Substrate-Induced Facilitated Dissociation of the Competitive Inhibitor

Article pubs.acs.org/biochemistry

Substrate-Induced Facilitated Dissociation of the Competitive Inhibitor from the Active Site of O‑Acetyl Serine Sulfhydrylase Reveals a Competitive-Allostery Mechanism Appu Kumar Singh, Mary Krishna Ekka, Abhishek Kaushik, Vaibhav Pandya, Ravi P. Singh, Shrijita Banerjee, Monica Mittal, Vijay Singh, and S. Kumaran* G. N. Ramachandran Protein Center, Institute of Microbial Technology (IMTECH), Council of Scientific and Industrial Research (CSIR), Sector 39-A, Chandigarh, India 160036 S Supporting Information *

ABSTRACT: By classical competitive antagonism, a substrate and competitive inhibitor must bind mutually exclusively to the active site. The competitive inhibition of O-acetyl serine sulfhydrylase (OASS) by the C-terminus of serine acetyltransferase (SAT) presents a paradox, because the C-terminus of SAT binds to the active site of OASS with an affinity that is 4−6 log-fold (104−106) greater than that of the substrate. Therefore, we employed multiple approaches to understand how the substrate gains access to the OASS active site under physiological conditions. Single-molecule and ensemble approaches showed that the active site-bound high-affinity competitive inhibitor is actively dissociated by the substrate, which is not consistent with classical views of competitive antagonism. We employed fast-flow kinetic approaches to demonstrate that substrate-mediated dissociation of full length SAT−OASS (cysteine regulatory complex) follows a noncanonical “facilitated dissociation” mechanism. To understand the mechanism by which the substrate induces inhibitor dissociation, we resolved the crystal structures of enzyme·inhibitor·substrate ternary complexes. Crystal structures reveal a competitive allosteric binding mechanism in which the substrate intrudes into the inhibitor-bound active site and disengages the inhibitor before occupying the site vacated by the inhibitor. In summary, here we reveal a new type of competitive allosteric binding mechanism by which one of the competitive antagonists facilitates the dissociation of the other. Together, our results indicate that “competitive allostery” is the general feature of noncanonical “facilitated/accelerated dissociation” mechanisms. Further understanding of the mechanistic framework of “competitive allosteric” mechanism may allow us to design a new family of “competitive allosteric drugs/small molecules” that will have improved selectivity and specificity as compared to their competitive and allosteric counterparts.

C

ation of the TNF-α trimer unit and IgE−receptor complexes by small molecule or protein inhibitors cannot be adequately described by either classical or allosteric models.7,8 It has been proposed that competitive antagonists facilitate the dissociation of the bound ligand by competing for subsites within the targetbinding site.4,5 In principle, facilitated or accelerated dissociative models would promote the idea that both competing antagonists should co-localize at the same binding site briefly before one facilitates the dissociation of the other. However, direct structural evidence to support the existence of a “nonclassical” facilitated dissociation mechanism is lacking. In this study, we investigate the dissociation of the OASS−SAT complex, known as the cysteine regulatory complex (CRC), by O-acetylserine (OAS) and provide direct structural evidence that reveals a “nonclassical” competitive allosteric mechanism

ompetitive inhibitors bind to the active site of an enzyme and diminish the rate of catalysis by decreasing the amount of enzyme−substrate complex formed. It is widely accepted that in reversible competitive inhibition, the active site can bind either substrate or inhibitor, but not both at the same time. The rationale for this notion is that reversible competitive inhibition can be relieved by increasing the substrate concentration.1,2 The mutually exclusive binding property of the classical model is also strengthened by structural observations that competitive inhibition occurs by steric hindrance.3 Recently, accelerated or facilitated dissociation of natural ligand or binding partners by competing inhibitors has been observed for a few systems.4−6 In one case, a small molecule inhibitor was able to displace one subunit of the TNF-α trimer and bound to the dimer of TNF-α subunits.7 Kinetic studies showed that the small molecule inhibitor first binds to the TNF-α trimer, forming the intermediate complex before one monomer is completely dissociated. Recently, an engineered protein inhibitor has been shown to accelerate the disassembly of IgE−receptor complexes.8 Accelerated dissoci© XXXX American Chemical Society

Received: May 24, 2017 Revised: July 27, 2017 Published: August 14, 2017 A

DOI: 10.1021/acs.biochem.7b00500 Biochemistry XXXX, XXX, XXX−XXX

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Biochemistry

Figure 1. Schematic of the cysteine biosynthesis step and illustration of competitive antagonism between the substrate of OASS and the C-terminus of SAT. (A) In the first step, SAT catalyzes the synthesis of OAS. In the next step, OASS catalyzes the formation of cysteine from OAS and sulfur. As shown above the scheme in cartoon representation, the C-terminus of SAT binds to the active site of OASS with a high affinity. However, OAS competes with the C-terminus of SAT and dissociates the C-terminus of SAT from the active site of OASS. (B) Ribbon diagram of OASS (Protein Data Bank entry 4HO1) with secondary structural elements labeled. The N-terminal and C-terminal domains and active site are labeled. The movable domain that responds to only OAS (substrate) binding moves to close the active site upon OAS entry (shown within the circle). Arrows at the bottom indicate that the last 10 residues of SAT C-terminal peptides, here known as C10 peptide inhibitors, and the substrate of OASS (OAS) bind to the same active site. Sequences of two different C10 peptide inhibitors and chemical structure of OAS are shown at the bottom.

Table 1. Crystallographic and Refinement Parameters for Structures Binary-1 Protein Data Bank entry

Binary-2

4NU8

Ternary-1

5DBH

4ORE

1.5 I41 112.32, 45.80 2.03−35.62 (2.06−2.19) 0.043 (0.16) 53.3 (11.5) 90.80 (99.80) 5.7 (5.5) 17594 Refinement 0.18/0.23 2425 2275 63 81 38.0 48.5 38.0 35.0

1.5 I41 112.66, 43.36 1.98−32 (1.98−2.05) 0.03 (0.08) 56.5 (21.5) 99.4 (99.74) 4.8 (4.7) 19145

1.5 I41 112.22, 46.03 2.03−39.68 (2.2−2.27) 0.08 (0.23) 3.9 (2.3) 98 (100) 7.2 (6.7) 14742

0.15/0.20 2418 2298 27 93 25.1 25.0 22.0 27.0

0.22/0.27 2423 2247 61 115 19.0 19.0 54.5 30.0

0.010 1.10

0.008 1.07

0.007 1.15

97.00 0.32

98.00 0.00

95.50 0.32

Data Collection wavelength (Å) space group cell dimensions [a = b, c (Å)] resolution (Å) Rmerge I/σ(I) completeness (%) redundancy no. of reflections (unique) Rwork/Rfree no. of atoms protein ligand/ion water average B factor protein ligand/ion water root-mean-square deviation bond lengths (Å) bond angles (deg) Ramachandran statistics (%) favored outliers

reaction intermediate, α-amino acrylate, in which acetate group of OAS has been eliminated represent the first half-cycle of the reaction (Figure 1). The first half-cycle is accompanied by conformational changes that switch the active site from the open state to the closed state.12,13 It is assumed that release of cysteine reverses the conformational effect, switching the active site back to the open state and PLP again forms a Schiff base with active site lysine. In addition, two consecutive steps of cysteine biosynthesis are connected through a regulatory protein−protein interaction step. OASS interacts with SAT to form the cysteine regulatory complex (CRC) that dissociates when the concentration of OAS increases (Figure 1A).

by which a high-affinity competitive inhibitor is actively dissociated by a low-affinity substrate molecule. Cysteine biosynthesis in bacteria and plants occurs in two steps.9,10 In the first step, SAT synthesizes OAS, and in the second step, OASS catalyzes the formation of cysteine from OAS and sulfide. OASS is a pyridoxal 5′-phosphate (PLP)dependent enzyme that catalyzes the β-elimination of acetate from OAS, forming cysteine. Conversion of OAS into Lcysteine involves two distinct steps; in the first step, the acetate group from OAS is eliminated through a β-elimination reaction, and in the second step, sulfide is added to produce L-cysteine.11 The reaction of OAS with active site PLP and formation of the B


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Biochemistry

Sigma-Aldrich. Atto dyes were obtained from Jena Biosciences. Sulfo-NHS-Biotin Reagent was procured from Pierce Thermo Scientific, and chemically synthesized C10 peptides of SAT were obtained from GenScript Scotch. Protein Purification and Activity Assay. HiOASS was purified as described previously, and the cysteine synthesis activity of the enzyme was monitored using a ninhydrin-based assay.34 Briefly, cysteine synthesized by OASS is allowed to react with ninhydrin under strongly acidic conditions. This reaction is more specific to cysteine under strongly acidic conditions, and hence, the progress of the reaction can be monitored at 560 nm. All assays were performed in 0.1 M HEPES (pH 7.0) at 30 °C, with concentrations of OAS and Na2S kept constant at 5.0 and 3.0 mM, respectively. In all assays, reaction components were mixed together first, and the enzyme (0.1−0.3 μg) was added last. The reaction was allowed to proceed for 25.0 min and then terminated by the addition of 5% TCA. The amount of cysteine produced by the enzyme was calculated using the standard curve. Steady-State and Competitive Inhibition Kinetic Studies. Steady-state kinetic parameters and inhibition constants were determined by initial velocity measurements in which either OAS concentration was varied (0.2−15 mM) with the Na2S concentration fixed at 3.0 mM or the Na2S concentration was varied (0.1−2 mM) with the OAS concentration fixed at 10.0 mM. Initial velocities plotted against substrate concentrations were fit to the Michaelis− Menten equation (eq 1) using the nonlinear least-squares analysis method.

Regulatory protein−protein interactions between OASS and SAT have been well characterized.14−18 In the CRC, the activity of OASS is significantly reduced because the last 10 amino acids of the C-terminus of SAT (C10 peptide) bind tightly to the OASS active site and block access to OAS.19,20 The competitive inhibition of O-acetyl serine sulfhydrylase (OASS) by the Cterminus of serine acetyltransferase (SAT) presents a paradox, because the C-terminus of SAT binds to the active site of OASS with an affinity 3−6 log-fold greater than the apparent affinity of OAS.16−18 Structural and computational studies predicted that binding of the C-terminus of SAT will render active sites of OASS “nonfunctional”, and therefore, development of peptide inhibitors against OASS is desired.20,21 These studies confirmed that the C-terminus of SAT is a natural inhibitor of OASS, but the physiological significance of CRC formation and competitive inhibition is unknown. However, our understanding of regulatory protein−protein interaction between these enzymes is complicated by the fact the substrate, OAS, binds to the OASS active site with a very low affinity (kM ∼ 1−3 mM) versus the high affinity of SAT C-terminal or C10 peptides (kd values from 1.0 nM to 1.0 μM) for the active site of OASS.16,18,21,22 Intracellular concentrations of both OAS and cysteine (product) have been estimated to be in the micromolar range.23,24 Because the affinity of OASS for its substrate (OAS) is 6 log-fold lower than that of the competitive inhibitor, substrate concentrations should be at least in the high millimolar range to compete with the competitive inhibitor. Very high nonphysiological substrate concentrations would be necessary to compete with the high-affinity inhibitor, and therefore, it is not clear how OAS gains access to the OASS active site or dissociates the C-terminus of SAT from it. At the outset, this scenario portrays the active site of OASS as being counterintuitive by design because it will be occupied by a highaffinity inhibitor under physiological conditions. As shown in Figure 1, OAS can also dissociate the CRC into OASS and SAT, but it is not clear how OAS dissociates the high-affinity Cterminus of SAT from the OASS active site, if both antagonists bind mutually exclusively according to the classical competitive binding model.25,26 To resolve this paradox, we employed both analytical and structural approaches aimed at understanding how the lowaffinity substrate, OAS, competes with the high-affinity inhibitor, the C-terminal tail of SAT. We first demonstrate through high-resolution analytical and structural studies that both the competitive inhibitor and the substrate bind to the active site of OASS and have overlapping binding sites. By single-molecule and kinetic experiments, we show that the substrate actively dissociates the OASS−inhibitor and full length OASS−SAT (SAT inhibitor of OASS) complexes through a nonclassical mechanism. Finally, we resolved binary enzyme·inhibitor and enzyme·reaction intermediate complexes and the first ever enzyme·substrate·competitive inhibitor ternary complexes in which both substrate and the competitive inhibitor are co-localized within the same active site (Table 1 and Table S1). All the observed structural changes can be mapped to the movable N-terminal domain (residues 45−135) that consists of a three-stranded parallel β-sheet (β3−β5) sandwiched by two helices (α3 and α4) on one side and helix α5 on the other (Figure 1B).

v = (Vmax[S])/(kM + [S])

(1)

where Vmax is the maximum velocity of the enzyme and kM is the apparent affinity of the substrate. In the competitive inhibition assays, initial velocities were determined in the presence of various concentrations of the inhibitor (0−16.0 μM) with increasing concentrations of OAS (0−10 mM) and at a fixed concentration of Na2S (3.0 mM). To estimate initial velocities, aliquots of reaction mixtures were assayed for cysteine formation. Experiments were performed in triplicate, and the inhibition constant (KI) for the inhibitor was estimated from double-reciprocal plots, as described previously.35 Equilibrium Binding Measurements. All binding studies were performed at pH 7.5 in 20 mM Tris, and the salt concentration (NaCl) was kept at 20 mM because of the apparent negative cooperativity of binding at higher salt concentrations. Binding of high-affinity C10 peptide inhibitors to OASS was examined by monitoring changes in the fluorescence of the active site pyridoxal 5′-phosphate (PLP). The excitation wavelength was set at 412 nm, and fluorescence was monitored at 507 nm. All experiments were performed at 23.0 ± 1 °C with the excitation and emission bandpass set to 5.0 nm. The OASS solution (0.5−2.0 μM) was equilibrated before adding aliquots of C10 peptide inhibitors, and the initial fluorescence value (F0) was noted. After each addition, the reaction mixture was equilibrated for 2−3 min before the PLP fluorescence was recorded, and data points from five such measurements were averaged to obtain Fave,i. The relative fluorescence quenching upon ligand binding is defined as Qobs,i = (Fave,i − F0)/F0. The formation of the inhibitor−OASS complex was analyzed to obtain the equilibrium binding constant [kobs = [PL]/([P][L])] using two independent site binding models

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MATERIALS AND METHODS Chemicals and Peptides. All chemicals and buffers used in this study were of reagent grade and were procured from C


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Biochemistry Q obs/Q max = n × (kobsL)/(1 + kobsL)

ratio. Prior to binding experiments, the effective confocal volume (Veff) was calculated by measuring the diffusion properties of Atto-655 (diffusion coefficient D ∼ 426 ± 8 μm2 s−1) as described previously.36 The diffusion time, τD, and effective volume can be calculated from the autocorrelation function [G(τ)] by eq 5

(2)

where n equals 2 because the OASS dimer has two binding sites, Qobs is the observed fluorescence quenching, and Qmax is the maximum fluorescence quenching at saturation. Isothermal titration calorimetry (ITC) experiments were performed using a VP-ITC calorimeter (Microcal, Inc.) under buffer conditions (buffer A) of 50.0 mM Tris-HCl (pH 7.5), 70.0 mM NaCl, and 5% glycerol at 25 C. C10 peptides (10 μL/ injection) were titrated into a cell containing OASS with an automated 250 μL microsyringe at an interval of 3−4 min. Control experiments were performed by injecting the same amount of C10 peptides in buffer into the cell to estimate the heat of dilution. Data obtained from titrations were analyzed using a single-site binding model using software provided by the instrument manufacturer (eq 3). Q itot = VoM totN[(KI )ΔH /(1 + KI )]

G(τ ) ∼ 1/NVeff {[1/(1 + τ /τD)][1/(1 + τD/S2τ )]1/2 } (5)

where S, the structure parameter, is the depth to diameter ratio of the Gaussian observation volume. The initial concentration of the labeled inhibitor was between 10 and 20 nM, and OASS was added incrementally to the solution containing the labeled inhibitor. Measurements were then taken. Upon saturation of formation of the enzyme−inhibitor complex, OAS was added incrementally from a 50 mM stock solution prepared in the same buffer. Because we separated the labeled peptide inhibitor from the free dye by RP-HPLC, we considered only two diffusing species, one free inhibitor and the enzyme−inhibitor complex. Therefore, the autocorrelation curve was fit to a twocomponent model using eq 6

(3)

Qtot i

where is total heat after the ith injection, Vo is the cell volume, Mtot is the OASS concentration in the cell, N = 1 (one site), K is the equilibrium constant, I is the concentration of the free inhibitor, and ΔH is the corresponding enthalpy change. Labeling of Inhibitor Peptides and Purification. Peptides (4 mg/mL) were dissolved in a 0.1 M sodium bicarbonate solution, and 10.0 μL of Atto-655 NHS ester dye (stock concentration of 10 mg/mL) was added to 100 μL of the peptide solution. The mixture was incubated at room temperature for 1 h while being shaken at 100 rpm. After reaction, the labeled peptide inhibitor (HiC10) was isolated from both the unlabeled peptide and the free dye by reverse phase high-performance liquid chromatography (RP-HPLC). The peptide was purified by using a Waters HPLC system (2489 UV-VIS detector), fitted with a reverse phase C8 column (250 mm × 4.6 mm, 5 μm). Before purification, the reaction mixture was diluted in HPLC buffer A (0.1% TFA; 20.0 μL of reaction mixture to 100.0 μL of buffer A), and 45.0 μL of this diluted sample was injected into the reverse phase column preequilibrated with 95% buffer A. The peptide was eluted at a flow rate of 1.0 mL/min using buffer A and buffer B (B, 80% acetonitrile in 0.1% TFA) using a linear gradient (from 0 to 80% over 60 min) (data not shown). The concentration of the HPLC-purified labeled peptide was calculated as given below (eq 4). C P = A 280 − A max (CF/MEC)A 280

G(τ ) ∼ 1/N ∑ {(1 + τ /τD)−1[1 + τ /(S2τD)]−1/2 }

(6)

Data were analyzed using picoquant software provided by the manufacturer. Biotinylation and Purification of the C10 Peptide. Two peptides, StC10 peptide (WHTFEYGDGI) and HiC10 (YIDDGMNLNI), were used for labeling with biotin in this experiment. The peptide (1−3 mg) was dissolved in 1 mL of 50 mM phosphate buffer (pH 6.5). Immediately prior to use, a 10 mM stock of Sulfo-NHS-Biotin was made in dimethyl sulfoxide. A 5-fold molar excess of this biotin reagent was added to the dissolved peptide, and the mixture was incubated at 4 °C for 24 h. The peptide/biotin mixture was then dialyzed extensively in 20 mM Tris-HCl (pH 7.5), 50 mM NaCl buffer to remove the nonreacted NHS-Biotin. The dialyzed peptide was then loaded on to HPLC and biotinylated and nonbiotinylated peaks were separated. The biotinylated fraction was lyophilized and dissolved in the experimental buffer for further use. Dissociation Kinetics of the OASS−SAT/C10 Peptide Complex Determined by Surface Plasmon Resonance. Real-time monitoring of the OASS-binding C10 peptide and its dissociation kinetics was performed with a BIAcore 3000 using a streptavidin (SA) sensor chip (GE HealthCare). All experiments were performed at 25 °C in the standard buffer [20 mM Tris-HCl (pH 7.5) and 50 mM NaCl]. The biotinlabeled C10 peptide was immobilized on one flow cell in which the binding and dissociation kinetics of OASS were monitored and another flow cell with no C10 peptide immobilized was used as the reference. The signal from first flow cell was substracted from the reference to account for nonspecific binding of OASS to the chip surface. The purified biotinylated peptide (0.5 μM) was immobilized on the chip by constant injection of 20 μL/min over 200−400 s. The peptide-bound chip was washed with buffer (20 μL/min over 300−500 s). Purified OASS (10−20 μM) was injected (20 μL/min) into both sample and reference cells, and the kinetics of association and dissociation were observed in the differential mode at various OASS concentrations. Dissociation of the OASS was initiated by injection of the buffer of OAS (0.5−10 mM) and monitored for 15 min. Real-time monitoring of the

(4)

where A280 is the absorbance of the sample at 280 nm, Amax is the maximum absorbance of the sample at the maximum excitation of the sample (∼662 nm), CF is the correction factor for the dye used, and MEC is the molar extinction coefficient of the peptide. The correction factor for Atto dye at A280 is 0.08 as recommended by the manufacturer. Fluorescence Correlation Spectroscopy (FCS). FCS measurements were performed with an inverted NikonA1R laser scanning microscope integrated with a Picoquant (LSMSPAD) unit. The CW red diode laser was used to excite the Atto-655-labeled inhibitor at 640 nm. The properties of the freely diffusing N-terminally labeled inhibitor and inhibitor− enzyme complexes were measured using LabTekII chamber slides with eight wells (Nunc, Wiesbaden, Germany). The fluorescence was filtered with a band pass filter (Semrock FF01732/68), and all measurements were taken in 0.1 M HEPES (pH 7.4) and 50 mM NaCl. The optimal pinhole diameter and laser intensity were calculated to maximize the signal-to-noise D


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Biochemistry

were deleted) were used as the search model. All structures are resolved in the I41 space group with one HiOASS monomer in the asymmetric unit. The second monomer of the dimer can be found by rotating along the 2-fold axis parallel to the crystal axis. Initial difference density maps of all structures resolved in this study showed that the PLP is bound to active site lysine. A topology file for PLP external aldimine was generated using the HIC-up server, and 5% of X-ray data were removed for calculation of Rfree during all refinements. Many iterative rounds of manual rebuilding in Coot41 and refinements using Phenix42 were performed to obtain the final models. Water molecules were added to unascribed electron densities on the basis of their shape and peak heights. Initial omit map analyses of Binary-2, Ternary-1, Ternary-2, and Tetra-nary structures showed significant structural changes in the N-terminal movable domain. Using unbiased difference densities (σweighted 2Fo − Fc and Fo − Fc density maps) and omit maps, we rebuilt the movable domain of the enzyme for Binary2, Ternary-1, Ternary-2, and Tetra-nary. Ligands were modeled into their respective densities only after the enzyme had been rebuilt and refined. Difference Fourier maps and simulated annealing omit maps were used to confirm the presence of competitive antagonists at their respective positions. R and Rfree factors converge to those reported values, and details of final models can be found in Table 1. Stereochemical features of structures were checked using Procheck,43 and all figures were made using Pymol.44 Stopped-Flow Kinetics. Stopped-flow kinetic experiments were performed with a Biologic rapid kinetics instrument equipped with four syringes (10 mL) set in a parallel fashion (SFM400). Fluorescence data were collected by a MOS-250 unit equipped with PMT450 (detector) fitted with a long pass filter (450 nm) (Semrock Inc.) and a 8 or 15 mm fluorescence quartz cuvette (FC8 or FC15, respectively). All the experiments were performed in 20 mM Tris and 20 mM NaCl (pH 7.5) as the running buffer in the flow lines. All proteins and peptide stocks were dissolved in the same buffer. Three syringes were filled with buffer (S2), OAS (S3), and protein (S4). The excitation wavelength was 412 nm, and the slit width of 4 nm was used after optimizing the fluorescence. Fluorescence light emitted from the reaction mixture (510 nm) was monitored after passing through an emission long pass filter (450 nm). Series of stopped-flow experiments were performed at 20 °C in the buffer. After rapid mixing, time course internal fluorescence intensity data were recorded from the PLP. The OAS concentration was in the range of 10−1500 μM. Each averaged set of data collected was analyzed with the Biokine analysis program using single-exponential fitting (eq 9)

OASS−SAT binding and dissociation kinetics was performed with experimental conditions similar to those discussed above. Purified SAT was immobilized on the NTA chip (GE HealthCare) by constant injection at a rate of 20 μL/min until ~750 R.U. was achieved. The SAT-bound chip was washed with buffer (20 μL/min over 300−500 s). Purified OASS (800 nM) was injected (20 μL/min) into both sample and reference cells, and kinetics of association and dissociation were observed in the differential mode at varied OAS concentrations. Dissociation of the OASS was initiated by injection of the buffer of OAS (10−500 μM) and monitored for 15 min. The data were processed and analyzed using SCIENTIS (Micromath). A single-exponential (eqs 7 and 8) model was used to analyze the binding data as described. R = (CkaR max {1 − exp[( −Ck a+kd)t ]}) /(Cka + kd)

(7)

R = R max exp( −kdt )

(8)

where C is the concentration of OASS, t is the time (seconds), R is the maximum response (response units), ka is the on rate, and kd is the off rate. Dissociation rate constants determined from fitting the dissociation phase were used as constraints while fitting the association phase data. Crystallization and Data Collection. We screened for crystallization conditions using commercially available screens (Hampton, NexTal, and Molecular Dimensions). The protein (1.0 μL, 20 mg/mL) was mixed with 1.0 μL of crystallization buffer [1.4 M sodium citrate and 0.1 M Na HEPES (pH 7.5)], and diffraction quality crystals were grown by the sitting drop method. For co-crystallization experiments, a 4.0-fold molar excess of the StC10 peptide inhibitor was incubated with OASS at room temperature for 15 min before being mixed with crystallization buffer [1.4 M sodium citrate and 0.1 M Na HEPES (pH 7.5)] in 1:1 ratio. The OASS·α-amino acrylate complex was prepared by soaking free OASS protein crystals into mother liquid containing OAS (120.0 mM). Crystals were picked up at various time intervals (30, 45, and 90 s), soaked in mother liquid containing cryoprotectant (20% glycerol), and flash-frozen for data collection. Ternary and tetranary enzyme· inhibitor·substrate complexes were prepared by soaking the enzyme·inhibitor complex crystals in the mother liquid containing OAS (120−200 mM). Soaking intervals were kept between 30 and 90 s and varied between trials. Multiple trials of soakings of HiOASS−HiC10 complex crystals into an OAS solution led to the enzyme·reaction intermediate binary complex due to fast dissociation of the HiC10 peptide. However, multiple trial soaks of HiOASS−StC10 crystals into a solution containing OAS at described concentrations led to the few successful experiments described in this study. Data were collected at 100 K using an in-house MAR345 image plate detector mounted on a Rigaku (MicroMax-007HF micro focus) rotating anode X-ray generator or beamline 14 at ESRF (Grenoble, France). Diffraction intensities were integrated, merged, and scaled with the HKL2000 suite.37 All crystals belonged to tetragonal space group I41 with the cell dimensions listed in Table 1. Structure Refinement and Validation. All structures reported in this study were determined by the molecular replacement method using Phaser, and initial rigid body refinements were performed using REFMAC5.38−40 Atomic coordinates of OASS from Haemophilus inf luenzae [Protein Data Bank (PDB) entry 1Y7L] without any bound ligand (atoms belonging to the inhibitor peptide and other ligands

F = A 0 − A[exp( −kobs, nt )]

(9)

where F is the fluorescence at time t, n is the number of exponential terms, A and kobs are the amplitude and the observed rate constant of the nth term, respectively, and A0 is the fluorescence intensity at time zero. The errors shown for the rate constants represent the means of the deviation of the reported rate constants from the values computed from the maximum and minimum kobs values. kobs values obtained from eq 9 were plotted versus the ligand concentration (C).

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RESULTS C10 Peptide Inhibitors Bind with High Affinity and Block Access to the Substrate. The crystal structure of E


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Biochemistry

Figure 2. Kinetic and thermodynamic characterization of binding of C10 peptide inhibitors to the OASS active site. (A and B) Competitive inhibition kinetic assays. Steady-state kinetic studies were performed to estimate inhibitory potentials of two C10 peptide inhibitors (StC10 and HiC10). Primary double-reciprocal plots of initial rates determined at fixed inhibitory concentrations of C10 peptide inhibitors, StC10 (A) and HiC10 (B). The inset shows the secondary plot of slopes of reciprocal plots (K/V) vs inhibitor concentrations. Estimation of their inhibitory potential yields KI values of ∼7.3 μM for StC10 and 6.5 μM for HiC10. (C) Determination of affinities. The change in the relative fluorescence of PLP of HiOASS upon C10 peptide inhibitor binding is plotted vs peptide concentration. Data fitting was performed using nonlinear least-square analysis with Scientist (Micromath, St. Louis, MO). Data were analyzed using a two-equivalent site binding model (solid fit to data), which yields kd values of ∼0.63 μM for StC10 and 1.65 μM for HiC10. (D−F) Isothermal titration calorimetry analyses of C10 peptide inhibitor−HiOASS interactions. In the top panel, raw data are plotted as the heat signal vs time. In the bottom panel, integrated heat responses per injection are plotted vs molar ratio. All titrations were performed in buffer A at 25 °C. Data were analyzed with nonlinear least-squares analyses using Microcal Origin 7.0. The solid line is the fit to experimental data using a single site binding model. (D) Titration of StOASS (4.0 μM, in the cell) with the StC10 peptide (40.0 μM, in the syringe). The fit to the single site binding model yields a kd of ∼60.0 nM. (E) Titration of HiOASS (4.0 μM, in the cell) with HiC10 (100.0 μM, in the syringe). The fit to the single site binding model yields a kd of ∼2 μM. (F) Titration of HiOASS (3.0 μM, in the cell) with StC10 (60.0 μM, in the syringe). The fit to the single site binding model yields a kd of ∼3.3 μM.

2A,B). Next, we employed fluorescence and isothermal titration calorimetry approaches to determine the thermodynamic parameters of the two inhibitory C10 peptides. HiOASS binds to its cognate HiC10 peptide with a kd of ∼2 ± 0.04 μM but recognizes its substrate, OAS, with a relatively low affinity (kM ∼ 2.0 ± 0.2 mM), consistent with earlier observations (Figure 2C). Interestingly, the StC10 peptide binds to StOASS with a very high affinity (kd ∼ 60 ± 5.1 nM), ∼6.0 × 104 times higher than the apparent affinity of the substrate, OAS (kM ∼ 4.0 ± 1.0 mM), for StOASS. Therefore, we tested whether the StC10 peptide can also be used as a high-affinity inhibitor that can bind to the active site of HiOASS and block access to OAS. A previous study evaluated the inhibitory potentials of designed C10 peptides and found that C10 peptides with varied sequences can inhibit the cysteine synthesis activity of OASS.20 It is also notable that C10 peptides that belong to the family of SATs from various species do not show any sequence similarity except the last C-terminal isoleucine that is invariably conserved. Structural studies showed that C10 peptides bind through a similar mechanism with their conserved isoleucine docked close to the reaction center

OASS from H. inf luenzae (HiOASS) in complex with the C10 peptide of SAT provided early insights into the structural mechanism of competitive inhibition of OASS by SAT Cterminal/C10 peptides.19,20 HiOASS is a homodimer that shares a common tertiary structure with other forms of OASS, ́ and one pyridoxal 5-phosphate (PLP) is bound per subunit. The active site cleft is formed between the movable N-terminal domain (residues 45−135) and the C-terminal domain (residues 136−316) (Figure 1B). Using HiOASS as our model system, we examined the binding and inhibitory potential of two C10 peptides (HiC10 and StC10), derived from two SAT proteins (HiSAT from H. inf luenzae and StSAT from Salmonella typhimurium). First, we examined the inhibitory potentials of these C10 peptides by performing competitive inhibition steady-state kinetics. We estimated the rate of cysteine synthesis by HiOASS in the presence of different concentrations of C10 peptides and determined the inhibitory constants of the peptides. Results showed that inhibitory potentials varied as a function of C10 peptide concentration and StC10 and HiC10 inhibited HiOASS activity with KI,in values of ∼7.3 and ∼6.3 μM, respectively (Figure F


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Biochemistry Table 2. Thermodynamic Parameters for Binding of the C10 Peptide Inhibitor to OASS kd (M)

no. of sites StC10 peptide−StOASS HiC10 peptide−HiOASS StC10 peptide−HiOASS

1.45 ± 0.0105 1.69 ± 0.26 0.916 ± 0.11

−8

−9

6 × 10 ± 5.16 × 10 2 × 10−6 ± 4 × 10−8 3.3 × 10−6 ± 9.18 × 10−8

ΔG (kcal mol−1)

ΔH (kcal mol−1)

TΔS (kcal mol−1)

−9.7 ± 0.2 −7.7 ± 0.04 −7.4 ± 0.03

−13.2 ± 0.13 −4.7 ± 0.9 −23.7 ± 3.2

−3.5 ± 0.23 3 ± 0.9 −16.3 ± 3.2

Figure 3. Facilitated dissociation of HiOASS−C10 peptide complexes by OAS. (A) Dissociation of the OASS−inhibitor complex as a function of OAS concentration examined by fluorescence correlation spectroscopy. Dissociation is biphasic, and the KI,OAS estimated from 50% dissociation is ∼35.0 μM. (B−D) Surface plasmon response experiments. Differential response (DR) (reference subtracted) plotted vs time. (B) Association and dissociation kinetics of binding of HiOASS to the HiC10 peptide immobilized on the streptavidin-BIAcore chip. The binding of OASS to the C10 peptide inhibitor is accompanied by an increase in DR and the dissociation of OASS by a decrease in DR. Experiments were performed at two different OASS concentrations (5.0 and 10.0 μM). Three independent binding and dissociation kinetic experiments performed at 10.0 μM OASS are overlaid. The concentration of OAS used in the dissociation buffer is indicated. Dissociation of OASS is accelerated in the presence of OAS (1 and 10.0 mM). (C) Analyses of association kinetics phase of panel B. Raw data can be described well with a single-exponential model with a kobs of (∼3.1 ± 0.1) × 103 s−1 M−1. (D) Analyses of the dissociation kinetic phase of panel B. Dissociation constants estimated from fitting the data to the singleexponential model: kd ∼ (3.0 ± 0.1) × 10−3 s−1 (0.5 mM OAS), and kd ∼ 1.1 ± 0.1 s−1 (10.0 mM OAS).

where the substrate molecule is expected to bind.12,19,20 Therefore, we anticipated that StC10 should also bind to HiOASS and block its activity. Results show that the StC10 peptide binds to HiOASS with an affinity (kd ∼ 3.3 ± 0.09 μM) similar to that of the HiC10 peptide but with an affinity 3−4 log-fold higher than that of substrate, OAS. We used isothermal titration calorimtry (ITC) as another independent approach to evaluate the binding affinities of the inhibitory peptides. Results show that although both StC10 and HiC10 peptides bind to their respective cognate binding partners, the StC10 peptide binds to StOASS with a higher affinity (∼3.5-fold higher), consistent with the results of the fluorescence-based approach (Figure 2D−F). Analyses of thermodynamic parameters, listed in Table 2, show C10 peptide−OASS interactions are accompanied by enthalpically favorable exothermic reactions. In summary, our results demonstrate that two C10 peptides derived from two different SAT enzymes inhibit the cysteine synthesis activity of OASS and could be for testing the facilitated dissociation mechanism as both bind HiOASS with an affinity 3 log-fold higher than that of the OAS. OAS Accelerates the Dissociation of the Enzyme− Inhibitor Complex. OAS-mediated dissociation of CRC has

been observed previously.14 However, the stability of OASS− C10 peptide inhibitor complexes was not examined, and the process by which OAS dissociates from C10 peptide inhibitors or the C-terminus of SAT from the active site of OASS is not known. We examined the dissociation behavior of the OASS− C10 peptide complex by fluorescence correlation spectroscopy (FCS) and surface plasmon resonance (SPR) methods. First, we examined the binding of HiOASS to the StC10 peptide in the buffer and then studied the dissociation of the enzyme− inhibitor complex in the presence of OAS by monitoring the diffusion properties of the C10 peptide inhibitor. The diffusion time of the purified Atto-655-labeled inhibitor in the observation volume was found to be ∼112 ± 5 μs. The increase in diffusion time (τ ∼ 225 ± 5 μs) noticed upon addition of OASS suggests the formation of the enzyme− inhibitor complex. We then monitored the dissociation of this preformed enzyme−inhibitor complex in the presence of OAS. Addition of OAS decreased the diffusion time back to 128 ± 5 μs, indicating that OAS dissociates from the enzyme−inhibitor complex. As discussed in the text, the estimation of the OAS concentration at which 50% of the enzyme−inhibitor complex was dissociated yielded a KI,OAS of ∼35.0 μM (Figure 3A). It is G


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Biochemistry also interesting to note that although the values of the apparent dissociation constant of OAS estimated from steady-state kinetics studies are in the low millimolar range (kM ∼ 2.0−4.0 mM), the KI,OAS estimated from FCS experiments shows that the affinity of OAS might be in the micromolar range. Because the FCS experiment is very sensitive to events that occur at the single-molecule level, we assume that OAS may initiate the dissociation of the inhibitor as soon as it binds to OASS. However, the steady-state kinetic experiments are not sensitive to initial events, and the Km is estimated as the substrate concentration at which the half-maximal velocity is achieved, which might underestimate the OAS affinity, as the conversion of OAS into cysteine proceeds through multiple reaction intermediates.11−13 Next, using the SPR method, we examined the binding of the StC10 peptide to HiOASS in the buffer and then monitored the dissociation of HiOASS as a function of OAS concentration (Figure 3B). The kinetics of association of OASS with the StC10 peptide can be described by a singleexponential phase, and three experiments performed at constant OASS concentrations yield a kobs of (∼3.1 ± 0.1) × 103 s−1 M−1 (Figure 3C). The dissociation of the OASS−C10 peptide complex in the buffer is extremely slow, indicating that the enzyme−inhibitor complex is very stable. However, small injections of OAS accelerate the dissociation of OASS as shown in Figure 3D. In addition, the dissociation phase of OASS in the presence of OAS cannot be fitted to a single-exponential model, suggesting that a more complex dissociative process is being used. Therefore, we used a two-exponential model to analyze dissociation kinetic data. The dissociation rate is extremely weak in the buffer but increases in the presence of OAS. The initial dissociation rate (kd) increased from (∼3.0 ± 0.1) × 10−4 to ∼1.1 ± 0.1 s−1 when dissociation buffer containing 10.0 mM OAS was used. Accelerated dissociation of the OASS−C10 peptide complex in the presence of OAS supports the facilitated dissociation mechanism.5,8 Both FCS and SPR results indicate that OAS actively dissociates the C10 peptides from the active site of OASS. In summary, dissociation rates of the OASS−C10 peptide inhibitor complex increase as the OAS concentration is increased, suggesting that OAS-mediated dissociation may follow a nonclassical competitive dissociative mechanism. The Substrate and C10 Peptide Inhibitor Occupy the S1 Site of the Active Site. We aimed to understand the accelerated dissociation mechanism through structural approaches. The crystal structure of HiOASS in complex with the HiC10 peptide has been resolved previously.19 To characterize the structural features of the HiOASS·StC10 peptide complex and compare them with those of the HiOASS·substrate complex, we first resolved the crystal structures of these two binary complexes. Our results show that HiOASS exists in two different conformational states, open and closed. Crystal structures of OASS in complex with either StC10 (Binary-1, 4NU8) or HiC10 show that the active site is in the open state. Superposition of HiOASS in complex with StC10 and HiC10 reveals that C-terminal isoleucines (I267 and I273, respective positions in SAT) occupy similar positions, suggesting that the modes of binding of these C10 peptides are very similar (Figure 4A). The HiOASS−HiC10 peptide complex structure is known, and therefore, we discuss the features of the HiOASS−StC10 complex structure here. The StC10 inhibitor occupies the entire active site channel with clear electron density for the last eight residues. The multipoint attachment platform of the inhibitor is S-shaped and consists of three subsites, S1−S3 (Figure 4B). The C-terminal I273 (I273,

Figure 4. Structural analyses of the C10 peptide inhibitor that binds in an open state. (A) Superposition of Binary-1 (green) with the structure of the HiOASS−HiC10 peptide (1Y7L, slate blue) shows that C-terminal isoleucines (I273 and I267) bind to the S1 binding pocket, which is adjacent to the reaction center, marked by PLP (yellow sticks). (B) Active site-bound inhibitor [magenta sticks, unbiased Fo − Fc map contoured at 2.8σ (green)] in the open state (Binary-1), showing the S-shaped binding trajectory (S1−S3 sites). (C) OAS binding traps the active site in the closed state. Close-up view of the reacted active site (Binary-2, closed state), showing the σAweighted Fo − Fc omit map (green mesh) of α-amino acrylate (yellow) contoured at 2.8σ. (D) Superposition of the closed state (Binary-2, cyan) with the open state (Binary-1, green), showing two distinct conformations. The overlay (circled) shows the reaction intermediate (RI) occupies the position of I273 of the inhibitor (magenta sticks) at the S1 site. The S70 loop and α-helix 5 move into the inhibitor-binding pocket in the closed state.

last of 273 amino acids in SAT) is held tightly at the S1 site, and as many as six hydrogen bonds lock I273 to the S1 site. The S2 site begins with the first β-turn of the inhibitor and ends at the second β-turn. Active site residues S70 and G228 fix the geometry of G272 and D271 of the inhibitor. The remaining Nterminal part of the peptide is bound to the S3 subsite, and residues H224 and Q226 from the conserved 222GPHKIQG228 loop stabilize the orientation of the F270−T269 peptide bond. In summary, the S1 site is primarily used for locking I273 and residues from two other sites provide further stability to the inhibitor−enzyme interaction. The pyridine ring of PLP, tilted by 13 Å along the 2′−5′ carbon axis, moved along with a majority of the movable domain (α-helix 4, α-helix 5, β-sheets 3 and 4, α4β5-turn, and α-helix 3) toward the active site channel to close the active site. Superposition of Binary-1 and Binary-2 structures shows that the reaction intermediate occupies the position of I273 of the inhibitor (Figure 4D). M120, M96, and S70 from three conserved loops move into binding pockets I273, G272, D271, and G270 of the inhibitor. Structures of Binary-2 and Binary-1 clearly show that OAS binding traps the active site in the closed state, as reported for MtOASS. The concerted movement of M120, M96, and S70 from conserved loops into binding pockets of the inhibitor suggests that the inhibitory C10 peptide can bind to H


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Biochemistry

Figure 5. Structural snapshot of the ternary enzyme·competitive inhibitor·substrate complex. (A) The 2Fo − Fc (blue mesh) map contoured at 1.0σ shows both the C10 peptide (magenta sticks) and OAS (gray sticks) in active site channel of HiOASS. (B) Interaction map of OAS with the protein and inhibitor (LigPlot). (C) Refolding of rotated α-helix 5 and ordering of the movable domain upon formation of the reaction intermediate. Cartoon representation of unfolding, translocation, and folding events of α-helix 5 capture from different crystal structures resolved in this study (PDB entries are given).

Interestingly, binding of OAS at the entrance triggers allosteric conformational changes that propagate from the site of binding to the S70 peptide-binding loop. Superposition of Ternary-1 (4ORE) with Binary-1 (inhibitor-bound open state, PDB entry 4NU8) and Binary-2 (substrate-reacted closed state, PDB entry 5DBH) shows that the N-terminal movable domain of OASS, including α-helix 5, the α4β5-turn, β-sheet 4, and α-helix 4, attains non-native-like structure. The multistage transition from the open to closed state can be illustrated by displaying OASinduced movements of α-helix 5 (Figure 5C). Following OAS entry, the N-terminus of α-helix 5 unfolds along with adjacent regions, and translocation of OAS to the S1 site unfolds α-helix 5 more while rotating it toward the channel. Reaction of the substrate refolds the rotated α-helix 5 and traps the active site in the closed state (Figure 5C). In summary, the Ternary-1 structure suggests that OAS can bind to the enzyme−inhibitor complex and the incoming OAS triggers allosteric conformational changes that propagate to the site of the inhibitor-binding loop. OAS-induced remodeling of the enzyme active site weakens enzyme−inhibitor stability by specifically targeting crucial interactions between the Cterminus of the inhibitor and the S70 inhibitor-binding loop. We compared the structure of Ternary-1 (PDB entry 4ORE) with other structures (PDB entry 4ZU6). In these structures, electron densities for the peptide inhibitor are clearly visible whereas density for OAS is less clear. In the fourth structure (PDB entry 5DBE), electron density for the reaction intermediate and external aldimine was interpretable but electron density for the peptide inhibitor was broken and noisy (Figure S2A,B). The structure of 5DBE consists of one OAS in the pre-reactive state, α-amino acrylate. Interestingly, active sites of 4ZU6 and 5DBE structures are in closed-state

only the open state, and the conformational state of the active site is sensitive only to substrate binding. Structure of the Ternary Enzyme·Inhibitor·Substrate Complex. Although not discussed previously, the complex dissociation kinetics of CRC in the presence of OAS indicates that OAS may bind to the CRC or OASS−C10 peptide complex and then may dissociate from the C-terminus of SAT.25,26 We used OASS−StC10 complex crystals to capture transient ternary enzyme·inhibitor·substrate complexes (see Materials and Methods). We resolved four more structures (PDB entries 4ORE, 4ZU6, 5DBH, and 5DBE), generated from crystals of the enzyme·inhibitor complex soaked in OAS (Table 1 and Table S1). Here, we discuss structural features of one of the four complexes, named Ternary-1 (PDB entry 4ORE). The structure of Ternary-1 reveals that both the inhibitor and the substrate co-localize at the active site channel of OASS. The structure of Ternary-1 shows that OAS would enter the active site channel of OASS bound to the C10 peptide inhibitor. The final electron density map confirms the presence of both the peptide inhibitor and OAS within the active site channel (Figure 5A). Electron density for the peptide is continuous, and the peptide backbone superposes well with that of Binary-1. One molecule of OAS is wedged inside a Vshaped channel, formed by the inhibitor on one side and the 118 KGMK121 loop of OASS on the other. The observed position of OAS not only reveals the entry position but also suggests that residues of the 118KGMK121 loop may play a role in the recruitment of OAS into the active site channel. OAS, bound to the S3 site, mediates a hydrogen bond with the N atom of M120 of the 118KGMK121 loop and engages in additional interactions with E268, F267, and T266 of the peptide (Figure 5B). I


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Figure 6. Stopped-flow kinetics of HiOASS−peptide complex dissociation in the presence of OAS. Dissociation events were monitored by quenching of PLP fluorescence of OASS. Data were analyzed using a nonlinear least-squares method with biologic software provided by the manufacturer (Bio-Kine 32 V4.66). (A and B) Stopped-flow kinetic traces of dissociation of preassembled HiOASS−HiC10 and HiOASS−StC10 complexes in the presence of varied concentrations of OAS as indicated. Solid lines are best fits of kinetic traces to a single-exponential model to determine kobs using eq 9. Dissociation rates increase as the OAS concentration increases (Table 2). (C and D) Observed rates (kobs) obtained from single-exponential fits of kinetic time traces in panels A and B are plotted vs OAS concentration. The solid line is the fit to data to show that dissociation rates increase linearly as a function of OAS concentration.

1:2 [OASS]dimer:[peptide] molar ratio. A final protein concentration (HiOASS) of 6−8 μM was maintained in the cuvette. Saturating the HiOASS with two different peptides gives different total fluorescence amplitudes, suggesting that there might be subtle conformational differences in the states of final enzyme−inhibitor complexes (Figure 6A,B). Enzyme− inhibitor (HiOASS−C10 peptide) complexes were loaded into the syringe, and OAS (dissociated ligand) is loaded into another syringe. We performed control experiments to quantify the amplitudes of the PLP fluorescence change of OASS upon formation of a complex with either the inhibitor or OAS. Binding of C10 peptide inhibitors increases PLP fluorescence, whereas OAS binding completely quenches the PLP signal (Figure S5A,B). Therefore, complete dissociation of the C10 peptide inhibitor in the absence of OAS would result in a decrease in PLP fluorescence, but the final fluorescence would be equal to that of free OASS. On the other hand, the PLP fluorescence of the OASS−inhibitor binary complex decreases more than >95%, similar to that of the enzyme·reaction intermediate complex. This is because the enzyme−inhibitor complex transforms into the enzyme·reaction intermediate complex in the presence of OAS (Figure S5C). Each dissociation kinetic experiment was performed by mixing a fixed amount of the enzyme−inhibitor complex with a fixed amount of OAS, and monitoring PLP fluorescence quenching resulting from OAS binding-mediated enzyme−inhibitor complex dissociation. Our control experiments showed that OAS binding quenches whereas C10 peptide binding increases the PLP fluorescence of HiOASS. Because the PLP fluorescence signals, upon binding of HiOASS to OAS and the inhibitory C10 peptide, move in the opposite direction, it is

site, similar to that of Binary-2. Analyses of open-state (PDB entries 4NU8 and 4ORE) and closed-state structures (PDB entries 5DBH, 4ZU6, and 5DBE) allow us to propose a structural model of OAS-induced conformational remodeling that weakens inhibitor affinity (Figures S3 and S4). In closed ternary structures, M96 and S70 from the S2 and S1 sites moved closer to the binding pockets of I273, G272, and D271 of the inhibitor, consistent with missing electron densities for I273, G272, and D271 of the inhibitor. This indicates that the Cterminus of the inhibitor is dissociated and the translocated OAS occupies the place of I273 of the inhibitor. Because the Cterminal isoleucine is crucial for inhibitor binding, OASinduced conformational changes dissociate the C-terminus first to weaken the interaction between the inhibitor and enzyme. M120, M96, and S70 moved into the inhibitor-binding pocket, suggesting that rebinding of the inhibitor is not possible in this state (Figure S4). These results suggest that binding and translocation of OAS to the reaction center facilitate the exit of the inhibitor. OAS-Induced Dissociation Kinetics of the HiOASSBound StC10 Peptide and HiC10 Peptide Determined by the Stopped-Flow Method. One of the kinetic signatures of competitive antagonism is the fact that the dissociation kinetic parameters of the inhibitor should not be affected in the presence of OAS. Although results of SPR experiments indicated that OAS facilitates the dissociation of the C10 peptide inhibitor, we used fast kinetics as another independent method to capture the trend of OAS-induced dissociation kinetics. We monitored the dissociation kinetics of the C10 peptide inhibitor, bound to the OASS active site. HiOASS was saturated with HiC10 and StC10 peptides by being mixed in a J


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dissociation rates increase linearly as a function of OAS concentration, suggesting that OAS facilitates the dissociation of the HiOASS·C10 peptide complex (Figure 6C,D). Estimation of rate constants from the plot shows that although the on rate constants are similar, the off rates differ by 1 logfold. It should be noted here that the change in signal associated with PLP quenching involves two molecular events, simultaneous binding of OAS and inhibitor dissociation. OAS-Induced Dissociation Kinetics of the Cysteine Regulatory Complex (S. typhimurium) Using SPR. Next, we extended the approach to examine whether the bienzyme complex, the OASS−SAT full length multienzyme complex, behaves like the OASS−C10 peptide system. We used highly purified StOASS and StSAT proteins and assembled them to form CRC under similar solution conditions. First, the Histagged SAT was immobilized on the Biacore chip (see Materials and Methods), and OASS was injected into the channel until the resonance signal was saturated, indicating the assembly of the SAT−OASS complex. The kinetics of dissociation of the SAT−OASS complex was initiated by injecting OAS into the buffer. The OAS-induced dissociation kinetics was performed in the saturating OASS-bound state, as a function of OAS concentration (Materials and Methods). The concentration of OAS was varied in the range of 50−500 μM. The rate of complex dissociation is greatly increased in the presence of OAS as reported previously.14 The dissociation was analyzed with a single-exponential model (Figure 7A,B). Consistent with the noncanonical facilitated dissociation mechanism and results of enzyme·C10 peptide complex dissociation, the dissociation rates increase as a function of OAS concentration (Figure 7C,D). Dissociation rates plotted versus OAS concentrations follow a hyperbolic model with half-saturation at ∼200 μM OAS.

possible to monitor the dissociation of the enzyme−inhibitor complex in the presence of OAS. Both dissociation of the peptide and binding of OAS should result in the complete quenching of PLP fluorescence. Therefore, by monitoring the PLP quenching kinetics in the presence of varying concentrations of OAS, we should be able to estimate the rates of dissociation of the C10 peptide inhibitor from the active site of HiOASS. The time kinetic traces of C10 peptide inhibitor dissociation in the presence of OAS were analyzed by single-exponential models (eq 9). PLP fluorescence quenching kinetic traces of the binary enzyme−inhibitor complex consist of two events: OAS binding-mediated quenching and quenching contributed by the OAS-induced dissociation of the C10 peptide inhibitor from the active site of OASS. However, analyses of time traces indicate no noticeable biphasic trend, suggesting that both processes occur simultaneously. Kinetic and structural studies have shown that the formation of the enzyme·reaction intermediate complex may proceed with a number of reaction intermediates, but many of them are yet to be characterized.11−13 Therefore, explicit modeling of all species is not practical, and it is not relevant to the question of interest. The results of SPR experiments clearly showed that dissociation of the enzyme·C10 peptide inhibitor complex follows a singleexponential decay model. Therefore, we analyzed PLP quenching time traces by fitting data to eq 9 and estimated the respective dissociation rates of the inhibitor (Table 3). The Table 3. Comparison of Dissociation Rates of the C10 Peptide Inhibitor [OAS] (μM)

HiOASS−HiC10 peptide kobs (s−1)

[OAS] (μM)

HiOASS−StC10 peptide kd (s−1)

10 13 15 20 25 37

0.75 0.84 1.25 1.55 1.65 1.87

10 35 50 100 125 150

0.1 0.21 0.34 0.71 0.9 1.01

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DISCUSSION The results described here demonstrate that OAS dissociates the high-affinity OASS·inhibitor complex through a noncanonical “facilitated dissociation” mechanism, which has previously been reported for a few systems.4,8,45,46 However,

Figure 7. Surface plasmon resonance (SPR) analysis of CRC dissociation. All experiments were performed at 25 °C. SPR dissociation kinetics was performed by first assembling the CRC in the absence of OAS and monitoring CRC dissociation in the presence of different concentrations of OAS. The StSAT (∼100.0 nM stock) solution was used for immobilization on the NTA chip. Formation of CRC was achieved by passing the StOASS (1.0 μM) solution, and dissociation of CRC was monitored in the presence of varying concentrations of OAS (10−500 μM) used for dissociation. The starting concentration of CRC assembled on the NTA chip is kept constant as it can be seen from all dissociation traces to start around ∼450 RU (A). (A) Dissociation of StOASS from SAT (dissociation of CRC), plotted as differential resonance units (RU) vs time. The solid line represents a fit to a double-exponential model and kinetic parameters. (B) Dissociation rate constants estimated from panel A are plotted as a function of OAS concentration. Dissociation rate constants do not follow a linear trend, and therefore, they were fit to Hill’s cooperativity function. K


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sulfur. Therefore, under sulfur limiting conditions, increasing levels of OAS due to the continued action of SAT may lead to the dissociation of the OASS·SAT complex. Recent studies in plants show that OAS signals to regulate expression of many proteins and its levels vary in a cyclic manner, peaking at night.27,28 Therefore, dynamic associative and dissociative cycles of CRC may be designed to control levels of OAS to economize expression of proteins involved in sulfate metabolism. However, systematic biochemical and genetic studies are necessary to unravel the relationship between the CRC cycle and gene regulation. Crystal structures reported here capture the features of the facilitated dissociation mechanism and also provide insight into the dissociative cycle of OASS·SAT C-terminal interaction. The crystal structures of OASS·StC10peptide·OAS ternary complexes clearly show that OAS enters the active site of OASS while the StC10 peptide inhibitor is bound tightly to the active site, and upon entering, OAS facilitates the dissociation of the StC10 peptide. Active dissociation of a competitive inhibitor from the active site by the substrate is assisted by substratespecific conformational changes that transform the active site of the enzyme from an “open binding competent state” (Binary-1) to a “closed binding incompetent state” (Binary-2). Crystal structures of ternary complexes resolved in this study (Ternary1, Ternary-2, and Tetra-nary) exist in transition states between open (Binary-1) and closed (Binary-2) states and provide insight into sequential allosteric conformational changes that decrease inhibitor binding affinity and transform the inhibitorbound open state into a substrate-bound closed state (Figure S4). Superposition of all structures allows us to predict trajectories of the substrate and inhibitor from their respective positions. This comparison shows that the incoming substrate and leaving inhibitor share the same active site channel but move in opposite directions (Figure 6A). Simultaneous movements of both ligands are facilitated by OAS bindinginduced conformational changes. The secondary structural elements of the movable domain undergo unfolding and refolding while moving toward the closed state (Figure 5C). The results presented in this study clearly indicate that OAS dissociates OASS−C10 peptide complexes by a nonclassical competitive binding mechanism. Structural snapshots presented here capture the dissociative cycle of CRC, which begins with binding of the SAT C-terminal/C10 peptide to OASS in the open state. In the next step, OAS enters the preoccupied active site of OASS and initiates the dissociation of the SAT Cterminal/C10 peptide. Analyses of contact maps between OAS and the protein show that M120 from the α5β4-turn may play a key role in recruiting OAS into the inhibitor-bound active site. The M120 loop encompassing residues 117AKGMK121 has lost its native conformation but now forms a V-shaped channel with the second β-turn of the inhibitor (G272 to D271). Interaction of OAS with M120 destabilizes other interactions within the α5β4turn, leading to the partial opening of α-helix 5 and opening of the cooperative neighboring α5β4-turn. The intra β-turn hydrogen bond between the A118 N atom and the carbonyl oxygen of E115 is disrupted, converting the α5β4-turn into a loop. The cooperative unfolding of α-helix 5 and the α5β4-turn is now transmitted to adjacent β-sheet 4, pulling β-sheet 4 away. Because of these changes, a long stretch between residues L107 and I124 (α-helix, β-sheet 4, and α5β4-turn between them) lose their native conformation. Sequential structural transitions weaken the interaction between the C-terminus of SAT and OASS, while simultaneously favoring the recruitment of OAS.

molecular underpinnings of such noncanonical processes of facilitated/accelerated dissociation have not previously been dissected. Equilibrium binding and steady-state kinetic studies showed that C10 peptide inhibitors bind to the OASS active site with an affinity 3−6 log-fold higher than that of the substrate. The variability in inhibitor affinity seems to arise from subtle sequence differences, both within the active site of OASS and at the C-terminus of SAT. For example, the StC10 peptide binds to StOASS with nanomolar affinity as compared to the affinity of HiOASS for HiC10 and StC10 peptides. Nonetheless, the results of multiple experiments performed using different approaches confirm that the OASS active site has evolved to bind to the C-terminus of its natural binding partner, SAT, which is also its inhibitor with an affinity that is higher than its affinity for its own substrate. Although the molecular reasons for high-affinity interaction between OASS and SAT remain to be understood, it is only natural to expect that OASS might have evolved an alternative mechanism for recruiting its own substrate in the presence of its natural competitive inhibitor, which is SAT. In this study, we designed experiments to uncover such a mechanism that enables the substrate to gain access to the active site of OASS. In this study, we first demonstrate that a high-affinity OASS· inhibitor complex is actively dissociated by the low-affinity substrate, and the mechanism of active dissociation is not consistent with features of classical competitive antagonism. Through a series of analytical experiments, we show that dissociation of the OASS·inhibitor complex in the presence of a substrate follows a noncanonical “facilitated dissociation” mechanism and then dissect out the mechanistic features of the facilitated dissociation mechanism by structural approaches. OASS has one single-binding site channel with the reaction center located at the deep end of the channel. The last 10 amino acids of the C-terminus of SAT, here named the C10 peptide, occupy the entire active channel and block the entry of the substrate. However, as we show here, the substrate enters the inhibitor-bound active site of OASS and causes allosteric conformational changes that break interactions between the inhibitor and enzyme, leading to the exit of the inhibitor. Concentration-dependent dissociation rates of protein− ligand complexes have been associated with the noncanonical accelerated or facilitated dissociation mechanism.4,6 Immune receptor−ligand complexes and transcription factor−DNA complexes have been shown to dissociate through facilitated dissociative mechanisms.8,45 Using single-molecule and surface plasmon resonance (SPR)-based kinetic approaches, these studies have shown that dissociation rates increase as the concentration of the competing ligand is increased.4 In this study, we employed single-molecule, SPR, and pre-steady-state kinetic approaches to demonstrate that both OASS·C10 peptide and OASS·SAT complexes dissociate through a facilitated dissociation mechanism. In the presence of OAS, the competing substrate, rates of dissociation of either the C10 peptide inhibitor or full length SAT, the natural inhibitor, increase. The dissociation rate increases in proportion to the OAS concentration and is saturated at higher OAS concentrations, suggesting that the OASS−inhibitor complex is actively dissociated in the presence of OAS. Furthermore, these observations also support the fact that OASS uses the facilitated dissociation mechanism as a way to disengage itself with the bound inhibitor and recruit its own substrate. Intracellular concentrations of OAS are expected to increase and decrease in response to changing levels of intracellular L


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Figure 8. Dissociative cycle of the enzyme−inhibitor complex. The inhibitor is shown as magenta sticks, and the enzyme is colored marine blue. (A) Free OASS in the open state, ready to bind the inhibitor. (B) OASS·inhibitor complex in the open state. (C) Binding of OAS to the enzyme· inhibitor complex, initiating conformational changes to weaken inhibitor−OASS interactions. (D) The inhibitor is expelled, and OAS is trapped in the enzyme·reaction intermediate complex in the final closed state, ready to react with the incoming sulfide to form cysteine. After cysteine release, OASS switches back to the open state (A) to start the cycle again.

not favor the binding of its natural partners. Further research on uncovering salient features of competitive allostery may be biotechnologically and pharmaceutically important.

In the presence of sulfide, the reaction intermediate is converted into cysteine, OASS switches back to the open state to bind SAT, and the cycle starts again (Figure 8). Although the channeling ability of OAS in the SAT Cterminally bound active site of OASS may suggest substrate channeling within the CRC, the crystal structure and detailed kinetic studies are necessary to validate this hypothesis. This study unravels a competitive allosteric mechanism by which a low-affinity substrate dissociates the high-affinity OASS−C10 inhibitor complex. Structural snapshots capture a mechanism, which works by principles of both competitive (orthosteric) and allosteric binding models. The exploitation of principles of orthosteric and allosteric binding has benefitted humankind to a great extent.29,30 Recent reports on facilitated and accelerated dissociation of ligands by their competitors suggest that “nonclassical” models are necessary to explain those mechanisms.5−8 Therefore, understanding salient structural and biochemical features of “nonclassical” dissociation mechanisms like the “competitive allosteric” mechanism demonstrated in this study would allow us to design nextgeneration competitive allosteric drugs that may work by dissociating protein−protein and protein−ligand complexes actively. Allosteric drugs are more capable of discriminating between closely related proteins than orthosteric drugs are, but a combination of orthosteric and allosteric drugs confer higher specificity.31−33 The discovery of a competitive allosteric mechanism opens doors to the possible design of competitive allosteric drugs and/or inhibitors that will have potential not only to compete with natural binding partners (protein, ligands, etc.) of the target protein and/or enzyme but also to facilitate the dissociation of prebound target−ligand complexes and trap the target protein and/or enzyme in a conformation that does

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.7b00500. Figures S1−S5 and Table S1 (PDF) Accession Codes

The atomic coordinates and structure factors reported in this paper have been deposited in the Protein Data Bank. PDB entries are listed in Table 1 [4NU8 (Binary-1), 5DBH (Binary2), and 4ORE (Ternary-1)] or Table S1 [4ZU6 (Ternary-2) and 5DBE (Tetra-nary)].

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AUTHOR INFORMATION

Corresponding Author

*Institute of Microbial Technology, Sector 39-A, Chandigarh, India 160036. E-mail: [email protected]. Telephone: +91-0172-6665474. Fax: +91-172-2690585. ORCID

S. Kumaran: 0000-0002-7972-1032 Author Contributions

A.K.S., M.K.E., and A.K. contributed equally to this work. S.K. conceived the concept, designed experiments and wrote the manuscript. A.K.S., M.K.E., A.K., V.P., and S.B. contributed to crystallization and structure determination. V.S., R.P.S., and M.M. performed dissociation experiments. M


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A.K.S., M.K.E., A.K., V.P., R.P.S., S.B., and M.M. received research fellowships from CSIR, India, and V.S. from the Department of Biotechnology (DBT), India. CSIR and DBT, India, funded this research and DBT, India, sponsored data collection at ESRF. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully acknowledge access to in-house X-ray and 3D structural biology facilities at IMTECH and ESRF. The authors thank Drs. R. P. Roy and Alok Mondal for their valuable comments and suggestions.

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Substrate-Induced Facilitated Dissociation of the Competitive Inhibitor

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