Propagation of the Allosteric Signal in Phosphofructokinase


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Propagation of the Allosteric Signal in Phosphofructokinase from Bacillus stearothermophilus Examined by Methyl-TROSY NMR Amy M. Whitaker, Mandar T. Naik, Rockann Mosser, and Gregory D. Reinhart Biochemistry, Just Accepted Manuscript • Publication Date (Web): 03 Sep 2019 Downloaded from pubs.acs.org on September 3, 2019

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is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Biochemistry

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Propagation of the Allosteric Signal in Phosphofructokinase from Bacillus stearothermophilus Examined by Methyl-TROSY NMR (SECOND REVISION) ‡

§

Amy M. Whitaker†, Mandar T. Naik , Rockann E. Mosser , and Gregory D. Reinhart

Department of Biochemistry and Biophysics, Texas A&M University and Texas A&M AgriLife Research, College Station, TX 77845-2128

Corresponding Author *Gregory D. Reinhart, Department of Biochemistry and Biophysics, Texas A&M University, 2128 TAMU, College Station, TX 77843-2128 Email: [email protected] Phone: (979) 862-2263

Present Address †Department

of Biochemistry and Molecular Biology, University of Kansas Medical Center,

Kansas City, KS. ‡

Department of Molecular Pharmacology, Physiology and Biotechnology, Brown University,

Providence RI 02903. §

2650 Old U.S. 431, Springfield, TN 37172

Abbreviations and Textural Footnotes BsPFK, Bacillus stearothermophilus phosphofructokinase; Fru-6-P, fructose-6-phosphate; PEP, phospho(enol)pyruvate

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Abstract Phosphofructokinase from Bacillus stearothermophilus (BsPFK) is a 136 kDa homotetromeric enzyme. Binding of the substrate, fructose-6-phospate (Fru-6-P), is allosterically regulated by the K-type inhibitor phospho-(enol)pyruvate (PEP). The allosteric coupling between substrate and inhibitor is quantified by a standard coupling free energy that defines an equilibrium with the Fru-6-P bound and PEP bound complexes on one side and the apo form and ternary complex on the other. Methyl-TROSY NMR was employed in order to gain structural information on BsPFK in all four states of ligation relevant to the allosteric coupling. BsPFK was uniformly

15N

and 2H labeled and specifically labeled with δ-[13CH3]-isoleucine utilizing an

isotopically labeled α-keto acid isoleucine precursor. Methyl-TROSY experiments were conducted on all four ligation states and all 30 isoleucines, which are well dispersed throughout each subunit of the enzyme, are well resolved in chemical shift correlation maps of 13C and 1H. Assignments for 17 isoleucines were determined through 3D HMQC-NOESY experiments with [U-15N,2H]; Ileδ1-[13CH3]-BsPFK and complementary HNCA and HNCOCA experiments with [U-2H,15N,13C]-BsPFK. The assignments allowed for the mapping of resonances representing isoleucine residues to a previously determined X-ray crystallography structure. This analysis, preformed for all four states of ligation, has allowed specific regions of the enzyme influenced by the binding of allosteric ligands and those involved in the propagation of the allosteric effect to be identified and distinguished from one another.

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Introduction Allosteric inhibition of enzyme activity can involve substantial conformational changes that relate to the basis of inhibition such as exhibited by the classical allosteric enzyme aspartate transcarbamoylase. The large conformational change this enzyme undergoes has been studied extensively with biophysical techniques ranging from analytical ultracentrifugation (1) to x-ray crystallography (2) and NMR (3, 4). However, some allosteric modifications of enzyme activity seem to occur without obvious causal structural perturbations.

A case in point is

phosphofructokinase from Bacillus stearothermophilus (BsPFK, UnitProt A7ZUC9), the enzyme that catalyzes the phosphorylation of fructose 6-phosphate (Fru-6-P) by MgATP to produce fructose-1,6-bisphosphate. BsPFK exhibits a decrease of over 2-orders of magnitude in the binding affinity of Fru-6-P upon the binding of the strictly K-type allosteric inhibitor phospho(enol)pyruvate (PEP).

Yet the two most obvious structural consequences of PEP

binding, the positioning of the negative charge carried by Glu161 in the Fru-6-P binding site prior to Fru-6-P binding and a 7° twist along a dimer-dimer interface, have been shown to be responsible for no more than a small percentage of the ensuing binding antagonism between PEP and Fru-6-P (5, 6). Of course, structural perturbations need not be dramatic in scale to be consequential in influencing functionality.

Cooper and Dryden (7) have calculated that

perturbations of only 1-2 percent in high frequency dynamics, if distributed uniformly over all the atoms of the protein could lead to allosteric effects with magnitudes typically observed. In such a scenario, the perturbations would be virtually impossible to detect experimentally. In addition, not all structural changes that result from ligand binding may be consequential to the structural conflict that needs to occur to create an allosteric inhibitory response. The challenge in these cases is therefore 2-fold: 1) to assess whether there are observable conformational "hot spots" of perturbations that are large enough that they that can be experimentally observed to

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arise as a consequence of ligand binding; and 2) to distinguish between those observable structural changes that are important to the binding antagonism between allosteric and substrate ligands and those that occur merely in response to ligand binding but are inconsequential to the allosteric communication. This investigation was undertaken in an effort to identify regions within the structure of BsPFK where observable structural perturbations might be particularly important for the transmission of the allosteric antagonism between the binding of PEP and Fru6-P despite being unexceptional when compared to other structural changes. In order to recognize regions and residues of BsPFK that are involved in the propagation of the inhibitory allosteric signal, we employed linkage analysis and methyl-TROSY NMR. Chemical shift changes of the relevant resonance frequencies were analyzed in response to ligand binding and the formation of the ternary complex containing both PEP and Fru-6-P bound. These resonance frequencies vary in response to changes in the local magnetic environment of the nuclei and therefore small changes in structure and dynamics of a particular nucleus are readily detectable by monitoring the change in chemical shift (8). This sensitivity of the nuclei to its local magnetic environment has led to the frequent use of 2D heteronuclear experiments to generate “fingerprints” by which protein conformational changes and ligand binding can be studied (9). Unfortunately, the utility of these traditional experiments is limited to proteins with a molecular weight of less than 50 kDa. BsPFK is a fairly large enzyme with a subunit molecular weight of 34 kDa. In its active form, the subunits form a homotetramer composed of a dimer of dimers where inter- and intra-dimer interfaces form the active and allosteric binding sites, respectively, with a total molecular weight of 136 kDa. The development of NMR experiments that enable the preservation of NMR signals that would otherwise rapidly decay for large protein systems (10, 11), including transverse relaxation-optimized spectroscopy (TROSY) coupled with selective isotope labeling strategies

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(12, 13), has allowed for the study of increasingly larger proteins by NMR spectroscopy; approaches that have been particularly useful in studying allosteric proteins; such as protein kinase (14, 15), glucokinase (16, 17), glycerol phosphate synthase (18), and aspartate transcarbamoylase

(3,

4);

that

are

often

large,

oligomeric

structures.

Aspartate

transcarbamoylase in particular is quite large, and Me-TROSY has been used effectively to study the conformational transition that enzyme experiences. The general approach that extends Me-TROSY utility to high molecular weight proteins, such as BsPFK, involves the use of

13C

and protonated methyl group probes on alanine,

methionine, isoleucine, leucine, and/or valine residues in an otherwise highly deuterated environment (4, 13, 19, 20). Selection of this labeling scheme is motivated firstly by the fact that methyl groups are prevalent throughout the enzyme, including in hydrophobic cores and at molecular interfaces, thereby serving as well distributed internal reporters of dynamics and structure (21). Secondly, exceptional spectral sensitivity results from the three equivalent protons in each methyl group, combined with the rapid rotation of the methyl about its threefold symmetry axis and its localization to flexible ends of side chains. Furthermore, the inherent spin physics of a methyl group enables the preservation of NMR signals, even in large biomolecular systems, via a methyl-TROSY effect that manifests in

13C–1H

heteronuclear multiple quantum

correlation (HMQC) spectra (10). This investigation utilizes methyl-TROSY NMR to examine the four enzyme species involved in PEP inhibition of BsPFK. The labeling strategy incorporated isoleucines that were selectively and

13C

labeled and protonated at the δ-methyl group in an otherwise

15N-labeled

12C-,

deuterated-,

enzyme. The labeling was accomplished by adding isotopically labeled α-

ketobutyrate, a metabolic precursor to isoleucine biosynthesis, to the cell culture prior to induction (19). Figure 1 shows the overall reaction by which 2-keto-3-d2-4-13C-butyrate, [12C,

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2H]

D-glucose,

15NH

4Cl,

and D2O are incorporated into the isoleucine residues of BsPFK as

carried out by E. coli metabolism.

Figure 1. Fate of isotopic labels when isoleucine is metabolically synthesized from 2-keto-3d2-4-13C-butyrate in isotopically-labeled minimal media producing [U-15N,2H];Ileδ1-[13CH3] BsPFK. Precursor was synthesized from 2-keto-4-13C-butyrate as described in Materials and Methods.

By measuring the perturbations in NMR chemical shifts upon substrate and inhibitor binding, the 13C-labeled

methyl groups can serve as local reporters on the allosteric coupling, the

functionality of which is quantitatively measured by the coupling free energy between Fru-6-P and PEP (22, 23). The present study attempts to identify regions of enzyme that likely contribute most strongly to the coupling free energy. More specifically, Methyl-TROSY HMQC experiments were conducted with [U-2H,15N]-BsPFK specifically labeled with Ileδ1-[13C,1H3] in order to gain structural information about BsPFK in all four states of ligation relevant to the inhibitory allosteric response. As a result of these experiments, specific residues of the enzyme where structural conflicts arise upon the binding of both ligands simultaneously were identified. Mapping these residues back to the crystal structure has allowed specific regions of the enzyme likely involved in the propagation of the allosteric signal to be localized.

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Materials and Methods Materials All chemical reagents used in buffers for protein purification, enzymatic assays, and NMR experiments were of analytical grade and were purchased from Sigma-Aldrich (St. Louis, MO), Fisher Scientific (Fair Lawn, NJ), or Research Products International (Mt. Prospect, IL) unless otherwise noted. Lyophilized creatine kinase, the ammonium sulfate suspension of glycerol-3-phosphate dehydrogenase, and the potassium salt of phospho(enol)pyruvate were purchased from Roche (Indianapolis, IN). The ammonium sulfate suspensions of aldolase and triosephosphate isomerase, the disodium salt of fructose-6-phosphate, and the disodium salt of phosphocreatine were purchased from Sigma-Aldrich (St. Louis, MO). The coupling enzymes were extensively dialyzed against 50 mM EPPS pH 8.0, 100 mM KCl, 5 mM MgCl2, and 0.1 mM EDTA before use. NADH and DTT were purchased from Research Products International (Mt. Prospect, IL). Mimetic Blue 1 A6XL resin used in protein purification was purchased from Promatic BioSciences (Rockville, MD). The Mono-Q HR anion exchange column used in protein purification was purchased pre-packed for FPLC use from Pharmacia (currently GE Healthcare, Uppsala, Sweden). Amicon Ultra centrifugal filter units (spin concentrators) were from Millipore Corporation (Billerica, MA) and poly(ethylene glycol)-3000 was from SigmaAldrich (St. Louis, MO). Minimal media was made using potassium phosphate, monobasic, and sodium phosphate, dibasic, from EMD Chemicals (Gibbsown, NJ). Additional components of the minimal media included D-glucose from Macron Chemicals (Center Valley, PA), magnesium sulfate and ferrous sulfate from Fisher Scientific (Fair Lawn, NJ), thiamine hydrochloride and calcium chloride dihydrate from Sigma-Aldrich (St. Louis, MO), and ammonium chloride from Acros Organics (NJ). Deuterated MES-d13 was from Sigma-Aldrich (St. Louis, MO). Ammonium chloride (15N, 99%), L-isoleucine (15N, 98%), L-isoleucine (13C6,99%;15N, 99%), α-

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ketobutyric acid, sodium salt (CH3-13C, 99%), D-glucose (1,2,3,4,5,6,6-D7,97-98%), D-glucose (U-13C6,99%; 1,2,3,4,5,6,6-D7,97-98%) and deuterium oxide (D, 99.9%) are from Cambridge Isotope Laboratories, INC (Andover, MA). Shigemi NMR tubes were purchased from Shigemi, Inc. (Allison Park, PA) and were used for all NMR experiments. Synthesis of 2-keto-3-d2-4-13C-butyrate 2-keto-3-d2-4-13C-butyrate

was

synthesized

from

2-keto-4-13C-butyrate

by

proton/deuterium exchange of C3 through incubation at pH 10.2 in 99.5% D2O for 12 - 14 hours (16). Protein Expression and Purification of Isotopically Labeled Wild-type BsPFK The plasmid pBR322/BsPFK (24) contains the gene for BsPFK behind the native Bacillus stearothermophilus promoter. This plasmid was modified to place the BsPFK gene behind an inducible lac promoter in pALTER-1. An inducible plasmid was necessary to express the enzyme in minimal media. BsPFK was expressed in E. coli RL257A cells, which are a T1 bacteriophage resistant derivation of RL257 cells (25). RL257 cells lack both the pfkA and pfkB genes. RL259A cells were made by P1 transduction (26) using RL257 cells as the recipient strain and RY12459 cells as a donor strain. RY12459 cells were obtained from Ryland Young (Texas A&M University, College Station, TX) and are a derivative of MC4100b (27) that contain a tonA gene disruption within a kanamycin cassette. Protein expression of the [U-15N,2H];Ileδ1-[13C1H3]-BsPFK was performed as described previously by Tugarinov, V. et. al., (13) with a few minor modifications. Following heat shock transformation, cells were picked from a single bacterial colony that was grown on solid Lysogeny Broth (LB)/tet/H2O media (Tryptone 10 g/L, yeast extract 5 g/L, and sodium chloride 10 g/L, tetracycline 15 μg/mL). These cells were transferred to a 5 mL culture of LB/tet/H2O media and allowed to grow in a shaking incubator at 37 °C until the cell density reached an

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OD600 of 0.7 - 0.8 (4 - 6 hours). The 5 mL culture was centrifuged at 1,200 × g at room temperature and the pellet was gently re-suspended in 1 mL of M9/H2O media (0.048 M Na2HPO4, 0.022 M KH2PO4, 9 mM NaCl, 19 mM NH4Cl, 0.2 % glucose, 2 mM MgSO4, 100 µM CaCl2, 10 µg/mL thiamine, 10 µg/ml FeSO4, 15 µg/mL tetracycline) which contained unlabeled glucose and NH4Cl. Aliquots of the re-suspension were added to 20 mL of the unlabeled M9/H2O media until the starting OD600 was between 0.05 and 0.1. The culture was grown until the OD600 reached 0.6, which took between 8 - 10 hours. The culture was then centrifuged and re-suspended in 100 mL of labeled M9/D2O media (containing [2H,C]-glucose and 15NH4Cl) so that the beginning OD600 was 0.1. These cells were grown until the OD600 was between 0.4 - 0.5 (8 - 10 hours), then the cells were diluted to 200 mL by the addition of 100 mL labeled M9/D2O media and were again grown until the OD600 reached 0.4 - 0.5 (4 - 6 hours). At this time the culture was diluted with labeled M9/D2O media to a volume that equaled 1 L once the α-ketobutyrate was added and allowed to grow until the OD600 was 0.25 (4 - 6 hours). At this time, 70 mg/L of 2-keto-3-d2-4-13C-butyrate was added to the culture. The culture was allowed to grow for approximately one hour until the OD600 was between 0.3 - 0.4. Protein expression was induced with the addition of 1 mM IPTG and the cells were allowed to grow for 8 hours. The final OD600 ranged between 0.7 - 1.0. For [U-2H];Ile-[15N]-BsPFK, the same procedure was followed with the following exceptions: minimal media was prepared with

14NH

4Cl

instead of

15NH

4Cl,

and Ile-[15N] was

added to the media instead of the α-ketobutyrate precursor. For [U-15N,2H,]; Ile-[15N,

13C]-

BsPFK the same procedure was followed except Ile-[15N, 13C] was added to the media instead of the α-ketobutyrate precursor. For [U-2H,15N,13C]-BsPFK minimal media was prepared with D[2H,13C] glucose and

15NH

4Cl

and no selective labeling of isoleucine methyl groups was

performed.

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Upon completion of growth, cells were harvested from the media by centrifugation and frozen at -20 °C for at least 12 hours before re-suspension in purification buffer (10 mM TrisHCl pH 8.0 and 1 mM EDTA) and sonication in a Fisher 550 Sonic Dismemberator at 0 °C in 15 second pulses at setting 6 for 12 minutes or until the OD600 was no longer decreasing. The crude lysate was centrifuged at 22,500×g for 30 minutes at 4 °C. The clarified supernatant was incubated in a water bath at 70 °C for 15 minutes, cooled on ice for 15 minutes, and centrifuged again for 30 minutes at 4 °C. The BsPFK supernatant was diluted to 1 L and loaded onto a Mimetic Blue 1 A6XL column that was equilibrated with purification buffer. The column was washed with 1 L of purification buffer, and the enzyme was eluted with a 600 mL 0 - 1.5 M NaCl gradient. Enzyme containing fractions were pooled and dialyzed into 20 mM Tris-HCl pH 8.5 and loaded onto a Pharmacia Mono-Q HR anion exchange column that had been equilibrated with the same buffer. The enzymes were eluted with a 200 mL 0 - 1 M NaCl gradient and PFK containing fractions were combined, concentrated, then dialyzed into MES Buffer (10mM MES, pH 6.0). Concentrated enzyme was further dialyzed and stored in MES-d13/D2O Buffer (10 mM deuterated MES pH 6.0 and 0.02 % NaN3) at 4 °C. The final enzyme was determined to be pure by SDS-PAGE and the concentration was ascertained using the absorbance at 280 nm (ε = 18910 M-1cm-1). The final enzyme concentration achieved for NMR experiments ranged between 0.4 0.5 mM in monomer. Approximately 325 μL of protein was added to a Shigemi NMR tube. Methyl-TROSY NMR Spectroscopy The temperature was set to 37 °C and the pH was adjusted to 6.0 for all NMR experiments. Methyl-TROSY experiments were performed on either a Bruker 600 MHz, or 800 MHz spectrometer, both instruments equipped with a cryo-probe. Two-dimensional 1H-13C HMQC methyl correlation experiments were acquired on samples of [U-15N,2H]; Ileδ1-[13CH3]BsPFK using the pulse schemes described previously (10). NMR data were processed and

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analyzed with TopSpin (Bruker, Inc.) and analyzed using Sparky 3 (T. D. Goddard and D. G. Kneller, University of California, San Francisco). Resonance Assignments Isoleucine assignments were determined through 3D HMQC-NOESY experiments with [U-15N,2H]; Ileδ1-[13CH3]-BsPFK and complementary HNCA and HNCOCA experiments with [U-2H,15N,13C] BsPFK. In addition a 2D 1H-15N TROSY spectrum of [U-2H];Ile-[15N]-BsPFK and triple resonance experiments with [U-15N,

2H];Ile-[15N,

13C]-BsPFK

confirmed the

assignments. Generation of the Four Enzyme-Ligand Species To generate the PEP-BsPFK species, PEP was added to BsPFK to a final concentration of 50 mM. For the generation of the substrate bound complex, Fru-6-P was added to BsPFK to a final concentration of 10 mM. The ternary complex (PEP:BsPFK:Fru-6-P) required BsPFK to be mixed with final concentrations of 10 mM Fru-6-P and 50 mM PEP. Based on the ligand dissociation constants and the allosteric coupling constant at 37 °C and pH 6.0, less than 2 % of other species were present. The ligand dissociation constants were determined using steady-state fluorescence assays with a tryptophan-shifted mutant. These assays were performed in the absence of the second substrate, MgATP, and hence turnover to match the conditions of the enzyme during the NMR experiments (data not shown). In addition, NMR titrations confirmed that spectra represent saturated complexes where indicated. Steady-State Kinetic Assays Activity measurements for BsPFK were carried out using a coupled enzyme system (28) in a 0.6 mL reaction volume of either 50 mM EPPS buffer (pH 8.0, 25 °C) or 10 mM MES buffer (pH 6.0, 37 °C), additionally containing 5 mM MgCl2, 100 mM KCl, 0.1 mM EDTA, 2 mM DTT, 0.2 mM NADH, 3 mM ATP, 250 μg aldolase, 50 μg of glycerol-3-phosphate

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dehydrogenase, and 5 μg of triosephosphate isomerase. 40 μg/mL of creatine kinase, and 4 mM phosphocreatine were added as an ATP regenerating system to avoid the accumulation of MgADP, which is an activator. Temperature was controlled using a NESLab RTE-111 circulating water bath. Fru-6-P and PEP were added at varied concentrations as indicated. Assays were started by the addition of 10 μL of appropriately diluted PFK. The rate of the reaction was measured on Beckman Series 600 spectrophotometers using a linear regression calculation to convert change in absorbance at 340 nm to PFK activity. One unit of PFK activity is described as the amount of enzyme needed to produce 1 μmol of fructose-1,6-bisphosphate per minute. Kinetic Data Analysis Data were fit using the non-linear least-squares fitting analysis of Kaleidagraph software version 4.5 (Synergy). For the steady-state kinetic assays the initial velocity data were plotted against concentration of Fru-6-P and fit to the Hill equation (29):

𝑣=

𝑉[𝐴]𝑛𝐻

(1)

𝐾𝑛𝑎𝐻 + [𝐴]𝑛𝐻

where 𝑣 is the initial velocity, [𝐴] is the concentration of the substrate Fru-6-P, 𝑉 is the maximal velocity, and 𝑛𝐻 is the Hill coefficient. 𝐾𝑎 is defined as the concentration of Fru-6-P at which the enzyme’s activity is half-maximal. Assuming that Fru-6-P achieves a rapid binding equilibrium, which was shown to be valid in EcPFK using a steady-state kinetic method (30), 𝐾𝑎 is equivalent to the dissociation constant for Fru-6-P from the binary enzyme-substrate complex (22, 23). Values of 𝐾𝑎 obtained from the initial velocity experiments were plotted against the concentration of opposing ligand and fit according to:

𝐾𝑎 =

𝐾𝑜𝑖𝑎

(

𝐾𝑜𝑖𝑦 + [𝑌] 𝐾𝑜𝑖𝑦 + 𝑄𝑎𝑦[𝑌]

)

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where 𝐾𝑜𝑖𝑎 is the dissociation constant for Fru-6-P in the absence of PEP, 𝐾𝑜𝑖𝑦 is the dissociation constant for PEP in the absence of Fru-6-P, and 𝑄𝑎𝑦 is the coupling coefficient (22, 23). 𝑄𝑎𝑦 describes the effect of the allosteric effector on the binding of the substrate, and is defined in equation 3. Analysis of Allosteric Interactions The effect the inhibitor PEP has on the subsequent binding of substrate Fru-6-P to BsPFK, and the equivalent reciprocal effect of Fru-6-P on the binding of PEP, can be quantified by the coupling constant, 𝑄𝑎𝑦, which is defined by a ratio of thermodynamic dissociation constants for Fru-6-P and PEP using the following equation (23):

𝑄𝑎𝑦 =

𝐾𝑜𝑖𝑎 𝐾∞ 𝑖𝑎

=

𝐾𝑜𝑖𝑦

(3)

𝐾∞ 𝑖𝑦

where 𝐾𝑜𝑖𝑎and 𝐾∞ 𝑖𝑎 are the dissociation constants for Fru-6-P (A) in the absence and saturating presence of PEP (Y), respectively. Analogously, 𝐾𝑜𝑖𝑦 and 𝐾∞ 𝑖𝑦 are the dissociation constants for PEP in the absence and saturating presence of Fru-6-P, respectively. When describing the nature and magnitude of the allosteric effect between the two ligands Fru-6-P and PEP, all possible ligation states of the enzyme must be considered. BsPFK (E) is free enzyme. BsPFK:F6P (EA) and PEP:BsPFK (YE) are the two binary complexes and PEP:BsPFK:F6P (YEA) is the ternary complex.

By substituting the definitions for the

dissociation constants into equation 3 it can be shown that the coupling constant serves as an equilibrium constant between the four species as they appear in the following disproportionation reaction: [𝐸𝐴] + [𝑌𝐸]

𝑄𝑎𝑦

[𝐸] + [𝑌𝐸𝐴]

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where the left side of the equilibrium contains the two binary complexes and the right side consists of free enzyme and the ternary complex. For this reaction, the standard allosteric coupling free energy (∆𝐺𝑎𝑦) is related to the coupling constant and its associated enthalpy and entropy components as follows: ∆𝐺𝑎𝑦 = ―𝑅𝑇𝑙𝑛(𝑄𝑎𝑦) = ∆𝐻𝑎𝑦 ― 𝑇∆𝑆𝑎𝑦

(5)

where R is the gas constant and T is the absolute temperature in Kelvin. [By past convention, the superscript “o” usually included to denote a standard free energy is omitted to avoid confusion with the meaning of the superscripts appearing in equation 3 (23).] In an effort to ascertain the molecular basis for the inhibition by PEP, for which ∆𝐺𝑎𝑦> 0 by definition, one must consider the four enzyme species depicted in equation 4 and how their differences might ultimately generate the thermodynamic values of ∆𝐻𝑎𝑦 and ∆𝑆𝑎𝑦 as shown in equation 5. A useful way in the present context to consider these ideas further is to recognize that the coupling free energy value associated with that equilibrium can be described in terms of the free energy of formation for the products minus the free energy of formation for the reactants (23): ∆𝐺𝑎𝑦 = (𝐺𝑌𝐸𝐴 + 𝐺𝐸) ― (𝐺𝐸𝐴 + 𝐺𝑌𝐸)

(6)

Equation 6 can be rearranged to emphasize the changes introduced by the binding of ligands by the following rearrangement: ∆𝐺𝑎𝑦 = ∂𝐺𝑌𝐸𝐴 ― (∂𝐺𝐸𝐴 + ∂𝐺𝑌𝐸)

(7)

where ∂𝐺𝐸𝐴 = 𝐺𝐸𝐴 ― 𝐺𝐸; ∂𝐺𝑌𝐸 = 𝐺𝑌𝐸 ― 𝐺𝐸; and ∂𝐺𝑌𝐸𝐴 = 𝐺𝑌𝐸𝐴 ― 𝐺𝐸. For example, the perturbations in free energy of formation arising from the binding of both A and Y simultaneously to previously unligated enzyme are described by ∂𝐺𝑌𝐸𝐴. When this value is equal to the sum of the perturbations that occur when the ligands bind individually, ∆𝐺𝑎𝑦 equals zero and by definition there is no allosteric effect. Inhibition or activation occurs when ∂𝐺𝑌𝐸𝐴 and the quantity (∂𝐺𝐸𝐴 + ∂𝐺𝑌𝐸) are not equal to each other. In other words, there must be

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Biochemistry

15

an energetic conflict arising from the binding of both ligands simultaneously that leads to the allosteric effect.

If the ternary complex can accommodate simultaneously the free energy

changes introduced by each ligand binding individually, then there is no conflict and hence no allosteric effect. These energetic conflicts can be manifested in ∆Hay and/or ∆Say:

∆𝐻𝑎𝑦 = ∂𝐻𝑌𝐸𝐴 ―(∂𝐻𝐸𝐴 + ∂𝐻𝑌𝐸)

(8)

∆𝑆𝑎𝑦 = ∂𝑆𝑌𝐸𝐴 ―(∂𝑆𝐸𝐴 + ∂𝑆𝑌𝐸)

(9)

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16

Results Methyl TROSY assignments As a result of the selective labeling, all 30 of the isoleucine residues per monomer of [U15N,2H];

Ileδ1-[13CH3]-BsPFK contain one methyl group that is potentially visible by NMR. As

shown in Figure 2A, the isoleucine residues in BsPFK are all well dispersed throughout each subunit providing excellent coverage of the enzyme structure. This includes many residues between the allosteric sites and the active sites in a BsPFK subunit. In Me-TROSY chemicalshift correlation maps of 13C and 1H, all 30 isoleucines are well resolved (Figure 2B). In addition, the amide backbones of all 320 amino acids are 15N labeled in [U-15N,2H]; Ileδ1-[13CH3]-BsPFK and Figure 2C shows a 1H-15N TROSY spectrum with good dispersion of resonances. However, because the entire backbone is

15N

labeled, there is quite a bit of crowding in the 1H-15N

TROSY. Both 2D spectra indicate the enzyme is well folded under the conditions of the experiments, which was confirmed by the sample remaining fully enzymatically active throughout the experiments. Specific activity (data not shown), apparent ligand dissociation constants, and the allosteric coupling parameters for the isotopically labeled enzymes were comparable to their unlabeled counterparts, as shown in Tables 1 and 2. These parameters were assessed at pH 8 and 25°C (Table 1) to be consistent with other similar characterizations in the literature and at 37°C and pH 6 because those conditions were optimal for performing the NMR studies reported herein. The similar values for the apparent dissociation constants and the allosteric coupling parameters indicate that the isotopic labeling did not disrupt the structure or function of the enzyme in any appreciable way.

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Biochemistry

17

Figure 2. [U-15N,2H];Ileδ1-[13CH3] labeling of BsPFK provided excellent coverage of the enzyme and well resolved 2D spectra. A) X-ray crystal structure of the BsPFK monomer (PDBID# 4PFK) with all 30 isoleucine residues represented by spheres. Yellow spheres are isoleucines we were able to assign, and black spheres are isoleucines that remain unassigned. ADP is shown in the allosteric site in blue, and Fru-6-P is shown in the active site in red. Lines depict the 4 unique pair-wise heterotropic allosteric interactions that can occur within each subunit. B) Methy-TROSY spectrum (37 °C; pH 6.0; 600 MHz; 10% D20/90% H20) C) 1H-15N TROSY spectrum (37 °C; pH 6.0; 800 MHz; 10% D20/90% H20).

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18

Table 1: Allosteric coupling parameters for unlabeled and the isotopically labeled BsPFK at 25 °C and pH 8.0.

BsPFK Labeling

𝑲𝒐𝒊𝒂 (µM)

𝑲𝒐𝒊𝒚 (µM)

𝑸𝒂𝒚

∆𝑮𝒂𝒚 (kcal/mol)

Unlabeled

39 ± 2

63 ± 4

0.0021 ± 0.0002

3.65 ± 0.06

[U-15N,2H];Ileδ1-[13CH3]

34 ± 2

40 ± 2

0.0022 ± 0.0002

3.63 ± 0.06

[U-2H,15N,13C]

44 ± 3

49 ± 4

0.0021 ± 0.0003

3.65 ± 0.08

[U-2H];Ile-[15N]

30 ± 2

33 ± 2

0.0021 ± 0.0001

3.65 ± 0.03

[U-2H,15N];Ile-[13C]

41 ± 1

56 ± 2

0.0022 ± 0.0002

3.63 ± 0.06

Table 2: Allosteric coupling parameters for unlabeled and the isotopically labeled BsPFK at 37 °C and pH 6.0.

BsPFK Labeling

𝑲𝒐𝒊𝒂 (µM)

𝑲𝒐𝒊𝒚 (µM)

𝑸𝒂𝒚

∆𝑮𝒂𝒚 (kcal/mol)

Unlabeled

35 ± 2

48 ± 7

0.010 ± 0.002

2.73 ± 0.12

[U-15N,2H]; Ileδ1-[13CH3]

32 ± 1

55 ± 4

0.008 ± 0.001

2.86 ± 0.07

[U-2H,15N,13C]

35 ± 4

42 ± 7

0.012 ± 0.002

2.62 ± 0.10

[U-2H]; Ile-[15N]

35 ± 4

82 ± 14

0.011 ± 0.001

2.67 ± 0.06

[U-2H,15N]; Ile-[13C]

31 ± 1

50 ± 3

0.011 ± 0.001

2.67 ± 0.06

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Biochemistry

19

Methyl-TROSY spectral maps reveal several cross peaks with chemical shifts unique to each state of ligation indicating unique structures for each of the enzyme forms appearing in equation 4. Apo BsPFK and F6P-bound BsPFK spectra have several overlapping resonances, whereas the PEP-bound BsPFK spectrum has numerous dissimilar peaks. Distinct peaks, not seen in any other spectra, are present in the spectrum of the ternary complex (BsPFK with both Fru-6-P and PEP bound). Complementary HNCA and HN(CO)CA experiments with [U2H,15N,13C]-BsPFK 1H-15N

both

were used to assign the resonances corresponding to the isoleucines in the

TROSY spectrum. In order to transfer these assignments to the methyl-TROSY spectrum

15N-edited

15N,2H];

and

13C-edited

Ileδ1-[13CH3]-BsPFK.

3D-HMQC NOESY experiments were performed with [UWe were able to unambiguously assign 17 out of the 30

isoleucine residues. One residue, Ile-61, was assigned in the 1H-15N TROSY but was unable to be transferred to the methyl-TROSY spectrum. Of the remaining 12 unassigned isoleucines, at least eight appear to be buried and were likely unable to undergo the H/D exchange with solvent required to be NMR visible in 1H-15N TROSY experiments. Fortunately, the Fru-6-P binding process and the formation of the ternary complex by either order of ligand addition are in the fast exchange regime, allowing us to see resonances move across the magnetic field in response to an increase in ligand concentration. Resonance assignments were transferred to the ligated forms of the enzyme using these titrations as demonstrated in Figure 3 with the binding of Fru-6-P. This behavior facilitated the assignments of the Fru-6-P bound binary and ternary complexes.

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Figure 3. A) Overlay of methyl-TROSY spectra (37 °C; pH 6.0; 800 MHz; 10% D20/90% H20) with Fru-6-P concentrations ranging from 0 (red) to 10 mM (Purple). B) Close up of boxed region of panel A. Arrows indicate the direction of chemical shift perturbation in response to increasing ligand concentration.

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Biochemistry

21

Perturbations relevant to allosteric communication To identify chemical shift changes that reflect conformational perturbations involved in the allosteric inhibition of BsPFK, it was essential to first determine those residues for which the chemical shift values for each ligation state differ from those of the apo-enzyme to a significant extent. We assessed this criterion by evaluating the chemical shift changes due to ligand binding in both the

13C

and 1H dimensions. The chemical shift changes due to the binding of Fru-6-P

([EA]), PEP ([XE]), or both ligands ([XEA]), relative to apo-enzyme, of all assigned isoleucine residues in both the 13C and 1H dimensions are listed in Table 3. A largest 1H shift observed is 0.209 ppm up-field in Ile-67 when either Fru-6-P or both ligands bind. The largest 13C shift is the 1.364 ppm up-field shift observed with Ile-28 when PEP binds. We have selected for further scrutiny those residues for which the absolute value of the 13C or 1H chemical shift perturbations, relative to apo-enzyme, are equal to at least 20% of either of these maximum (absolute value) chemical shift perturbations in order to focus attention on those residues that exhibit the most substantial effects from ligand binding.

Applying this criterion results in 10 residues of

particular interest that are highlighted in Table 3 in boldface font, and they are the residues shown in Figure 4. Second, the residues involved in the energetic conflict that leads to the allosteric inhibition were identified by examining the nature and magnitude of these chemical shift changes in these 10 residues deemed to have substantial perturbations from ligand binding as just described. If we presume that the magnitude of the chemical shifts due to the binding of either or both ligands roughly relates to the magnitude of the energetic perturbation introduced by that ligand, we can make this assessment as follows. The issue becomes whether the chemical shift change when both ligands bind is comparable to the sum of the chemical shift changes

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22 Table 3: Chemical shifts EA], [YE], [YEA], and the net value of [YEA] - ([EA] + [YE]), in the 1H

and 13C dimensions for assigned Ile residues. Bold numbers are those shifts greater than 20% of

the maximum observed for either nucleus. Green denotes net values greater than 20% of the maximum for either nucleus.

1H

(ppm)

13C

(ppm)

[EA]

[YE]

[YEA]

[YEA] – ([EA] + [YE])

[EA]

[YE]

[YEA]

[YEA] – ([EA] + [YE])

Ile-4

-0.004

-0.001

0.001

0.006

0.005

0.018

0.020

-0.003

Ile-20

0.008

0.028

0.019

-0.017

0.025

-0.398

0.022

0.395

Ile-28

-0.010

0.169

-0.049

-0.208

0.243

1.364

0.287

-1.320

Ile-49

0.002

0.010

0.039

0.027

0.030

-0.210

-0.170

0.010

Ile-67

-0.008

0.209

0.209

0.008

0.015

0.136

0.286

0.135

Ile-86

0.014

0.010

0.011

-0.013

-0.003

0.002

0.011

0.012

Ile-94

0.000

0.010

0.016

0.006

0.028

0.116

0.139

-0.005

Ile-126

-0.016

-0.059

-0.005

0.070

0.065

-0.444

0.215

0.594

Ile-130

-0.012

-0.029

-0.019

0.022

0.015

-0.120

-0.164

-0.059

Ile-137

-0.030

0.014

-0.003

0.013

-0.273

0.691

0.002

-0.416

Ile-147

-0.034

0.012

-0.033

-0.011

-0.046

-0.160

-0.272

-0.066

Ile-150

-0.003

-0.154

-0.157

0.000

0.032

0.320

0.158

-0.194

Ile-153

-0.007

-0.015

-0.010

0.012

-0.021

0.031

0.080

0.070

Ile-166

-0.005

-0.041

0.007

0.053

0.024

-0.366

0.020

0.362

Ile-176

-0.037

-0.025

-0.002

0.060

0.077

0.491

0.240

-0.328

Ile-202

-0.019

0.006

-0.022

-0.009

-0.135

-0.161

-0.192

0.104

Ile-286

-0.024

-0.077

-0.065

0.036

-0.059

-0.312

-0.376

-0.005

Residue

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Biochemistry

23

Figure 4. A) 13C and B) 1H chemical shift perturbations of Ile methyl groups of BsPFK in response to the binding of Fru-6-P (red), PEP (blue) and both simultaneously (purple) for those residues which had a at least one chemical shift perturbation greater than 20% of the maximum perturbation. Residues identified in green exhibit a marked distinction between the purple bar and the sum of the red and blue bars in either the 1H or 13C chemical shifts (see explanation in text).

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24

introduced by the binding of each ligand alone. These comparisons can be visualized in Figure 4 for the 10 residues most influenced by ligand binding. A more quantitative way to make this comparison is by calculating the net values of the chemical shifts that occur upon the binding of both ligands ([YEA]) less the sum of the chemical shifts obtained for each binary complex individually ([EA] + [YE]). These values are given for each residue for both 13C and 1H dimensions in Table 3. The residues predicted to be involved in the allosteric communication are those for which this difference is significantly different from zero. We can assess this significance by seeing for which residues this net difference in either 1H or

13C

chemical shift perturbations is greater than 2 standard deviations

away from the 10% trimmed mean of values for all 17 residues appearing in Table 3 (Figure 5). Based on this analysis, we conclude that although isoleucine residues 67, 147, 150, and 286 exhibit substantial individual chemical shift perturbations in response to ligand binding, those perturbations seem to suggest only a minor, if any, contribution to the overall coupling that defines the allosteric communication between Fru-6-P and PEP binding sites. By contrast, 6 residues meet this criterion in the 13C dimension, and 4 of these 6 also meet this criterion in the 1H

dimension. These 6 isoleucine residues are in positions 20, 28, 126, 137, 166, and 176 and

are denoted in green in Table 3 and in Figure 4. Figure 6 shows an X-ray crystallography structure of the BsPFK tetramer with the residues color-coded to distinguish between all three categories of chemical shift changes in response to ligand binding. Positions Ile-4, Ile-49, Ile-86, Ile-94, Ile-130, Ile-153 and Ile-202 show no substantial changes in chemical shift upon ligand binding and are depicted in pink. Therefore, we predict that these resonances do not detect structural changes that directly contribute to the coupling free energy that defines the inhibition of BsPFK by PEP. Positions Ile-

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Biochemistry

25

Figure 5. Changes in 1H (black) and 13C (red) chemical shifts introduced by the binding of both Fru-6-P and PEP simultaneously minus the sum of the shifts associated with the respective binary complexes. Solid horizontal lines indicate 2 standard deviations above and below the respective means, which are near zero in each case. producing the net values outside these 2 standard deviations are labeled.

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26

Figure 6. Two views of the BsPFK homotetramer (PDBID# 4PFK) displaying the locations of isoleucine residues with no shifts (pink spheres), additive shifts (yellow spheres), and nonadditive shifts (green spheres). Positions previously identified by fluorescence spectroscopy to play a role in allosteric coupling are shown with orange spheres. ADP is shown in the allosteric site in blue, and Fru-6-P is shown in the active site in red. Three subunits of the homotetramer are represented as gray ribbons and one is shown in cyan spacefill for visual clarity.

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Biochemistry

27

67, Ile-147, Ile-150 and Ile-286, depicted in yellow, show substantial changes in chemical shift upon ligand binding, but the ternary complex appears to be able to accommodate the perturbations brought on by the ligands at these locations, indicating a lack of structural conflicts at these sites. We interpret this behavior as suggesting that these residues are also not likely involved in the structural conflict that gives rise to allosteric inhibition of BsPFK. By contrast, positions Ile-20, Ile-28, Ile-126, Ile-137, Ile-166 and Ile-176, shown in green, possess the greatest difference between the chemical shifts of the ternary complex and the sum of the chemical shifts resulting from the binary complexes. We feel that these residues, where a conflict is reported in response to the binding of both ligands simultaneously, mark regions crucial to the transmission of the allosteric signal for inhibition. These residues are located generally in the regions near the two interfaces and directly between the substrate and effector binding sites. Four isoleucines; Ile-20, Ile-28, Ile-126, and Ile-137; lie between Fru-6-P and PEP binding sites that are 30 Å away from each other. The other two isoleucine residues, Ile-166 and Ile-176, both fall between another pair of binding sites that are located 32 Å apart. This implies the lack of a single discrete structural pathway by which the allosteric signal is propagated. Ile-153 is located between the binding sites 22 Å apart, which we have previously determined is the strongest pair-wise inhibitory coupling (31), and it does not seem to experience structural changes involved in the propagating of the allosteric signal based on these NMR data. However, the conservative mutation of Ile-153 to valine has an almost 4-fold effect on allosteric inhibition (32), suggesting that this residue is involved in a way not accounted for by this NMR analysis. In addition, previous studies identified regions of the BsPFK enzyme that likely contribute to the entropy component of the coupling free energy (33, 34). This was accomplished by measuring the rotational correlation time of engineered fluorescent tryptophan probes

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28

throughout the enzyme under all four ligation states. The positions identified were Y164 and F240, and they are mapped as orange residues onto the structure in Figure 5. It is clear that a more thorough assessment of the changes in dynamics will be required to obtain a fuller picture of the nature, and locations, of all of the conflicts that give rise to the allosteric inhibition of BsPFK by PEP.

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Discussion The essential challenge and promise of structural biology is to relate structure to function. In the case of K-type allosteric behavior, where 'function' involves the perturbation of ligand (including substrate) binding affinities, the issue is how structural perturbations relate to free energy changes resulting from ligand binding. We propose that perturbations to the enzyme reported by chemical shifts can be qualitatively related to the energetics of allosteric coupling between Fru-6-P and PEP in the broadest possible terms by elaborating on ∆𝐺𝑎𝑦 as described previously (23) and summarized in equations 6 and 7. This restatement of the disproportionation equilibrium reaction given in equation 4 emphasizes the contrast between the summation of chemical potentials associated with the binary complexes versus the chemical potential of the ternary complex, each normalized to free enzyme, as the determinant of the value of ∆𝐺𝑎𝑦. This formulation demonstrates the need to include structural information on all four species, with a focus on changes introduced to free enzyme, in order to interpret any structural changes even in the context of allosteric inhibition. In particular we note that the consideration of only substratebound and the inhibitor-bound forms is insufficient. It is useful to consider how this analysis compares with other uses of NMR to study allosteric behavior. For example, in the case of V-type allosteric activation, an analysis similar to that performed here has been utilized to study the activation of imidazole glycerol phosphate synthase

by

N'-[(5'-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-

ribonucleotide (35). The differences between the changes in chemical shift of the ternary complex and the sum of the chemical shifts for the binary complexes were assessed as done here and [non-additive] [unique] features of the ternary complex were deduced from the fact that several residues produced a net value significantly different from zero. Consequently even in the

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30

case of activation, where a ternary complex must form, evidence for more mechanism for allosteric behavior more complex than that predicted by a 2-state model was obtained. Most structural studies of K-type allosteric inhibition using NMR have focused on differences in the structure of the protein introduced by the binding of an allosteric inhibitor compared to a form considered to be active (3, 4, 14-18). A particular strength of NMR in these studies is that it can reveal changes in dynamics as well as static conformations introduced by the allosteric ligand binding. In the case of an allosteric K-type inhibitor, however, for these changes to explain the reason for inhibition one must presume the nature of the binding interaction of substrate while the inhibitor remains bound (and vice versa). It is important to appreciate that an allosteric K-type inhibitor may not (and certainly in the case of BsPFK does not) function as a pure competitive inhibitor – which by definition totally prevents the binding of substrate unless the inhibitor first dissociates. In the case of the allosteric inhibition by PEP of BsPFK both PEP and substrate can bind simultaneously at high concentrations of both ligands. The resulting ternary complex exhibits an enhanced dissociation proclivity for both inhibitor and substrate when compared to either single-ligated species. The ternary complex is therefore neither the 'inhibited' form nor the 'active' form, but rather a unique form that uniquely manifests the mutual antagonism between the bound ligands that underlies the basis for the inhibition. When a single ligand binds, the resulting protein component of the binary complex usually differs in various respects from the apo-protein as a consequence of the free energy of binding that has been introduced into the system. It is not surprising when these perturbations are localized in the vicinity of the now-occupied binding site. In the case of allosteric proteins in particular, these perturbations can also extend to regions far removed from the active site. The same is true for the second ligand (or substrate) binding to a spatially distinct (i.e. allosteric) site. If a residue perturbed by the binding of the first ligand is in a region unaffected when the second

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residue binds, one would expect that that region would retain the perturbations associated with the first ligand when the ternary complex is formed. Several residues examined in BsPFK behave in this manner. For example, the proton chemical shift change exhibited by the ternary complex is comparable to that of the binary PEPbound complex for residues Ile-67 and Ile-150, while the proton chemical shift change associated with the ternary complex is virtually identical to that of the Fru-6-P binary complex for Ile-147. Similar patterns can be seen in the 13-C chemical shifts of residues (eg. Ile-286). While these changes might be interpreted as identifying residues sensitive to unique attributes of 2 hypothetical allosteric states, we prefer an alternative interpretation that expands on our view of the conformational changes introduced by the ligands of this enzyme in a way that is complementary to the analysis above. We feel that the real source of the energetic impact of the binding of one ligand on the binding of the other arises from the regions, suggested by the non-additive chemical shift changes, where the structural, and hence energetic, conflict occurs. Schematically we have represented this idea in Figure 7B, where we contrast it with the more conventional model in Figure 7A. Although this idea has been discussed previously (36), we believe the analysis we have performed points to where the regions of potential conflict are in BsPFK, as well as those that are dominated by the binding of one ligand or another. It is of future interest to use the methyl probes and the power of NMR spectroscopy to further explore the dynamics and more fully characterize all of the regions and interactions of the enzyme that contribute to the entropy component of the coupling free energy. Changes in the line width and intensities are seen between the ligation states for several of the isoleucine residues in our study, indicating that dynamics may be present on the ms-µs time scale. Relaxation dispersion experiments need to be performed to further probe these internal dynamics involved

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in propagating the allosteric signal between binding sites in BsPFK. In addition, experiments probing side chain dynamics on a faster time scale may also provide additional information on how the allosteric effect is propagated in BsPFK.

Figure 7. Alternative models illustrating contrasting expectations regarding the structural influence of binding of either the substrate, Fru-6-P, or the allosteric inhibitor, PEP, to BsPFK. The regions where the structure is predominantly influenced by Fru-6-P are shown in blue, and those predominantly influenced by PEP are shown in red. A) Result anticipated by a typical 2-state model where subsequent to ligand binding a protein would adopt either an active (blue) or inhibited (red) configuration. B) A model that accommodates regions dominated by either Fru-6-P or PEP to varying extents across the protein with a region in which the structural perturbations of both ligands cannot be accommodated when bound simultaneously depicted in purple.

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Accession Code BsPFK A7ZUC9

Acknowledgement We thank Ryland Young (Texas A&M University, College Station, TX) for the generous gift of RY12459 cells. This work was supported by National Institutes of Health Grant GM033216, Robert A. Welch grant A1543, and funds from Texas A&M AgriLife Research.

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Propagation of the Allosteric Signal in Phosphofructokinase from Bacillus stearothermophilus Examined by Methyl-TROSY NMR Amy M. Whitaker, Mandar T. Naik, Rockann E. Mosser, and Gregory D. Reinhart

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