Antidiabetic Disruptors of the GlucokinaseâGlucokinase Regulatory

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Antidiabetic disruptors of the glucokinase-glucokinase regulatory protein complex reorganize a coulombic interface Juliana Martinez, Qing Xiao, Armen Zakarian, and Brian G. Miller Biochemistry, Just Accepted Manuscript • Publication Date (Web): 18 May 2017 Downloaded from http://pubs.acs.org on May 19, 2017

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Biochemistry

Antidiabetic disruptors of the glucokinase-glucokinase regulatory protein complex reorganize a coulombic interface

Juliana A. Martinez1, Qing Xiao2, Armen Zakarian2 and Brian G. Miller1*

1

Department of Chemistry and Biochemistry, Florida State University, Tallahassee, Florida,

USA 2

Department of Chemistry and Biochemistry, University of California, Santa Barbara, Santa

Barbara CA 93106, USA

*Corresponding author: 4005 Chemical Sciences Lab, Department of Chemistry and Biochemistry, Florida State University, Tallahassee FL 32303; [email protected]; 850-645-6570.

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ABSTRACT The glucokinase regulatory protein (GKRP) plays an essential role in glucose homeostasis by acting as a competitive inhibitor of glucokinase (GCK) and triggering its localization to the hepatocyte nucleus upon glucose deprivation. Metabolites such as fructose 6-phosphate and sorbitol 6-phosphate promote assembly of the GCK-GKRP complex, whereas fructose 1phosphate and functionalized piperazines with potent in vivo antidiabetic activity disrupt the complex. Here, we establish the molecular basis by which these natural and synthetic ligands modulate the GCK-GKRP interaction. We demonstrate that a small-molecule disruptor of the protein-protein interaction utilizes a two-step conformational selection mechanism to associate with a rare GKRP conformation constituting 3% of the total population. Conformational heterogeneity of GKRP is localized to the N-terminus and deleting this region eliminates the ability of sorbitol 6-phosphate to promote the GCK-GKRP interaction. Stabilizing ligands favor an extended N-terminus, which sterically positions two arginine residues for optimal coulombic interaction with a pair of carboxylate side chains from GCK. Conversely, disruptors promote a more compact N-terminus in which an interfacial arginine residue is stabilized in an unproductive orientation through a cation-π interaction with tyrosine 75. Eliminating the ability to sample this binding impaired conformation enhances the intrinsic inhibitory activity of GKRP. Elucidating the molecular basis of ligand-mediated control over the GCK-GKRP interaction is expected to impact the development and future refinement of therapeutic agents for diabetes and cardiovascular disease, which result from improper GKRP regulation of GCK.


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INTRODUCTION Glucokinase (GCK) is a central metabolic enzyme that participates in glucose homeostatic maintenance by governing the rates of glucose catabolism in pancreatic β-cells and glycogen storage in hepatocytes.1–3 Dysfunction or misregulation of GCK causes a variety of disease states including maturity onset diabetes of the young type II and persistent hyperinsulinemia of infancy.4–8 In hepatocytes GCK is regulated by direct association with the glucokinase regulatory protein (GKRP), a 68 kDa polypeptide that functions as a competitive inhibitor of glucose binding to GCK.9–13 Under low glucose conditions GKRP forms an inhibitory complex with GCK and sequesters the enzyme within the nucleus. When glucose concentrations rise, GKRP releases GCK into the cytosol where it can participate in metabolism.9 The affinity of GKRP toward GCK is regulated by natural phosphorylated carbohydrates that bind to a site on GKRP distant from the protein-protein interface. Fructose 6-phosphate and sorbitol 6-phosphate stimulate formation of the inhibitory complex, whereas fructose 1-phosphate reduces the affinity of GKRP for GCK.12, 14–16 The mechanistic origin of these ligand-mediated effects on complex stability remain unresolved. The importance of the GCK-GKRP complex in hepatic glucose homeostasis, combined with the observation that mutations in GKRP are associated with an increased risk of cardiovascular disease,16, 17 suggests the GCK-GKRP complex as a putative target for therapeutic design. In December 2013, Amgen reported the discovery of a novel class of functionalized piperazines that disrupt the GCK-GKRP interaction (Figure 1A).18 In vitro and in vivo studies demonstrate that these molecules stimulate GCK activity by interfering with GKRP-mediated regulation, thereby reducing blood glucose levels in diabetic model organisms.18,

19

Crystal

structures reveal that GCK-GKRP disruptors bind to a site in GKRP that is composed of residues


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from three distinct structural regions: the N-terminus, the cap domain and the sugar isomerase I domain (Figure 1B).18 Interestingly, the binding site for functionalized piperazines is more than 30Å away from the GCK interaction interface and resides near, but does not overlap with, the binding site of phosphorylated carbohydrates. To date, co-crystal structures of GKRP in complex with more than a dozen structurally distinct piperazine derivatives have been determined. Despite this wealth of structural data, the mechanism by which these putative therapeutic agents act to disrupt the inhibitory complex with GCK remains unclear. In this study, we elucidate the structural and mechanistic basis of ligand-mediated regulation of the GCK-GKRP interaction.

Figure 1. (A) Structure of AMG-3969. (B) AMG-3969 (yellow) binds to GKRP at the confluence of the Nterminus (red), the cap domain (dark grey) and the sugar isomerase domains (light grey). (C) Expanded view of the AMG-6939 binding site, demonstrating the proximity of Trp517 and the formation of a putative cation-π interaction between Trp19 and Arg525 in the presence of AMG-3969.


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MATERIALS AND METHODS Synthesis of AMG-3969 Details of the synthesis, characterization and purification of AMG-3969 are described in supporting information.

Protein expression and purification Recombinant human GCK and variants bearing N-terminal hexahistidine, were produced and purified using previously described methods.13,

20

Recombinant full length, and N-terminally

truncated variants of rat GKRP were produced as C-terminal hexahistidine tagged proteins from pET-22b(+) in BL21(DE3) cells. An auto-induction minimal media expression protocol was developed in which a Luria-Bertani (LB) agar plate supplemented with ampicillin (150 µg/mL) was streaked from a frozen stock of cells. A single colony from the plate was inoculated in 50mL of LB broth supplemented with ampicillin (150 µg/mL), which was incubated overnight at 37 ºC. Five hundred milliliters of auto induction minimal media was inoculated to an initial OD600 of 0.02 and this culture was grown at 37 ºC. The auto induction minimal media contained M9 salts (Na2HPO4 (30 g/L), KH2PO4 (15 g/L), NaCl (2.5 g/L), pH 8.0), ampicillin (150 µg/mL), NH4Cl (2.8 mg/mL), glycerol (0.5 %), glucose (0.05%), lactose (0.2 %), thiamine (0.025 mg/mL), MgSO4 (1 mM), FeSO4 (0.01 mg/mL), Metal Mix (FeCl3 (16.2 mg/L), ZnCl2 (1.44 mg/L), CoCl2 (1.2 mg/L), Na2MoO4 (1.2 mg/L), CaCl2 (0.6 g/L), CuSO4 (190 mg/L), H3CO3 (5 mg/L) and 0.37 mL of HCl) and an amino acid mixture (200 µg/mL of each essential amino acid except tyrosine and cysteine). When the OD600 reached 2.0 the temperature was reduced to 16 ºC to induced gene expression, and growth was continued for 40 hours. Cells were harvested by centrifugation at 8,000g, yielding 10 g of wet cell pellet. Cell pellets were resuspended in GKRP load buffer


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containing Hepes (50 mM, pH 7.1), imidazole (25 mM), dithiothreitol (1 mM), and glycerol (10%, w/v) at a ratio of 5 mL per gram of cell pellet. Cells were lysed using french press and subjected to centrifugation at 25,000g and 4 ºC for 30 min. The supernatant was immediately loaded onto a 5 mL HisTrap fast flow affinity column (GE Healthcare) previously equilibrated in GKRP load buffer. The column was then washed with 20 columns volumes of GKRP load buffer to remove non-specifically bound proteins. GKRP was eluted with GKRP load buffer containing 250 mM imidazole, and the protein was dialyzed at 4 ºC against 1 L of GKRP SEC buffer containing Hepes (50 mM, pH 7.1), KCl (25 mM), and dithiothreitol (1 mM). Following dialysis GKRP was concentrated and injected onto a Superdex™ 200 column (GE Healthcare) pre-equilibrated in GKRP SEC buffer. The gel filtration column was run at a flow rate of 0.12 mL/min, and fractions containing the highest A280 nm readings were pooled and retained for analysis.

Steady-state kinetics analysis of GCK inhibition by GKRP For steady state analysis, GCK was used at a final concentration of 20-40 nM such that the uninhibited rate of glucose conversion corresponded to ~30 nanomoles of G6P produced per minute. GCK was mixed with GKRP at various concentrations (0-271 µM) and the proteins were incubated in GKRP SEC buffer containing 10 mM dithiothreitol for five minutes at 25ºC to allow binding. High concentrations of GKRP were necessary only when assaying the most inactive/insensitive variants. When assays were conducted in the presence of any GKRP ligand, the ligand was added at this stage. These mixtures were then transferred to a cuvette containing Hepes (250 mM, pH 7.1), KCl (25 mM), NADP+ (0.5 mM), dithiothreitol (10 mM), G6PDH (10 units), MgCl2 (6 mM) and glucose at the S0.5 value (5 mM) and the samples were incubated for


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an additional 3 minutes prior to initiating the GCK reaction with ATP (5 mM). This procedure allows GCK activity to be measured spectrophotometrically at 340 nm by coupling the production of glucose 6-phosphate to the reduction of NADP+ via glucose 6-phosphate dehydrogenase (G6PDH). When mixed with GKRP, GCK is inhibited, and its rate of glucose conversion to glucose 6-phosphate is decreased. The rate of glucose conversion was plotted as a function of GKRP concentration and the IC50 values were calculated using SigmaPlot 10.0. When indicated, sorbitol 6-phosphate was added to a final, saturating concentration of 0.5 mM.

Transient-state kinetic assays Equilibrium investigations of the spectroscopic consequences of AMG-3969 association with GKRP were performed by monitoring the change in GKRP fluorescence (1 µM) at 340 nm upon addition of AMG3969 following excitation at 280 nm. Rat GKRP contains 6 tryptophan residues that are evenly distributed througout the tertiary structure of the protein.13 Data were collected at 25˚C on a Cary Eclipse spectrofluorometer using a 10 nm slit width. An Applied Photophysics SX-20 stopped-flow spectrometer was used to measure the association of AMG-3969 with GKRP at 25 ºC, by monitoring the decrease in intrinsic protein fluorescence upon ligand binding. GKRP was excited at 280 nm, and an emission wavelength filter of 320 nm was used. The monochromator slit width was set at 0.26 nm and the mixing pressure was 60 psi. GKRP at 4 µM was mixed at a 1:1 ratio with concentrations of AMG-3969 ranging from 0.2 to 2.0 µM. Solutions of protein and AMG-3969 were prepared in a buffer containing sodium phosphate (50 mM, pH 7.1), dithiothreitol (1 mM) and DMSO 3% (v/v). A minimum of five individual binding traces were collected on a logarithmic scale until equilibrium was reached (0.005-20 s) for each AMG-3969 concentration. Binding traces were analyzed and fit to a sum of exponentials


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equation using the software provided by the manufacturer as previously described,21, 22 to yield rate constants and amplitudes for each AMG-3969 concentration. When present, the concentration of fructose 6-phosphate was 2 mM. AMG-3969 displays an absorbance value of less than 0.005 at 280 nm at the highest concentration employed in this study. AMG-3969 also displays a slight fluorescence signal, which did not contribute significantly to the overall change in fluorescence observed upon mixing with GKRP across the range of concentrations used in this study.

RESULTS AND DISCUSSION GCK-GKRP Disruptors Utilize a Conformational Selection Binding Mechanism To investigate the mechanism of action of GCK-GKRP antagonists, we sought to establish the quantitative binding mechanism for AMG-3969, a potent disruptor that has served as a lead agent for therapeutic refinement (Figure 1A). Based on a previously developed method,18, 19, 23 we produced 60 mg of racemic AMG-3969 in 10 steps from ethyl chloroacetate with an overall yield of 7%. Chiral HPLC was used to separate the active S-enantiomer from the inactive R-enantiomer. In vitro assays with purified recombinant human GCK and rat GKRP confirmed the effectiveness of AMG-3969 in disrupting the GCK-GKRP complex and restoring the enzymatic activity of GCK. Throughout our mechanistic studies we used rat GKRP, which is 89% identical to human GKRP. The binding site for AMG-3969 is conserved in both proteins and rat GKRP can be produced in large amounts from bacteria, unlike human GKRP. A highresolution crystal structure of the complex formed between rat GKRP and human GCK, determined in the presence of fructose 6-phosphate, is also available.13 Analysis of the crystal structure of human GKRP in complex with AMG-3969 suggested


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that changes in intrinsic protein fluorescence might provide a useful method to monitor ligand binding. Two tryptophan residues, Trp19 and Trp517, are located within close proximity of the AMG-3969 binding site (Figure 1C). In particular, Trp19 forms a cation-π interaction with Arg525, the side chain of which participates in a hydrogen bond with the hydroxyl group of AMG-3969. Based on these observations, we postulated that association of AMG-3969 with GKRP might cause an observable change in intrinsic protein fluorescence. To test this hypothesis, we examined the fluorescence spectrum of GKRP in the presence and absence of AMG-3969. Equilibrium binding experiments demonstrate that association of AMG-3969 with GKRP causes a substantial decrease in intrinsic protein fluorescence (Figure 2A). A

200

GKRP

Fluorescence (A.U.)

Figure 2. Binding studies of AMG-3969 and GKRP. (A) Saturating concentrations of AMG-3969 produce a decrease in intrinsic GKRP fluorescence. (B) Transient state spectrofluorometric traces of GKRP (4 µM) upon

150

GKRP + AMG-3969

100

50

0 3 00

320

340

36 0

380

Wavelength (nm)

B

2

Normalized Fluorescence (A.U.)

addition of AMG-6939 at 0.2 µM (black), 0.4 µM (red), 0.6 µM (blue), 0.8 µM (purple) 1.0 µM (green), 2.0 µM (grey). (C) AMG-3969 binding curve (2.0 µM) fit to an analytical solution comprised of a single exponential term. The experimental data is shown in

1

+ 0.2 µM AMG-3969 0

-1

-2

-3

+ 2.0 µM AMG-3969

-4 0

inset.

C

10

15

20

2 Residuals

red and the fit is black, with the residuals shown in the

5

Time (s) Normalized Fluorescence (A.U.)

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1

1 .0 0 .0 -1 .0 0

5

0

10

15

20

Time (s)

-1

-2

Experimental Data Fit

-3

-4 0


5

10

Time (s)

15

20

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We used the time-dependent change in GKRP fluorescence to monitor the kinetics of ligand binding via stopped-flow spectrofluorometry. Transient state binding experiments were performed following 1:1 mixing of GKRP (4 µM final concentration) with a range of AMG3969 concentrations (0.2–2.0 µM final concentrations) (Figure 2B). Fluorescence traces were collected over a time window of 0.005–20 seconds, at which point equilibrium was reached. The resulting binding curves were averaged, normalized and fitted to a sum of exponentials equation to determine how many kinetically distinguishable events were observable during the AMG3969 binding process. An analytical solution comprised of a single exponential function provided a good fit for binding curves across the entire range of AMG-3969 concentrations tested. These experiments yielded both the rate of approach to equilibrium, kobs, and the amplitude of the fluorescence change at each ligand concentration (Figure 2C). The rapid equilibrium approximation considers ligand binding and dissociation processes to be more rapid than protein conformational changes. Under these conditions, the dependence of kobs upon ligand concentration can be used to establish the nature of the binding mechanism.24, 25 Our transient state experiments reveal that the kobs value for AMG-3969 association with GKRP decreases with increasing ligand concentration (Figure 3A, B). This behavior supports a two-step, conformational selection binding mechanism (Figure 3C).25 A direct one-step binding mechanism, as well as a two-step induced fit process in which ligand binding precedes protein isomerization, are not supported by our data as both require the value of kobs to increase with increasing ligand concentration.


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Figure 3. Value of kobs as a function of AMG-3969

A

1.4 1.0

Amplitude

1.2

concentration determined in the absence (A) or 1.0

0.8 0.6 0.4

kobs

0.2

presence (B) of saturating concentrations of fructose 6-

0.0

0.8

0

0.5

1.0

1.5

2.0

[AMG-3969] (µM)

0.6

phosphate. The magnitude of the amplitude for each

0.4

transient as a function of AMG-3969 concentration is

0.2 0

0.5

1.0

1.5

2.0

[AMG-3969] (µM)

shown in the inset. (C) Two-step conformational

B

1.6 1 .0

binding

mechanism

for

AMG-3969

Amplitude

selection

1.4 1.2

0 .8 0 .6 0 .4 0 .2

association with GKRP.

kobs

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Biochemistry

1.0

0 .0 0

0.5

1.0

1.5

2.0

[AMG-3969] (µM)

0.8 0.6 0.4 0.2 0

0.5

1.0

1.5

2.0

[AMG-3969] (µM)

C k1 GKRP

k-1

k2 GKRP* + AMG3969

k-2

GKRP* AMG3969

Fitting the plot of kobs versus AMG-3969 concentration to an inverse hyperbolic function provides values for the microscopic rate constants describing the forward (k1) and the reverse (k1)

conformational change, as well as the ratio between the forward (k2) and reverse (k-2) rate

constants for binding (Table 1). Our experimental data yield values of 0.10 s-1 for k1 and 3.4 s-1 for k-1, and the ratio of these values provides the equilibrium constant for the conformational change (K1). The value of K1 is 0.03, indicating that AMG-3969 associates with a conformation of GKRP that constitutes 3% of the total solution population. Thus, AMG-3969 recognizes a rare form of the protein, denoted here as GKRP*, and produces a shift in the conformational distribution upon binding. Our data establish a value of 9.9 x 106 for k2/k-2, the reciprocal of


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which, 1.0 x 10-7 M, yields the intrinsic dissociation constant of AMG-3969 from the rare GKRP* conformation (Table 1). Attempts to resolve individual values for k2 and k-2 via global fitting analysis were unsuccessful.26 Nevertheless, an apparent dissociation constant for AMG3969 association with GKRP can be calculated from our experimentally determined microscopic rate constants using the relationship Kd,

app

= k1k-2 /k-1k2. The value of Kd,

app

determined in this

manner is 3.0 x 10-9 M, which is consistent with the experimentally observed dissociation constant of a structurally related functionalized piperazine determined by surface plasmon resonance.18

Table 1. Microscopic rate constants describing the association of AMG-3969 with GKRP in the absence and presence of fructose 6-phosphate.

- fructose 6-phosphate

+ fructose 6-phosphate

k1 = 0.10 ± 0.03 s-1

k1 = 0.16 ± 0.03 s-1

k-1 = 3.4 ± 0.55 s-1

k-1 = 2.3 ± 0.13 s-1

K1 = 0.03 ± 0.01

K1 = 0.07 ± 0.01

k2/k-2 = (9.9 ± 2.6) x 106 M-1

k2/k-2 = (4.3 ± 0.6) x 106 M-1

Kd, intrinsic = (1.0 ± 0.26) x 10-7 M

Kd, intrinsic = (2.3 ± 0.32) x 10-7 M

Kd, apparent = (3.0 ± 1.3) x 10-9 M

Kd, apparent = (1.6 ± 0.39) x 10-8 M

Our transient state binding studies detect only one kinetically observable event during the AMG-3969 binding process, however the conformational selection mechanism is a two-step process. Several possibilities could explain our inability to detect both steps. First, the binding


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event between AMG-3969 and GKRP* may be too rapid for detection via the stopped-flow instrument. Second, the association of AMG-3969 and GKRP* may be undetectable because it does not cause a measurable change in the fluorescence signal of the rare protein conformation. To probe these possibilities, we examined the dependence of the initial fluorescence signal of each binding curve as a function of AMG-3969 concentration. If association of AMG-3969 with GKRP* causes a change in intrinsic fluorescence, but is simply too rapid to be detected by stopped-flow, one would expect to observe a ligand-dependent change in the initial fluorescence value. We observed no such change across the range of AMG-3969 concentrations investigated herein (Figure S1). Based on this observation, we conclude that the observed fluorescence change results from the conformational isomerization step, rather than formation of the proteinligand complex. This conclusion does not preclude the possibility that formation of the complex between AMG-3969 and GKRP* is also very rapid on the stopped-flow timescale. Although we did not directly detect the initial encounter between AMG-3969 and GKRP*, our experimentally determined value for k2/k-2 is of sufficient magnitude to support the rapid equilibrium approximation. Association with AMG-3969 impairs the ability of GKRP to interact with GCK, however other ligands act in an opposite manner to promote complex formation. For example, the affinity of rat GKRP for human GCK increases 20-fold in the presence of fructose 6-phosphate.15 To investigate the extent to which fructose 6-phosphate might impact the mechanism and/or kinetics of AMG-3969 association, we performed transient state binding experiments in the presence of saturating concentrations of this molecule. The presence of fructose 6-phosphate does not change the dependence of kobs upon AMG-3969 concentration, indicating a conservation of the conformational selection binding mechanism (Figure 3B). However, our data indicate that prior


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association of fructose 6-phosphate alters the conformational ensemble of GKRP in a manner that facilitates recognition by AMG-3969, although with a slightly decreased affinity. The calculated apparent dissociation constant of AMG-3969 determined in the presence of F6P (1.6 x 10-8 M) is 5-fold higher than in its absence. These findings are consistent with a previous report indicating that functionalized piperazines bind to GKRP irrespective of the presence of phosphorylated sugars.18 The magnitudes of k1 and k-1 are slightly altered by fructose 6phosphate, with the value of k1 increasing to 0.16 s-1 and the value of k-1 decreasing to 2.3 s-1 (Table 1). As a result, the concentration of the binding competent species increases from 3% to 7% in the presence of fructose 6-phosphate.

Ligand-Mediated Regulation of GKRP Activity Involves Restructuring of the N-terminus The observation that AMG-3969 binding follows a conformational selection mechanism indicates that GKRP is capable of adopting at least two distinct conformations in solution. To understand the nature of this structural heterogeneity, we examined the AMG-3969 binding site in the currently available crystal structures of GKRP.18, 19, 27–29 These structures demonstrate that the N-terminus of GKRP, which comprises part of the AMG-3969 binding site, can adopt two distinct conformations (Figure 4A). When the GCK-GKRP-fructose 6-phosphate ternary complex is assembled the N-terminus adopts an extended structure in which two arginine residues of GKRP, Arg297 and Arg301, are sterically positioned for favorable coulombic interactions with Asp247 and Glu245 of GCK.13 Conversely, crystal structures of GKRP determined in the presence of ligands that disrupt complex formation, such as AMG-3969 or fructose 1-phosphate, show the N-terminus distant from the arginine pair (Figure 4B).12, 18 In this binding impaired conformation, Arg297 appears to be stabilized in an unproductive orientation


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Biochemistry

by an intermolecular hydrogen bond with Glu300 and a cation-π interaction with Tyr75. These observations provide a putative model for ligand-mediated control of GCK-GKRP that involves reorganization of interfacial coulombic residues by structural alterations at the N-terminus of GKRP.

Figure 4. Structural model for ligand-mediated control of the GCK-GKRP complex. (A) Stabilizing ligands such as fructose 6-phosphate (F6P) or sorbitol 6-phosphate (S6P) promote an extended GKRP N-terminus (red), whereas destabilizing ligands such as AMG-3969 or fructose 1-phosphate (F1P) promote a compact N-terminus. (B) The extended N-terminus sterically positions R297 and R301 for favorable coulombic interactions with E245 from GCK (yellow). (C) The compact N-terminus allows interfacial arginine residues to adopt a non-optimal conformation that is stabilized by a putative hydrogen bond between R297 and E300 and a cation-π interaction between R297 and Y75.


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To test this structural model, we constructed several variants to examine the impact of removing specific interactions upon the strength of the protein-protein interaction. Our investigation began by evaluating the importance of coulombic interactions between GKRP guanidino groups from Arg297 and Arg301 with GCK carboxylates from Asp247 and Glu245. Notably, these putative contacts are the only coulombic interactions found within the 2060 Å2 of buried surface area in the mammalian GCK-GKRP crystal structure (Figure 4B). Replacement of GKRP Arg297 with alanine resulted in a modest 2.3-fold decrease of the IC50 value compared with wild-type GKRP. The ability of sorbitol 6-phosphate to enhance the inhibitory action of this variant was also reduced (Table 2). Sorbitol 6-phosphate produced a 7-fold enhancement in the IC50 value of the R297A variant, compared to the 25-fold enhancement in inhibitory activity elicited by this ligand upon association with wild-type GKRP. Removing Arg301 resulted in a 12-fold decrease in the inhibitory activity of GKRP in the absence of sorbitol 6-phosphate (Table 2). Interestingly, the inhibitory activity of the R301A variant was insensitive to enhancement by sorbitol 6-phosphate, demonstrating the importance of this residue in communicating the occupancy of the carbohydrate binding site on GKRP to the protein-protein interaction interface. Replacing the Glu245 side chain of GCK, which is located within hydrogen bonding distance of both Arg297 and Arg301, causes a 14-fold reduction in susceptibility to GKRP inhibition. A smaller 3-fold decrease in GKRP inhibitory activity was observed when GCK Asp247 was replaced with alanine. The inhibitory activity of wild-type GKRP toward both E245A and D247A GCK was enhanced by sorbitol 6-phosphate, albeit to a slightly lesser degree than observed with wild-type GCK. Together these results confirm the importance of coloumbic interactions between GKRP and GCK in dictating the strength of the protein-protein interaction,


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an observation than is consistent with previous reports indicating that inhibition of GCK by GKRP is highly sensitive to ionic strength.10, 11 Our data indicate that Arg301 of GKRP and E245 of GCK play the largest role in dictating affinity. These results also clearly demonstrate that interactions mediated by Arg301 are essential to the enhancement in inhibitory action produced by sorbitol 6-phosphate.

Table 2. Mutational analysis of residues involved in mediating the GCK-GKRP interaction.

Protein

Apparent IC50 - S6P

Apparent IC50 + S6P

WT GKRP

4.9 ± 0.1 µM

0.20 ± 0.05 µM

R297A GKRP

11.1 ± 0.1 µM

1.5 ± 0.1 µM

R301A GKRP

59.6 ± 0.1 µM

52.2 ± 0.1 µM

E245A GCK

70.5 ± 0.2 µM

3.9 ± 0.1 µM

D247A GCK

14.9 ± 0.2 µM

1.1 ± 0.02 µM

∆1-20 GKRP

3.8 ± 0.1 µM

5.5 ± 0.1 µM

Y75A GKRP

1.2 ± 0.1 µM

0.20 ± 0.06 µM

E300A GKRP

5.6 ± 0.1 µM

0.30 ± 0.09 µM

To examine the role of the GKRP N-terminus in modulating the strength of the proteinprotein interaction, we constructed a variant in which the first 20 amino acids were deleted. In the absence of ligand, the truncated variant inhibited the catalytic activity of GCK to the same extent as full-length GKRP, with respective IC50 values of 3.8 µM and 4.9 µM. This result


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demonstrates that the N-terminus itself is not required for complex formation (Table 2). Similar to the R301A GKRP variant, removal of the N-terminus completely eliminated the ability of phosphorylated sugars to modulate affinity of GKRP toward GCK. For wild-type GKRP, saturating concentrations of sorbitol 6-phosphate reduces the IC50 value from 4.9 µM to 0.2 µM, whereas the IC50 values of the ∆1-20 variant in the absence and presence of sorbitol 6-phosphate are 3.8 µM and 5.5 µM, respectively. Importantly, N-terminal truncation does not impact the affinity of GKRP for sorbitol-6-phosphate.18 The impact of N-terminal truncation on the effectiveness of AMG-3969 could not be evaluated since residues in this region comprise part of the binding site for synthetic disruptors. Collectively, our data are consistent with a model in which the N-terminus of GKRP is responsible for communicating occupancy of the distant ligand binding sites of GKRP to the GCK interface by positioning Arg297 and Arg301 for optimal interaction with GCK. Our structural model also implies that the binding impaired conformation of GKRP is stabilized by intramolecular interactions between Arg297 and Tyr75 and/or Glu300 (Figure 4C). Accordingly, removing these interactions is expected to increase the inhibitory activity of GKRP and strengthen the GCK-GKRP complex. To test this hypothesis, we replaced both Tyr75 and Glu300 with alanine. Neither of these residues is located near the GCK interface nor are they contained within the binding sites for small-molecule effectors. In the absence of a GKRP ligand, the E300A variant inhibited GCK to the same extent as wild-type; however the Y75A variant showed a 4-fold improvement in inhibitory activity in the absence of any stabilizing carbohydrate ligand. In the presence of sorbitol 6-phosphate, both the Y75A and E300A variants inhibited wild-type GCK with an IC50 value that is comparable to wild-type GKRP. These results are consistent with our structural model whereby Tyr75 uses a cation-π interaction with Arg297


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to stabilize a GKRP conformation in which the interfacial arginine residues are positioned in a non-optimal orientation for interaction with GCK.

CONCLUSION The present findings provide a unified mechanism for understanding how natural and synthetic small-molecules modulate the GCK-GKRP complex (Figure 4). Our transient state kinetic data demonstrate that GKRP adopts at least two structurally distinct states and our combined structural/mutational analysis suggest that the N-terminus is the major site of ligandmediated reorganization. We postulate that antidiabetic disruptors such as AMG-3969 exclusively target the rare, binding impaired GKRP* conformation, which is characterized by a non-optimal positioning of Arg297 and Arg301 (Figure 4C). In contrast, promoting ligands, such as sorbitol 6-phosphate or fructose 6-phosphate, favor an extended N-terminus that sterically reinforces the coulombic interaction at the GCK-GKRP interface (Figure 4B). Differential ligand stabilization of the GKRP or GKRP* conformers dictates whether a given effector molecule will either promote or antagonize GCK association. Moreover, antidiabetic small-molecule disruptors will only be effective if they display an affinity for GKRP* that exceeds the product of K1 and the intrinsic affinity of GCK for the dominant GKRP conformation. Our results also demonstrate that the presence of stabilizing ligands such as fructose 6phosphate does not substantially alter the binding mechanism of AMG-3969, despite the fact that these two compounds have opposite effects upon the stability of the GCK-GKRP complex. Nevertheless, we find that fructose 6-phosphate has a small, deleterious impact upon the affinity of AMG-3969 for GKRP. This finding suggests that the efficacy this drug might be affected by fructose 6-phosphate or other, yet to be identified, sugars that may play a physiological role in


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stabilizing the GCK-GKRP complex. Such considerations are important for future efforts to refine GCK-GKRP disruptors as therapeutic agents to treat diseases including type 2 diabetes.

Supporting Information Available: Details of the synthesis, characterization and purification of AMG-3969. IC50 activity profiles for GCK and GKRP variants.

Acknowledgements The authors acknowledge K. Johnson for assistance in global fitting analysis and C. Mundoma of the Protein Biophysics Laboratory in the Institute for Molecular Biophysics at FSU for assistance with stopped-flow experiments.

Funding Sources This work was supported by grants from the National Institutes of Health (DK081358 and GM115388 to BGM and GM077379 to AZ) and a Developing Scholar Award from Florida State University to BGM.

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Antidiabetic Disruptors of the GlucokinaseâGlucokinase Regulatory

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