K. K. A. D., S. Z., F. P., and P. L. designed, performed, and analyzed the experiments. All authors were involved in the interpretation of the data. All authors critically revised the article, reviewed the results, and approved the final version of the manuscript.
We thank Grant Pearce and Jeremy Keown for graphic assistance. We acknowledge Jackie Healy for effervescent technical support.
Allostery: an illustrated definition for the second secret of life.