Two Distinct Assembly States of the Cysteine Regulatory Complex of

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Two Distinct Assembly States of Cysteine Regulatory Complex of Salmonella typhimurium are Regulated by Enzyme-Substrate Cognate Pairs Abhishek Kaushik, Mary Krishna Ekka, and Sangaralingam Kumaran Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b01204 • Publication Date (Web): 17 Apr 2017 Downloaded from http://pubs.acs.org on April 20, 2017

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Two Distinct Assembly States of Cysteine Regulatory Complex of Salmonella typhimurium Are Regulated by Enzyme-Substrate Cognate Pairs Abhishek Kaushik#, Mary Krishna Ekka#& & Sangaralingam Kumaran* G. N. Ramachandran Protein Center, Council of Scientific and Industrial Research (CSIR), Institute of Microbial Technology (IMTECH), Sector 39-A, Chandigarh, India, 160036. #

Authors contributed equally

&

Present Address: Council of Scientific and Industrial Research (CSIR), Institute of Genomics

and Integrative Biology, Mathura road, New Delhi-110025 *Address for correspondence to: Dr. S. Kumaran, Institute of Microbial Technology, Sector 39-A, Chandigarh, India, 160036.

E-mail: [email protected], Tel: +91 -0172 -6665474;

FAX: 91-172-2690585. Abbreviations: OASS: O-acetylserine sulfhydrylase, SAT serine acetyltransferase, CRC: Cysteine Regulatory Complex, OAS: O-acetylserine, Na2S: sodium sulfide.

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ABSTRACT Serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS), which catalyze last two steps of cysteine biosynthesis, interact and form Cysteine Regulatory Complex (CRC).The current model of Salmonella typhimurium predicts that CRC is composed of one [SAT]hexamer unit and two molecules of [OASS]dimer. However, it is not clear why [SAT]hexamer can not engage all of its six high-affinity binding sites. We examined the assembly state(s) of CRC by size exclusion chromatography (SEC), analytical ultracentrifugation (AUC), isothermal titration calorimetry (ITC), and surface plasmon resonance (SPR) approaches. We show that CRC exists in two major assembly states, low-molecular-weight (CRC1; 1[SAT]hexamer + 2[OASS]dimer) and high-molecular-weight (CRC2; 1[SAT]hexamer + 4[OASS]dimer). Along with AUC results, ITC and SPR studies show that [OASS]dimer binds to [SAT]hexamer in a step-wise manner but the formation of fully saturated CRC3(1[SAT]hexamer + 6[OASS]dimer) is not favorable. The fraction of CRC2 increases as [OASS]dimer/[SAT]hexamer increases to more than 4-fold, but CRC2 can be selectively dissociated into either CRC1 or free enzymes, in the presence of OAS and sulfide, in a concentration-dependent manner. Together, we show that CRC is a regulatable multi-enzyme assembly, sensitive to OASS-substrate(s) levels but subject to negative cooperativity and steric hindrance. Our results constitute the first report on dual assembly state nature of CRC and suggest that physiological conditions, which limit sulfate uptake, would favor CRC1 over CRC2


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INTRODUCTION Sulfur is an essential micronutrient for growth and development of all organisms

1, 2

. Bacteria

and plants meet major portion of their sulfur needs by reducing inorganic sulfate using a wellstructured sulfate assimilation, reduction, and cysteine biosynthesis pathway

3, 4

. Cysteine

biosynthesis is at the branch point of sulfate metabolism and plays a key role in converting the sulfur into cysteine, a precursor for all thiol containing metabolites 1. The key feature of cysteine biosynthesis step is the reversible protein-protein interaction between SAT and OASS to form a hetero-oligomeric multienzyme complex, referred to as cysteine synthase complex (CSC)

5-7

. In

the CSC, the cysteine synthesis activity of OASS is significantly reduced but the activity of SAT is not altered significantly

8, 9

. Further, biochemical studies ruled out any metabolic channeling

within the CSC 8, 10, 11. Since growing experimental evidences indicate that regulation of cysteine synthesis activity of OASS is found to be the key feature of protein-protein interaction between SAT and OASS, a recent study suggested that CSC should be referred to as Cysteine Regulatory Complex (CRC) to better reflect its physiological significance

12

. In this study, we henceforth

refer to CSC as CRC, Cysteine Regulatory Complex.

Cysteine regulatory complex (CRC), purified from Salmonella typhimurium, was characterized as a multi-enzyme assembly, that composed of two oligomeric enzymes, serine acetyltransferase (SAT) and O-acetylserine sulfhydrylase (OASS), which catalyze the last two steps of cysteine biosynthesis 5, 6. SAT produces O-acetyl serine (OAS) from L-serine and Acetyl CoA, and OASS catalyzes the formation of cysteine from OAS and sulfide

6, 13

.The molecular

weight of CRC from Salmonella was estimated at ~ 310 kDa and stoichiometric analyses revealed that CRC was composed of one SAT hexamer (MW ~ 160 kDa) and two OASS dimers (MW ~ 68 kDa) 5. Further, the study showed that cysteine synthesis activity of OASS in the 3 ACS Paragon Plus Environment

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CRC is reduced to 5%, suggesting that SAT binding inhibits OASS activity.

The crystal

structure of OASS from H. influenza, resolved in complex with SAT C-terminal peptide revealed the structural basis of OASS activity inhibition by SAT 14. This structure showed that the last 10 residues of SAT bind into the active site pocket of OASS and block the substrate from entering the active site. Biochemical and structural studies on OASS from across species (bacteria and plants) have established that SAT C-terminal binds to the active site of OASS with high affinity15-20. Further, it was shown that either deletion of the last 10 residues of SAT C-terminal or replacement of the last ILE, reduces the affinity of SAT towards OASS significantly 22

16, 17, 21,

. Systematic docking and structural based approaches identified key residues of SAT C-

terminal, including the invariant ILE, that are crucial for OASS interaction

19

. Although the

structural and molecular basis of OASS-SAT interactions has been elucidated in detail, information regarding the quaternary structure of CRC, regulatory mechanisms of CRC cycle, and the physiological significance remain unknown due to lack of structural and systematic analytical studies. CRC occupies a key position in the sulfate transport and cysteine biosynthesis cycle and the assembly of CRC is favored under normal physiological conditions. However, CRC is sensitive to the levels of OAS and it dissociates into free enzymes when OAS levels increase 8. Under sulfur limiting conditions, OASS cannot convert OAS into cysteine, and therefore, OAS levels increase. OAS dissociates CRC through competitive inhibition mechanism by displacing SAT C-terminal from the OASS active site. In the absence of crystal structure of CRC, biochemical and computational approaches have been employed to understand the quaternary structure/assembly states of CRC

7, 23-25

. Except for one study, the other four studies support a

model of CRC, which consists of two OASS dimers bound to one SAT tetramer/hexamer

23-25

.


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Studies on SAT from Glycine max indicated the possibility that CRC may exist in other assembly states in which one SAT hexamer is bound to more than two OASS dimers 7. By employing equilibrium and kinetic methods, this study showed that CRC formation follows a step-wise binding process in which the binding of successive OASS dimers are characterized by three distinct set of binding parameters. The study showed that the second and third OASS dimers bind with weaker affinity due to negative cooperativity between SAT binding sites 7. A recent study showed that SAT from Glycine max exhibits a propensity to undergo concentration dependent oligomerization 26. Therefore, it is not clear that the different assembly state observed for CRC from Glycine max is influenced by the oligomerization propensity of SAT or due to a pre-existing equilibrium between different assembly states. Interestingly, based on the crystal structure of SAT from E.coli, Pyle et. al. proposed a high-molecular-weight model of CRC, which would consist of a central SAT-hexamer, docked to six OASS dimers 27. This model is also supported to some extent by results of protein-protein interaction studies on different OASS/SAT systems. These studies showed that OASS binds to SAT with very high affinity (Kd < 10.0 nM) and suggested that SAT C-terminal tails are potential high-affinity binding sites for OASS

7, 8, 19, 23, 28, 29

. Thermodynamically, high-affinity

interaction and six free sites should favor the formation of CRC with more than two OASS dimers bound to one SAT-hexamer. Pre-steady-state kinetic studies showed that the binding of OASS to SAT follows two stage and three stage binding processes

19, 28

. Following the rapid

encounter step between OASS and SAT, the encounter complex was shown to undergo a slow isomerization step that stabilized the final complex. However, these studies have focused on monitoring the binding of single OASS dimer to one SAT hexamer unit and therefore, they provide insights only into a set of molecular events associated with the 1:1 binding event.


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Therefore, it is not clear why the other four tails of SAT hexamer do not engage OASS, although they all should, in principle, exhibit high-affinity towards OASS. Unlike multiple studies that aimed to build a model for the plant CRC, the assembly states of bacterial CRC have not been studied systematically 7, 23-26. In this study, we examined the assembly of CRC from Salmonella typhimurium for studying the CRC assembly state. We show here for the first time that the formation of CRC by multiple approaches in building a reliable model of CRC In this study, we investigated the equilibrium properties of bacterial CRC assembly in solution by multiple methods. For this study, we used SEC-purified SAT and OASS of Salmonella typhimurium and examined the assembly state(s) of CRC under defined solution conditions. We show here for the first time that the formation of Salmonella typhimurium CRC is described by binding of four successive OASS dimers to one hexameric unit of SAT. Our molecular size analyses result clearly show that CRC exists in at least two major states, a lower-molecularweight species, CRC1 (Mw ~ 310 kDa) and a higher-molecular-weight species, (Mw ~ 490 kDa). In contrast to previous models, we provide multiple pieces of evidence for the existence of higher-molecular-weight complex, CRC2 which is composed of one SAT hexameric unit bound to four OASS dimers. Further, we show that these two quaternary states exist in equilibrium and the fraction of each species can be changed by changing concentrations of OASS or its substrates, OAS and sulfide.


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MATERIALS Chemicals and buffers: All general chemicals used in the study were of analytical grade and purchased from Sigma-Aldrich, Merck, and HiMedia. Buffers were made with double-distilled water and filtered with 0.22 µM filters before use. EXPERIMENTAL PROCEDURES Protein expression and purification. Genes for OASS and SAT from Salmonella typhimurium (LT2 strain) were cloned at SalI and XhoI for OASS and BamHI and XhoI for SAT in pET28a (+) vector and were transformed into BL21 (λDE3) and Rosetta cells respectively. Cells were grown in LB media containing kanamycin (50.0 µg/mL) at 37.0 °C to an O.D. of 0.6 at 600nm. Cultures were induced with 1mM IPTG and 0.4 mM IPTG respectively and kept at 22˚C with shaking for 16 hours. Cells were harvested by centrifugation at 6000 rpm for 15 min and the cell pellet was resuspended in lysis buffer (50.0 mM Tris-Cl pH 8.0, 300 mM NaCl, 10% glycerol, 0.1% Tween-20) and lysed by sonication. The supernatant was recovered by centrifugation at 12000 rpm (16904 g) for 30 min. N-terminal His-tagged proteins were affinity purified using NiNTA agarose resin and were eluted with elution buffer (50.0 mM Tris-Cl pH 8.0, 300 mM NaCl, 10% glycerol, 250 mM imidazole). Eluted proteins fractions were analyzed on 12% SDS-PAGE gel. The fractions containing protein were pooled and further purified using size exclusion chromatography (SEC). Analyses of purified OASS and SAT on 12% SDS-PAGE showed that purity of proteins were >98%. All the experiments were performed using the concentrations determined using UV-Visible Spectrophotometer. The Molar extinction coefficients of StOASS


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and StSAT, estimated from their amino acid composition are 19940 and 26850 L mol-1cm-1 respectively 30.

Purification of the OASS, SAT and CRC. Affinity purified OASS and SAT were further purified by Size exclusion chromatography (SEC) using Hi load Superdex 200 pg 16/600 GL gel filtration column, at 4-5 °C. For CRC analysis, SEC purified OASS and SAT were mixed in different molar ratios (1 to 6) and incubated for 1-2 hours at 4 °C followed by equilibration at 20 °C for 5 minutes before injecting into Superdex 200 10/300 GL gel filtration column for the analytical SEC experiments at 20 °C. The quantities were mixed in a volume of 1.0 mL, filtered through small volume column filter (0.22 µm), and injected into gel filtration column preequilibrated with the buffer (50.0 mM Tris-HCl, pH 7.5, 100 mM NaCl, and 5% glycerol). The eluted peaks were collected and analyzed on 12% SDS-PAGE.

Steady-state kinetic studies.The enzyme assay for OASS and CRC was carried out using the acid ninhydrin assay for quantification of cysteine 31. This assay is based on the specific reaction of ninhydrin with cysteine under strongly acidic conditions. OASS hydrolyzes the substrate OAS in the presence of Na2S and synthesizes cysteine. The standard buffer was 0.1 M HEPES, pH 7.0. The assay for SAT and CRC was performed by monitoring the absorbance of acetyl-CoA while being consumed in the enzyme reaction. Both assays were performed as previously described

5, 32, 33

. The serine acetyltransferase activity was determined by Ellman’s reagent

(DTNB) reaction protocol, which reacts with the CoA group and gives increased absorbance at 412nm34. All reactions were carried out at 25°C; under similar buffer condition (50mM Tris-Cl, 100mM NaCl, 0.1mM EDTA) and enzyme concentration was in the range of 21-30 ng. The


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volume of the reaction mixture containing 1mM DNTB reagent was kept at 200 µL and L-serine concentration was varied from 0.001mM-3mM while keeping acetyl-CoA concentration constant at 0.2 mM. Two sets of controls were used; one buffer containing 2.0 mM L-serine and enzyme, and the other, 0.2 mM acetyl CoA with the same amount of enzyme in the reaction buffer. Absorbance readings were monitored with a microplate reader (Tecan Scientific instruments, Switzerland) at 412nm. Initial velocities vs. substrate concentrations were analyzed using classical Michaelis-Menten model by non-linear regression method to obtain Km and Vmax. The rate of product formation was calculated using the molar extinction coefficient of DTNB (14000 M-1cm-1). Analytical Ultracentrifugation. Sedimentation velocity experiments were performed using an Optima XL-I analytical ultracentrifuge equipped with absorbance optics with an An-50Ti 8 place rotor (Beckman Inc.). Purified OASS, SAT, and CRC (either collected from SEC elution or assembled from SEC-purified OASS and SAT) were extensively dialyzed in the standard buffer (50 mM Tris-Cl pH 7.5, 100 mM NaCl, 5% glycerol). Sedimentation velocity studies were carried out at 40,000 rpm (129048 x g) at 20 °C using two-channel charcoal-filled centerpieces with Sapphire glass windows. The velocity data was collected by scanning samples at a wavelength of 280 nm with a spacing of 0.003 cm and an average of three scans per step. The partial specific volumes at 20° C were determined from amino acid composition and solvent density was calculated using SEDNTERP 35. Assembly states of OASS, SAT, and two different CRC complexes were analyzed by direct curve fitting of sedimentation boundaries using Sedfit 36

. Fit to data were selected based on the root mean square deviations (RMSD) in the 0.003 to

0.006 range. Sedimentation coefficients were corrected to standard conditions at 20 °C in water, S20,w. Frictional ratio (f/fo) of [SAT]hexamer was fixed at 1.5 as calculated previously 37. The f/fo of 9 ACS Paragon Plus Environment

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[OASS]dimer is estimated to be ~ 1.35, from the limiting sedimentation coefficient, S20, w ~ 4.3 (Mol. wt ~ 68000 Daltons). In order to analyze c(s) patterns of samples containing complexes, first we analyzed the velocity data of SEC-purified fractions using continuous c(s) distribution with bi-modal f/fo model. Since both SEC fractions (9.9 and 8.5 mL) contain no free enzymes and velocity data of these fractions showed biphasic behavior, it is possible to optimize two frictional ratios, f/fo , using bi-modal f/fo option of Sedfit. By using bi-model f/f0 method, we were able to resolve f/fo for both species, CRC1 (f/fo ~1.5) and CRC2 (f/fo ~1.3). The goodness of fit was assessed from the RMSD of fit and analyses yielded sedimentation coefficients and corresponding normalized sedimentation coefficients (S20,w) of two complexes, CRC1 (S20,w ~ 9.5 -11.5 s) and CRC2 (S20,w ~ 16.5 -18.5 s). For analyzing the velocity data of assembled mixtures, we floated the frictional ratio and selected the best fit which resolved both CRC1 (S20,w ~ 9.5 11.5 s) and CRC2 (S20,w ~ 16.5 -18.5 s) species. Analyses of all velocity data of mixtures yielded the weight averaged f/fo which was in the range ~ 1.4-1.45. By constraining f/fo between 1.41.45, we analyzed the rest of the experiments described in our study. Fits to different data, using non-interacting continuous distribution c(s) model consistently yielded correct limiting sedimentation coefficients for free [OASS]dimer (S20, w ~ 4.3 s) and other two, CRC1 (S20,w ~ 9.5 11.0 s) and CRC2 (S20,w ~ 16.5 -18.5 s) species. Isothermal titration calorimetry. Isothermal titration calorimetry (ITC) experiments were performed using a VP-ITC calorimeter (Microcal, Inc). SEC-purified proteins were extensively dialyzed against buffer (50.0 mM HEPES, pH 7.5, 70.0 mM NaCl, 5% glycerol). Temperature dependent experiments were performed in 50 mM Tris pH 7.5, 70 mM NaCl, 5% glycerol. All samples and buffers were degassed at room temperature before use. OASS (10 µL/injection) was titrated into a cell containing SAT with an automated 250 µL microsyringe at an interval of 3-4


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minutes. Control experiments were performed by injecting the same amount of buffer into the cell to calculate the heat of dilution for each injection. Data obtained from titrations were analyzed using either a single-site binding model (Equation 1) or a three-site sequential binding model (Equation 2 & 3), Qitot = VoMtot ((K1P) ∆H1/ (1 + K1P)) Qi Total = Vo Mtot {[∆H1K1P+ (∆H1+ ∆H2) K1K2P2] / (1+K1P+K1K2P2)}

(Eq. 1) (Eq. 2)

Qitot = VoMtot ((∆H1K1P + (∆H1 + ∆H2) K1K2P2) + (∆H1 +∆ H2+∆ H3) K1K2K3P3) / (1 + K1P + K1K2P2 + K1K2K3P3)

(Eq. 3)

where Qitot is total heat after the ith injection, Mtot, total amount of SAT in the cell, Vo is the volume of the calorimetric cell, K1, K2, and K3 are the observed equilibrium constants for each site, P is the concentration of free OASS dimer, and ∆H1, ∆H2, and ∆H3 are the corresponding enthalpy changes. The corresponding Kobs and ∆H were obtained by fitting the experimental data to either model using software provided by the instrument manufacturer (Microcal, Inc.). The observed binding constants were converted to change in free energy (∆G) using ∆G= -RTlnKobs, where R is the gas constant (1.9872 cal K-1 mol-1) and T is the absolute temperature. Changes in entropy (∆S) were calculated using ∆G=∆H-T∆S. Surface Plasmon Resonance kinetics. Using Biacore 3000 (GE healthcare), we monitored the kinetics of OASS binding to SAT. SAT was immobilized on the Ni-NTA Biacore sensor chip and OASS (His-tag cleaved) was flown onto chip channels using the auto-controlled injector. All experiments were performed at 25° C and under similar buffer conditions (50.0 mM Tris pH 7.5, 100.0 mM NaCl). Stock of 200 nM SAT was used for immobilization on the chip to reach an R.U. of 1350-1400 and 10-20 uL of OASS at varied concentrations (0.05 - 0.8 µM), was injected at the flow rate of 20.0 µL/min and binding was monitored over 6 minutes. Dissociation of


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OASS was followed in the running buffer for 15.0 minutes at a flow rate of 30.0 µL/min. Both kinetics of association and dissociation were monitored in the differential mode (samplereference). The data were processed and analyzed using SCIENTIST (Micromath, USA). Single exponential or two-exponential models (Eq. 7) were used to analyze kinetic data (Equations 4-6). (4) (5) (6) Where C is the concentration of [OASS], t is time (secs), R and Rmax, response and maximum response units, ka is the on-rate and kd is the off-rate constants. Off-rate constants determined from fitting the dissociation phase were used as constraints while fitting the association phase data. RESULTS Characterization of CRC assembly by size exclusion chromatography CRC assembles from SAT and OASS which catalyze two consecutive steps of cysteine biosynthesis in bacteria. Although it has been shown that bacterial SAT is a hexameric enzyme and OASS is a dimer in solution, the stoichiometry of [SAT]hexamer to [OASS]dimer remains unresolved (Figure 1). In this study, we aimed to address this issue by systematically analyzing the solution properties of OASS, SAT, and CRC. First, we examined the kinetic properties of size exclusion purified OASS and SAT. The estimated kinetic parameters (Km,OAS =1.3 ± 0.7 mM, kcat= 20.0 ± 3 s-1 and for SAT, Km,ac = 0.26 ± 0.04 mM and kcat,ac = 19.23 ± 1.75 s-1; Km,ser = 0.46 ± 0.04 mM and kcat,ser = 59 ± 1.75 s-1 ) under our solution conditions show that both enzymes are active and kinetic parameters are comparable to those reported previously, except 12 ACS Paragon Plus Environment

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the kcat,ser of StSAT (17, 39-41). Next, we characterized the assembly states of free enzymes and CRC by size exclusion chromatography (SEC) method. Elution volume of proteins was monitored at 280 nm and the presence of pyridoxal phosphate (PLP) at the active site of OASS allowed us to monitor the elution profile of OASS at 412 nm.

Figure 1. Schematic cartoon illustrating two steps of cysteine biosynthesis and formation of CRC. The question mark indicates the unresolved stoichiometry of CRC.

To estimate the respective molecular masses of the enzymes and CRC, we compared their elution profiles with that of molecular weight standards, run under identical conditions. SEC results show that SAT elutes as a homo-hexamer (elution volume ~ 12.2 mL and estimated Mw ~ 160 kDa) and OASS elutes as a homodimer (elution volume ~ 14.0 mL and estimated Mw ~ 66 kDa) respectively (Figure 2A). We mixed purified free enzymes at different molar ratios and analyzed their respective SEC elution profiles. We mixed [SAT]hexamer with [OASS]dimer (concentrations normalized to hexamer and dimer states) at 1:1 and 1:2 molar ratios and incubated at 4 °C for 1-2 Hrs (see methods) before loading on to the size exclusion column. The overlay of elution profiles of 1:1 and 1:2 mixtures with that of free enzymes shows an additional peak (9.9 mL) which elutes as high-molecular-weight species (Figure 2A). It is intriguing to note that the apparent molecular weight of the 9.9 mL fraction was estimated to be ~ 480 kDa


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(Elution peak of protein standard: Ferritin supplementary Figure S1). Analyses of 9.9 mL fraction on the SDS-PAGE showed the presence of both SAT and OASS, suggesting that the 9.9 mL fraction represents the CRC (Figure 2A, inset). The elution profile of 1:1 mixture also showed an additional shoulder peak, centered close to the elution peak of SAT (Figure 2A: green curve). The broad distribution of 11.8 mL peak indicates that more than one species are present in equilibrium with one another and resolution of the column/conditions is not sufficient to separate the two species. The SDS-PAGE analyses showed that 11.8 mL fractions consisted mostly of SAT. The absence of any prominent peaks, corresponding to either SAT or OASS indicates that most of the OASS is in complex with SAT, i.e., in the form of CRC.


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Figure 2. Analytical characterization of StOASS, StSAT, and Cysteine Regulatory Complex (CRC). A) Elution volumes of proteins are plotted against absorbance units. Elution profiles of 1:1 and 1:2 mixtures of [SAT]hexamer to [OASS]dimer are superposed with that of free enzymes. Elution positions of CRC (9.9 mL) and free enzymes are observed indicated. Inset: - analyses of 9.9 mL fraction on 12 % SDS-PAGE gel shows the presence of OASS and SAT. Lane 1: Molecular weight marker, Lane 2: OASS, Lane 3: SAT, Lane 4: CRC (9.9 mL peak). B) Sedimentation coefficient distribution, c(S) analyses shows that OASS (25µM) and SAT (19 µM) sediment with S20,W ~ 4.3 (mw ~ 68 kDa, dimer) and S20,W ~ 7.6 (mw ~ 168 kDa, hexamer) respectively. C) Elution profiles of 1:3 and 1:4 mixtures of [SAT]hexamer to [OASS]dimer, monitored at 280 and 412 nm, are superposed with each other. Elution of a new higher-molecular-weight complex (8.5 mL) is observed in addition to low-molecularweight complex (9.9 mL). The two fractions, eluting at 8.5 and 9.9 mL represent two CRC complexes.

To assess the oligomeric states of free enzymes (OASS and SAT) rigorously, we performed sedimentation velocity (SV) method as an independent experiment to determine the molecular masses of OASS and SAT (Figure 2B). Estimated molecular masses (Mw of OASS ~ 68 kDa and SAT ~168 kDa respectively) from sedimentation coefficients are similar to the molecular masses obtained from the SEC method.

Results from two independent (SEC and AUC)

approaches performed at different total protein concentrations show that OASS is a stable dimer whereas the SAT is a stable hexamer under solution conditions examined here. These


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observations preclude any assumption of pre-existing equilibrium that may affect the symmetry of either OASS or SAT upon complex formation. Having determined the assembly states of free enzymes, we examined the assembly state of CRC under identical conditions. Since SAT is hexamer in solution and it has six equivalent binding sites for OASS, addition of substoichiometric amount of [OASS]dimer (only 2-fold excess) of OASS, as compared to six available binding sites in the [SAT]hexamer results in the complete binding of [OASS]dimer to [SAT]hexamer. To further understand the effect of molar ratio on CRC assembly state, the proteins, [SAT]hexamer and [OASS]dimer were mixed at 1:3 and 1:4 molar ratios, and performed SEC under identical conditions. Analyses of elution profiles indicated that in addition to 9.9 mL peak, another higher-molecular-weight fraction with a peak centered at 8.5 mL was observed (Figure 2C). However, the estimated molecular mass of this CRC was close to ~ 600 kDa (Elution Peak of Protein Standard: Thyroglobulin Figure S1). The SEC results provide direct evidence for the existence of two types of CRC which differ by mass, suggesting that the 8.5 mL fraction of CRC species is likely to consist of more [OASS]dimer molecules than the number of [OASS]dimer bound to CRC eluting in the 9.9 mL fraction. These results also suggest that the assembly state of CRC is sensitive to the relative molar ratio of [SAT]hexamer to [OASS]dimer and increasing OASS concentration while keeping SAT concentration fixed leads to the formation of CRC with higher order assembly state. Analyses of CRC assembly states by sedimentation velocity SEC results clearly showed that the molecular mass of CRC depends on the stoichiometric ratio of [SAT]hexamer to [OASS]dimer. In general, molar-ratio dependent change in the assembly state would indicate the presence of more than one type of CRC complexes. Further, two types of


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Biochemistry

CRCs, characterized by the number of [OASS]dimer bound to [SAT]hexamer, are expected to vary in shape and hence, each complex is expected to have different stokes radius. Therefore, estimation of molecular weights of CRCs from the standard calibration curves could be difficult38. Further, if these two types of CRCs are in equilibrium, the molecular mass estimated would be the weight-averaged molecular mass of each species. Sedimentation velocity (SV) has been used to resolve multiple oligomeric species that are in equilibrium 39. Therefore, we examined the CRC assembly by sedimentation velocity method. We analyzed the sedimentation profiles of SECpurified 8.5 mL and 9.9 mL CRC fractions. The c(s) analyses show that both 8.5 and 9.9 mL CRC fractions consist of two different species that sediment with different rates (Figure 3A, B). The 9.9 mL fraction consists of a major small molecular weight species (> 88 %) which sediments with S20,w ~ 9.6 s (Mw ~ 290 kDa) and a minor fraction (< 10 %) that sediments with S20,w ~ 16.4 s (Mw ~ 450 kDa) (Figure 3C). On the contrary, the 8.5 mL fraction consists of a larger molecular weight species (S20,w ~ 16.9 s and Mw ~ 450 kDa) which accounts for > 80 % of the total protein content and a minor small molecular weight fraction (S20,w ~ 10.1 s and Mw ~ 290 kDa) (Figure 3D). The presence of two types of CRC in each of SEC-purified fraction supports the assumption that these two types co-exist or in equilibrium with each other.


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Figure 3. Analytical ultracentrifugation characterization of SEC-purified Cysteine Regulatory Complex (CRC). Sedimentation velocity data monitored at 280 nm were fit to a continuous distribution of non-interacting species model using Sedfit and sedimentation coefficient distribution, c(S) as a function of normalized sedimentation coefficient (S20,W). A) Velocity data of SEC-purified 9.9 mL complex was fit (continuous lines) to a continuous distribution of non-interacting species model and residuals shown at the bottom. D) Velocity data of SEC-purified 8.5 mL complex fit (continuous lines) was to a continuous distribution of non-interacting species model and residuals shown at the bottom. C) Sedimentation coefficient distribution, c(S) analyses show that 9.9 mL fraction consists of two species; a major species (77 %, S20,W ~ 9.6 s and mw~ 290 kDa) and a minor species (22 %, S20,W ~ 16.4 s and mw~ 450 kDa). D) Sedimentation coefficient distribution, c(S) analyses show that 8.5 mL fraction also consists of two species; a major species (70 %, S20,W ~ 16.9 s and mw ~ 450 kDa) and a minor species (22 %, S20,W ~ 10.1 s and mw ~ 290 kDa).

The observation that low-molecular-weight fraction (9.9 mL) consists predominantly of CRC species with lower sedimentation coefficient (S20,w ~ 9.6-11.5) whereas high-molecular-weight fraction (8.5 mL) consists predominantly of CRC species with higher sedimentation coefficient (S20,w ~ 16.4 -18.5 s) not only supports the assumption that these two types co-exist or in equilibrium but also the sequential assembly process. Therefore, we conclude that CRC exists in two states or two distinct configurations, which differ only by the number of [OASS]dimer molecules bound to one [SAT]hexamer. In this study, we refer to the low-molecular-weight CRC which sediments with S20,w ~ 9.5 -11.5 s (mw ~ 290 kDa) as CRC1 and to the high-molecular18 ACS Paragon Plus Environment

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Biochemistry

weight CRC which sediments with an average S20,w ~ 16.5 -17.5 s (mw ~ 450 kDa) as CRC2. It is important to note here that symmetry of either OASS or SAT would not change. Under all conditions, the free enzymes (both OASS and SAT), which are in equilibrium with CRC complexes (whether CRC1 and CRC2) remain in their original oligomeric state. OASS remain as a homo dimer ([OASS]dimer), either in the free form or when bound to SAT. Similarly, SAT remains as a hexamer ([SAT]hexamer) either in the free form or when in the CRC (Table1). Table 1: Sedimentation velocity analyses of hydrodynamic parameters of free enzymes and CRC Protein

f/fo

Smax

Sexp

Sobs

S20,w

Svedberg

Svedberg

StOASS

4.9

3.6 s

StSAT

9.1

CRC (1:2) CRC (1:4)

Svedberg

Svedberg

1.35 ± 0.03

3.7 ± 0.2

4.3 s

67

6.0 s

1.5 ± 0.03

6.2 ± 0.2

7.5s

170

13.2

8.4 s

1.56 ± 0.03

9.0 ± 0.2

10.4 s

300

17

13.07 s

1.3 ± 0.04

17.5 s

480

13.5 ± 0.1

Mol. Wt. (kDa)

The CRC1 may correspond to [SAT]hexamer bound to two [OASS]dimer molecules of theoretical molecular weight (180 + 140 = 320 kDa) whereas the CRC2 may represent [SAT]hexamer bound to four [OASS]dimer molecules (180 + 280 = 460 kDa) which are very close to the calculated molecular weights from SV experiments. Under all conditions, the free enzymes (both OASS and SAT), which are in equilibrium with CRC complexes (whether CRC1 and CRC2) remain in their original oligomeric state. Therefore, stoichiometry of individual components within the CRC complexes will be multiples of [OASS]dimer to one [SAT]hexamer. SAT as the core, remain as the hexamer in both complexes, and two CRC complexes (CRC1 and CRC2) differ only in the number of OASS dimers bound to one SAT hexamer (Table1).


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Figure 4. AUC characterization of CRC assembled from free StOASS and StSAT and the effect of OAS. Sedimentation velocity data collected at 280 or 412 nm for CRC assembled from OASS and SAT, mixed in the ratio of 1:2, 1:4, and 1:6 ([SAT]hexamer to [OASS]dimer). Sedimentation coefficient distribution, c(S) analyses were performed by fitting velocity data to continuous distribution of non-interacting species model using Sedfit. A) Superposition of c(S), patterns of CRC assembled from 1:2 (black) and 1:4 (red) mixing ratios. Each consists of low-molecular-weight (CRC1, S20,W ~ 9.6-11.5 s) and high-molecular-weight (CRC2, S20,W ~ 16.5-18.5 s) species but the ratio of CRC2/CRC1 increases from 0.1 to 1.7 while increasing the stoichiometric ratio from 1:2 to 1:4. B) c(S) analyses of CRC assembled from the mixing free enzymes in the 1:6 ratio ([SAT]hexamer to [OASS]dimer). Overlay of c(S) patterns obtained at three different total protein concentrations; red ([SAT]hexamer ~ 1.3 µM and [OASS]dimer ~ 7.8 µM]); green ([SAT]hexamer ~ 1.6 µM and [OASS]dimer ~ 9.6 µM]); black ([SAT]hexamer ~ 2.5 µM and [OASS]dimer ~ 14.4 µM]). Fractional ratios of CRC1, (S20,W ~ 9.5-11.5 s) and CRC2, (S20,W ~ 16.5-18.5 s) do not depend on the total protein concentration. C) CRC assembled from 1:4 mixing ratio ([SAT]hexamer ~ 1.3 µM and [OASS]Dimer ~ 5.1 µM]) was incubated in the absence and presence of OAS (5.0 mM) and c(S) patterns were overlaid. Reduction in CRC2, (S20,W ~ 16.5-18.5 s) population is accompanied by increase in CRC1, (S20,W ~ 9.511.5 s) and free OASS (S20,W ~ 4.3 s) fractions. D) Similar experiment as described in (C) but performed in the presence of 15.0 mM OAS. Both CRC1, (S20,W ~ 9.5-11.5 s) and CRC2, (S20,W ~ 16.5-18.5 s) peaks disappeared and fractions of free OASS (S20,W ~ 4.5 s) and SAT (S20,W ~ 8.0 s) are only present.

We examined the hydrodynamic properties of pre-mixed OASS and SAT complexes, in order to understand the molar ratio dependent assembly of CRC. SAT and OASS were mixed at different molar ratios (1:2, 1:4, 1:6, and 1:10, [SAT]hexamer to [OASS]dimer) and sedimentation profiles of mixtures were analyzed. Bypassing the SEC step would allow us to capture all types of complexes that are formed under our experimental conditions. We performed three independent experiments at a given molar ratio and analyzed the sedimentation profiles using the continuous 20 ACS Paragon Plus Environment

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Biochemistry

distribution of non-interacting species model as described in methods. The c(s) analyses of sedimentation profiles of multiple experiments showed that only CRC1 (S20,w ~ 9.5-11.5 s) and CRC2 (S20,w ~ 16.5-18.5 s) complexes were consistently observed at low as well as high molar ratios studied here (Figure 4A). We believe that the narrow spread of S20,w observed for mixed but not for purified CRC by SEC might result from mixing-related variations in stoichiometry and on-going interactions between SAT and OASS. The mean values of S20,w of CRC1 and CRC2 are 10.1 s and 16.9 s, consistent with S20,w values of CRC1 and CRC2 obtained from SECpurified samples. Therefore, we conclude that CRC exists in two different quaternary states, CRC1 and CRC2 which are in equilibrium and the position of the equilibrium is dictated by OASS concentration. It is interesting to note that increasing the molar ratio further up to 1:6 ([SAT]hexamer to [OASS]dimer) does not lead to the formation of additional higher-order CRC complex in which one [SAT]hexamer] molecule is bound to six molecules of [OASS]dimer (Supplement Figure S2). We also examined the effect of total protein concentration on the assembly state of CRC to check whether any other intermediate species exist by keeping the relative molar ratio of [SAT]hexamer to [OASS]dimer 1:6 at 412 nm and 280 nm. The influence of reaction kinetics (fast and slow reactions) on sedimentation coefficient distribution can be examined from concentration dependent experiments (We varied the concentrations of both enzymes proportionately ~ four-fold range). Concentration-dependent peak positions or their relative abundance would serve as indicators. Superposition of three c(s) curves, obtained at three different total protein concentrations shows no shift in either peak positions or relative abundances of each species (Figure 4B). In summary, these results clearly show that the CRC exists in two different quaternary states, CRC1 (1 [SAT]hexamer + 2 [OASS]dimer) and CRC2 (1 [SAT]hexamer + 4 [OASS]dimer) which are at


Biochemistry

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equilibrium, determined by the concentration of [OASS]dimer. Although one molecule of [SAT]hexamer possesses six C-terminal binding sites, a maximum of only four [OASS]dimer can bind to one [SAT]hexamer, even in the presence of 10-fold excess [OASS]dimer, suggesting that binding of fifth and sixth [OASS]dimer are very unfavorable events. Effect of metabolites on CRC assembly states OAS, the substrate of OASS has been shown to disrupt the CRC5, 8. We examined the effect of OAS on both CRC1 and CRC2 by sedimentation velocity approach. The specific objective of this experiment was to understand whether CRC1 and CRC2 are equally sensitive to OAS and also, whether sulfide, the other substrate of OASS, would also have an effect on CRC stability. First, we examined the effect of OAS on stabilities of CRC1 and CRC2 over the concentration range of OAS (0-15 mM). The pre-mixed 1:3 molar ratio mixture ([SAT]hexamer to [OASS]dimer) was incubated in the absence and presence of OAS at a given concentration. Interestingly, at lower concentrations (≤ 5.0 mM), OAS selectively dissociates CRC2 without disrupting CRC1 significantly (Figure 4C). The area under the peaks of CRC1 and free OASS increase whereas the area under CRC2 peak decreases significantly. On the contrary, at higher concentrations (≥ 12.0 mM), OAS dissociates both CRC1 and CRC2 completely into free OASS and SAT. Incubation of assembled CRC in the presence of 15.0 mM OAS leads to complete disappearance of CRC1 and CRC2 peaks, leaving only free OASS and SAT (Figure 4D). These results clearly indicate that the two assembly states of CRC, CRC1 and CRC2 are in equilibrium, and CRC2 is converted into CRC1 before dissociating completely into free enzymes. Next, we examined the effect of sulfide on the CRC assembly state. We performed similar experiments and analyzed the sedimentation profiles obtained in the absence and the presence of Na2S (sodium sulfide), used as the substrate in the OASS-catalyzed cysteine synthesis reaction. First, we tested the effect of


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Biochemistry

sulfide (10.0 and 20.0 mM) on the 1:2 molar ratio complex ([SAT]hexamer to [OASS]dimer). At both concentrations, sulfide selectively destabilizes CRC2 (S20,w ~ 16.5-18.5 s) and promotes the dissociation of CRC2 to CRC1. The area under CRC2 peak is decreased significantly, accompanied by an increase in area under CRC1 peak (Figure 5A).

Figure 5. AUC analyses on the effect of sulfate and sulfide on two assembly states of CRC. All experiments described here performed using CRC assembled by mixing free enzymes at indicated ratios and velocity data were obtained by monitoring at 280 nm. A) an Overlay of c(S) analyses of 1:2 mixture ([SAT]hexamer ~ 1.5 µM and [OASS]dimer ~ 3.0 µM]) in the absence and presence of Na2S (10 and 20 mM). Reduction in CRC2, (S20,W ~ 16.5-18.5) fraction is accompanied by increase in CRC1 (S20,W ~ 9.5-11.5 s). B) Overlay of c(S) analyses of 1:4 mixture ([SAT]hexamer ~ 1.3 µM and [OASS]dimer ~ 5.2 µM]) in the absence and presence of Na2S (10.0 mM). Reduction in CRC2, (S20,W ~ 16.5-18.5 s) fraction is accompanied by increase in CRC1 (S20,W ~ 9.5-11.5 s) and StOASS (S20,W ~ 4.3 s). C) Overlay of c(S) analyses of 1:2 and 1:4 mixtures ([SAT]hexamer ~ 1.3 µM and [OASS]dimer ~ 2.6 / 5.2 µM) in the presence (10.0 mM) of ammonium sulfate (NH4SO4). No reduction in either CRC2, (S20,W ~ 16.5-18.5 s) or CRC1 (S20,W ~ 9.5-11.5 s) fractions are observed.

Next, we tested the effect of sulfide (20.0 mM) on the 1:4 molar ratio complex ([SAT]hexamer to [OASS]dimer). Once again, it is observed that sulfide destabilizes selectively CRC2 (S20,w ~ 16.518.5 s), leading to increase in CRC1 (S20,w ~ 9.5-11.5 s) fraction (Figure 5B). These results are consistent with the fact that disassembly of CRC is a sequential and step-wise process. Sulfide


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selectively dissociates CRC2 at physiological concentrations. Since sulfate is the original substrate for cysteine biosynthesis during sulfur assimilation, we also tested the effect of sulfate on CRC stability. Results show that sulfate has no effect on CRC assembled from 1:2 or 1:4 molar ratio ([SAT]hexamer to [OASS]dimer) mixtures (Figure 5C).

Energetics of sequential assembly of CRC Results of both SEC and AUC studies revealed the OASS-dependent step-wise/sequential assembly and metabolite-dependent step-wise dissociation of CRC. These two molecular massbased methods provided clear and consistent evidence for the existence of two distinct CRC species. Isothermal titration calorimetry (ITC) method allows complete thermodynamic profiling of molecular interactions to be determined in a single experiment. We performed ITC experiments to understand the energetics of CRC assembly. We titrated OASS into the cell containing SAT at a constant temperature and measured the heat released during the binding reaction. We estimated the thermodynamic parameters by analyzing the heat change as a function of the molar ratio of [SAT]hexamer to [OASS]dimer. Since the assembly of CRC may involve the binding of a maximum of four [OASS]dimer molecules to one [SAT]hexamer, we performed stoichiometric titrations at 25 °C to estimate net enthalpy change (⧍H) associated with the binding of first [OASS]dimer. By limiting the final molar ratio of [SAT]hexamer to [OASS]dimer to 1.5 and accommodating 20 incremental injections of OASS, we were able to obtain reliable ⧍H for the binding of the first [OASS]dimer (Figure 6A). The binding isotherm was fit to the single-site binding model and enthalpies of binding, as well as the lower limit of the binding constant, were estimated. This reaction is exothermic, with ⧍H1 ~ -18.1 ± 0.2 Kcal/mol and K1,obs ~ 1.3 ± 0.2 × 108 M-1. We used these two values as constraints for analyzing the binding isotherms of titrations performed at the higher molar ratios. 24 ACS Paragon Plus Environment

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Biochemistry

Figure 6. Isothermal titration calorimetry analyses of CRC formation. Raw data of titration (upper panel) is plotted as heat signal versus time. In the lower panel, integrated heat responses normalized per moles of the ligand are plotted against molar ratio and heat responses from reference titrations of OASS into the buffer were subtracted. In all titrations, SAT was placed in the cell and OASS was loaded into the injection syringe. A). Stoichiometric titration of concentration ([OASS]dimer ~ 9 µM and [SAT]hexamer ~ 1.0 µM) yields reliable estimates of binding enthalpy and affinity of the first OASS dimer binding. The solid line represents the best fit of the data to single site binding model, yielding Kobs = 1.3 ± 0.2 × 108 M-1, and ∆Hobs = -17.9 ± 0.2 Kcal/mol. B) One of the four independent titrations, performed to examine CRC formation under excess OASS concentration ([OASS]dimer ~ 40.0 µM and [SAT]hexamer ~ 1.0 µM) is shown. Data to different models and the three-site binding model describes the data better than the other two. Estimated parameters; K1,obs = 1.5 ± 0.8 × 108 M-1, ∆H1,obs = -18.0 ± 0.7 Kcal/mol, K2,obs = 1.4 ± 0.3 × 107 M-1, ∆H2,obs = -18.0 ± 0.9 Kcal/mol, K3,obs = 2.4 ± 0.3 × 104 M-1and ∆H3,obs = -16.0 ± 30 Kcal/mol. C) Temperature dependent analyses: Titration performed at different temperatures showed that binding enthalpies are exothermic at over the temperature range (15-30 ºC). The plot of reaction enthalpies for the binding of first two binding events (∆H1 and ∆H2) against temperature is linear. Heat capacities estimated from slopes yield ∆Cp,1 ~ 1.1 ±0.1 and ∆Cp,2 ~ 1.0 ± 0.1 Kcal/mole respectively. D) Thermodynamic parameters of the first binding event plotted as a function of temperature. Unfavorable changes in reaction entropy (T∆S) is compensated by enthalpy (∆H) change and therefore net free energy change (∆H) of binding is constant.

We performed three independent titrations at higher molar ratios (6-8 fold excess of OASS) in order to cover the entire binding range (a maximum of four [OASS]dimer binding).

A 25

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representative titration, performed at 25 °C is shown (Figure 6B). We also performed titrations at different total protein concentration (concentration varied in the syringe and cell) (Supplement Figure S3 A, B, and C). In both cases, estimated binding enthalpies are very similar and the average enthalpy change (⧍H1) for the binding of first [OASS]dimer is ~ -17.6 Kcal/mol, similar to that estimated from stoichiometric titration described above. Therefore, we used ⧍H1 as one of the constraints during our data analyses. Binding isotherms obtained at high molar-ratios (> 6-fold excess OASS) cannot be described adequately by single-site binding model. We used two-site and three-site sequential binding models to fit the data. Although twosite model describes the data well, the three-site sequential model fits the data well with better errors (Figure 6B). Comparison of binding parameters indicates that the first two sets of [OASS]dimer bind with similar intrinsic affinities and the third OASS dimer binds with weaker affinity. The binding enthalpies of two [OASS]dimer are also similar (~ -17.8 Kcal/mol). However, the binding of third OASS dimer was accompanied by a 3-fold reduction in the enthalpy change and 20-fold less affinity. Although both AUC and SEC provided clear evidence, the binding of fourth OASS dimer could not be observed through ITC due to low signal to noise ratios. Estimation of heat capacity (⧍Cp) provides useful information on possible structural and thermodynamic changes that accompany during CRC assembly. We performed calorimetric titration experiments at different temperatures and analyzed the thermodynamic parameters as a function of temperature. We used the two-site sequential binding model for interpreting the thermodynamic behavior of CRC assembly, as it was not possible to control six parameters while using the three-site model. Thermodynamic parameters that describe the first two [OASS]dimer binding steps suggest that CRC assembly is an exothermic reaction and the binding enthalpies


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Biochemistry

become more negative as temperature increases, exhibiting a linear tendency on temperature (Figure 6C). Table 2: ITC analyses of CRC formation at constant and different temperature. T (oC )

25

Kobs (kA1,kA2,kA3) (M-1) 1.50 ± 0.8(x10 )

KA2

1.4 ± 0.3(x10 )

KA3

2.4 ± 0.2(x10 )

T (oC)

15

20

25

30

8

KA1

7

4

Kobs (kA1,kA2) (M-1) 8

KA1

8.0 ± 0.8(x10 )

KA2

1.7 ± 0.2(x10 )

KA1

3.2 ± 0.15(x10 )

KA2

4.7 ± 0.3(x10 )

KA1

1.2 ± 0.1(x10 )

KA2

3.2 ± 0.2(x10 )

KA1

8.3 ± 0.4(x10 )

KA2

1.6 ± 0.2(x10 )

7

8

7

8

7

7

7

Affinity (KD) (nM)

∆G (Kcal/Mol)

∆H (Kcal/Mol)

T∆S (Kcal/Mol)

6.6

-11 ± 0.3

-18 ± 0.7

-7.82

7.1

-9 ± 0.1

-18.1 ± 0.9

-8.35

4x104

-5 ± 0.5

-16.3 ± 0.7

-10.3

Affinity (KD) (nM)

∆G (Kcal/Mol)

∆H (Kcal/Mol)

T∆S (Kcal/Mol)

1.2

-11.5 ± 0.1

-9.2 ± 0.3

2.4

58

-9.4 ± 0.1

-10.0 ± 0.3

-5.0

3.1

-11.3 ± 0.1

-15.0 ± 0.3

-3.6

21

-10.2 ± 0.1

-15.1 ± 0.7

-5.2

1.6

-10.9 ± 0.1

-20.0 ± 0.3

-9.0

31

-10.1 ± 0.1

-19.2 ± 0.3

-9.0

1.2

-10.9 ± 0.2

-28.0 ± 1.0

-17.1

62

-9.9 ± 0.1

-27.0 ± 1.2

-17.0

The linearity of enthalpy also indicates that heat capacity (⧍Cp) of CRC formation is constant within the temperature range studied. The heat capacities of binding of first and second [OASS]dimer are very similar, ⧍Cp1, ⧍Cp2, ~ -1.1 Kcal/mol/K indicating the polar solvation of groups. The analyses of temperature dependent thermodynamic parameters indicate that both binding enthalpies (⧍H) and entropies (T⧍S) decrease as temperature increases while the


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binding free energies (⧍G) do not change within the range of temperatures studied suggesting that binding is mostly enthalpically driven (Figure 6D & Table 2).

Kinetics of CRC formation and disassembly Knowledge on the kinetics of binding helps us to identify the steps involved and the mechanism of the binding event. We studied the kinetics of interaction between OASS and SAT by surface plasmon resonance (SPR) method (Figure 7A). We followed the assembly of CRC by monitoring the binding of [OASS]dimer to [SAT]hexmer. SAT was immobilized on a Ni2+nitrilotriacetic acid chip (NTA chip) (see methods) and OASS was flown through channels with SAT. The dissociation of CRC was monitored by allowing the SAT-bound [OASS]dimer to dissociate into the buffer. We studied the association and dissociation kinetics of OASS as a function of OASS concentration (Figure 7B, C). Between each experiment, the amount of SAT immobilized on the chip was controlled (by monitoring the difference between initial and final signal) by injecting the right amount of SAT into the chip channel. Initially, we analyzed kinetics data collected at different OASS concentrations (OASS ~ 0.05 to 0.8 µM and SAT~ 0.2 µM] using single exponential binding models (one OASS dimer binding to one SAT hexamer). However, both association and dissociation data obtained at 0.1 µM of [OASS]dimer or more display bi-phasic kinetics and could not be described well by the single exponential model. We analyzed these data using two-exponential binding model and determined the kinetic parameters (Figure 7B, table 3). Both rate constants show no dependency on OASS concentrations except, the concentrations below 100 µM have relatively lower association rate constants. (Figure 7D). Since SPR analyses of formation of CRC of bacterial species are not available, we compared kinetic parameters obtained in this study with parameters reported for plant system.


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Biochemistry

Figure 7. Surface Plasmon Resonance (SPR) analysis of CRC formation. All experiments were performed at 25º C and estimated kinetic parameters are presented in table 3. A) Raw SPR kinetic data of OASS binding to SAT at varied OASS concentrations. B) Analyses of dissociation of CRC or OASS from SAT. Data obtained at lower OASS concentrations are well described by single-site, suggesting that one OASS dimer was bound to SAT but dissociation kinetics of OASS at higher concentrations requires double and three site models, suggesting that more than one OASS dimer were bound. C) Analyses of association phase of OASS binding and CRC formation using single and multi-exponential models. Kinetic data collected at lower concentrations range (OASS~ 50.0 and 100 nM) can be well described by single exponential model but data obtained at higher concentrations are fit to two or three exponential models. D) The plot of kinetic parameters against total OASS concentrations. Both dissociation and association rate constant show no dependency on OASS.

In this study, the average ka determined for CRC of Salmonella (ka ~ 6.2 x 104 M-1 S-1, table 3) is similar to that of plants (Glycine max; ka ~ 2.5 x 105 M-1 S-1 (ref 7) and Arabidopsis; ka ~ 5.1 x104 M-1 S-1 (ref 23); The estimated equilibrium dissociation constants (Kd,1 ~ 30.0 nM and Kd,2 ~ 70 nM) from averages of association and dissociation rate constants are very similar to values determined from ITC experiments. Two distinct phases of kinetics may indicate two separate binding events such as the formation of CRC1 and CRC2 but more detailed fast kinetics studies are needed to characterize the steps and intermediates. 29 ACS Paragon Plus Environment

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Table 3: Surface Plasmon Resonance analyses of CRC formation. ka1 (M-1s-1)

ka2 (M-1s-1)

kd (s-1)

KD (nM)

50

(3.3 ± 0.1) x104

-

(1.4 ± 0.1) x10-3

42

100

(3.5 ± 0.1) x104

-

(1.3 ± 0.1) x10-3

37

200

(9.4 ± 0.2) x104

(1.4 ± 0.1) x104

(1.3 ± 0.1) x10-3

13

400

(8.8 ± 0.1) x104

(8.0 ± 0.1) x104

(1.4 ± 0.1) x10-3

15

600

(5.9 ± 0.1) x104

(8.9 ± 0.1) x104

(1.4 ± 0.1) x10-3

23

800

(5.4 ± 0.1) x104

(8.4 ± 0.1) x104

(1.4 ± 0.1) x10-3

25

OASS C (nM)

ka= Association rate constants; kd= Dissociation rate constants; C= concentration of OASS monomer KD= Equilibrium Dissociation constant

Activity of SAT in the CRC Cysteine synthesis activity of both bacterial and plant OASS in the CRC have been shown to be reduced significantly. The reduction is due to the blocking of OASS active site to its substrate by the C-terminal tail of SAT. However, it is not clear whether the activity of bacterial SAT changes upon forming CRC. We compared the steady-state kinetics of free SAT activity with the activity of SAT in the presence of varying concentrations of OASS (Figure 8 A-D). The kcat increase to 1.6 fold as the stoichiometry of [SAT]hexamer to [OASS]dimer is increased to 1:2 and thereafter, it does not show any further increase (Table 4). Table 4: Kinetic parameters of SAT activity (experiments performed in triplicate) Enzyme

kcat (s-1)

Km(mM)

kcat/Km(Ms-1)

SAT

SAT

59.5 ± 2.0

0.4 ± 0.1

149000

CRC

1:2

98.0 ± 2.0

0.6 ± 0.2

164000

CRC

1:4

92.8 ± 1.8

0.5 ± 0.1

186000

CRC

1:6

96 .0 ± 3.2

0.7 ± 0.1

135000


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Biochemistry

Figure 8. Steady-state kinetic analyses of SAT within the assembled CRC. Steady-state kinetics of free SAT and SAT complexed with OASS at molar ratios of 1:2, 1:4, and 1:6, ([SAT]hexamer to [OASS]dimer) were performed under identical conditions (method). All experiments were done in triplicates and errors plotted for each data point. Initial velocity data plotted against [L-Serine] for free SAT ([SAT]hexmer ~ 21 ng) (A), SAT complexed with OASS at 1:2 ([SAT]hexamer ~ 21 ng and [OASS]dimer ~ 42 ng) (B), and SAT complexed with OASS at 1:4 ([SAT]hexamer ~ 21 ng and [OASS]dimer ~ 85 ng) (C), and SAT complexed with OASS at 1:6 ([SAT]hexamer ~ 21 ng and [OASS]dimer ~ 125 ng) (D) are shown. Kinetic parameters are listed (table 4). Both Vmax increase by ~ 1.6-fold when one molecule of [SAT]hexamer is in complex with 2 molecules of [OASS]dimer but further increasing the molar ratio of ([SAT]Hexamer to [OASS]dimer to either 1:4 or 1:6 do not change kinetic parameters of SAT. The curve fitting was performed with Origin®13 and goodness of fit is shown (Supplementary Table S1).

DISCUSSION Formation of CRC has been linked to the regulation of sulfate transport, reduction, and cysteine biosynthesis in bacteria and plants6, 8, 40. In this study, we examined the features of CRC as a function of protein and metabolite concentrations using multiple biophysical approaches. Results presented in this study show, for the first time, that CRC exists in dual assembly states and stability of each state is controlled by OASS and its two substrates, OAS and sulfide. Molecular mass based analyses methods clearly show that CRC exists in both low-molecular31 ACS Paragon Plus Environment

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weight (CRC1) as well as in high-molecular-weight state (CRC2). The presence of both CRC1 and CRC2 in each SEC fraction (9.9 and 8.5 mL) gives clear indication that both CRC species exist in equilibrium. Therefore, in the absence of any dissociating ligands, both types of CRC states, CRC1 (two [OASS]dimer bound to one [SAT]hexamer) and CRC2 (four [OASS]dimer bound to one [SAT]hexamer) are present in solution. In the absence of dissociating ligands, assembly of CRC begins as soon as [OASS]dimers are added to [SAT]hexamer in solution. The ITC and surface plasmon kinetic measurements show that the first stage of assembly, possibly the binding of two molecules of [OASS]dimer is accompanied by high affinity and slow dissociation rate (off-rates) constants, consistent with previous observations. The next phase of binding which involves the binding of the second set of [OASS]dimer binding are accompanied with high affinity but increased off-rates. However, the assembly of CRC under physiological conditions proceeds in the presence of dissociator ligands and therefore, stabilities of the two CRC species would depend on the net concentrations of these ligands in the cell. We showed that both substrates of OASS are dissociators of CRC but sulfide is the weaker of the two. CRC2 is more sensitive to both OAS and sulfide, suggesting that CRC2 is a short-lived complex. The dynamics of CRC to alternate between CRC1 and CRC2 is primarily dictated by OASS and its two substrates. In the forward reaction, the formation of either CRC1 or CRC2 is facilitated by an increase in OASS concentrations while in the reverse reaction, dissociation of CRC2 to CRC1 and CRC1 to free enzymes are facilitated by OAS and sulfide. OAS is undoubtedly a better dissociator as it is able to dissociate CRC2 to CRC1 and CRC1 to free enzymes at much lower concentrations. The sulfide preferably stabilizes the CRC1 by selectively dissociating CRC2 over a concentration range (2.0-20.0 mM) studied here. Similarly, OAS also selectively dissociates the CRC2 at lower


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concentrations. Concentrations of OAS in plants has been estimated to vary between 10.0 and 60.0 µM during normal conditions, and between 60-200 µM during sulfur limiting conditions, suggesting that a maximum of 20-fold change is possible7, 41. Therefore, dramatic changes in OAS and sulfide would shift the equilibrium in favor of CRC1 or free enzymes. Interestingly, the steady-state enzyme kinetics performed using assembled CRC suggest that the activity of [SAT]hexamer increases only 2-fold in the presence of an excess of [OASS]dimer suggesting that addition of more than two [OASS]dimer molecules do not result in the functional efficiency of the complex. This is interesting because although a maximum of four molecules of [OASS]dimer can interact with [SAT]hexamer to form CRC2, CRC2 does not have any functional kinetic advantage over CRC1. The fact that the assembly and kinetic parameters are not correlated beyond the formation of CRC1 suggests that active sites of all six SAT molecules within the [SAT]hexamer may have been tuned to work with maximum velocity (~ 10 % monomer). The most probable configuration in the CRC1 state is the one in which each trimer of [SAT]hexamer molecule is bound to one molecule of [OASS]dimer. Therefore, it is possible that binding of a single [OASS]dimer to one trimer of [SAT]hexamer triggers cooperative interactions between three monomers in the trimer and subsequently, optimizes SAT activity. None of the three kinetic curves of SAT activities of CRC determined at different [SAT]hexamer to [OASS]dimer molar ratios exhibit any sign of hysteresis, suggesting that conformational changes and associated kinetic changes are very nominal as observed here. However, more detailed studies are needed to dissect out cooperativity in CRC assembly as well as SAT kinetics. The current model of bacterial CRC assembly state is derived primarily from the pioneering studies of Kredich et. al5. This study proposed that one molecule of [SAT]hexamer is in complex with two molecules of [OASS]dimer. Other studies that focused on understanding the


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assembly state of plant CRC also supported this model. The stoichiometry of the plant CRC, addressed by a computational approach, suggested that binding of the third and fourth [OASS]dimer are not favored due to changes in the electrostatic potential of SAT after binding to two [OASS]dimer. The study concluded that since SAT exists as a dimer of trimers, binding of one [OASS]dimer to each [SAT]trimer is more favorable as the electrostatic potential of SAT is not changed24. The other experimental study which investigated assembly state of CRC from Arabidopsis thaliana observed only one CRC that is equivalent to CRC1 reported in this study25. Interestingly, results presented in the original study of Kredich et al. shows that indeed they observed two types of CRC complexes; a major low-molecular-weight species that sedimented with S20,w of 11.5 s (similar to CRC1 reported here) and a minor high-molecular-weight species which had S20,w of 16.0 s, present only at protein concentration above 5 mg/mL (similar to CRC2 reported here) 5. However, they characterized only the low-molecular-weight species (CRC1) and proposed the above CRC model. Our systematic analyses by multiple approaches allowed us to characterize both type of complexes (CRC1 and CRC2) and to propose a dynamic equilibrium model. We also agree with the previous study that CRC1 should structurally mimic a configuration that each [SAT]trimer is in complex with one [OASS]dimer 25. In addition, we believe that the configuration of CRC2 should mimic a state in which [SAT]trimer is in complex with two [OASS]dimer. Based on the current information and results presented here on CRC, we used two major parameters to build a model of a dynamic cycle of CRC. The first parameter is the concentration of [OASS]dimer and the second parameter is a net concentration of OAS and sulfide, both functions of sulfate flux in the cell. We employ following constraints to build the model: 1intracellular concentration of OASS will always be higher than that of SAT (reported previously,


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Biochemistry

ref 28,41) 2- OASS binds to SAT with high-affinity (as reported here and previously); 3- CRC exists in two assembly states (CRC1 and CRC2) (as reported here); 4-OAS, sulfide, and sulfate are dissociators of CRC but OAS is the strongest of the three; 5- Only OAS has the ability to dissociate both CRC1 and CRC2 under physiological conditions (1-15 mM) and other ligands can dissociate only CRC2. We envisage that the assembly of CRC may occur at three distinct reversible stages and the lifetime of CRC species formed at each stage would be dictated mainly by the levels of the two dissociators (OAS, Na2S). In the step1, the formation of CRC1 is favored under normal conditions, contributed mainly by three factors; 1-excess OASS over SAT: 2normal levels of all two dissociators: 3-availability of six free high-affinity binding sites in the SAT. On the contrary, dissociation of CRC1 will be favored only when OAS levels rise high under sulfur limiting conditions. In step-2, (Figure 9) the formation of CRC2 may be favored if; 1-levels of ligands reduce temporally: 2-availability of 4-high-affinity binding sites in the SAT. The reverse reaction, dissociation of CRC2 would be favored, if levels of dissociator ligands increase. The transformation from CRC2 to free enzymes directly occurs when OAS levels increase significantly (>8 mM) under sulfate limiting conditions (diagonal arrows). In step-3, the formation of CRC3 (six [OASS]dimer bound to one [SAT]hexamer) may be favored only if: 1-OASS levels rises to very high concentrations; 2-levels of dissociative ligands reduce significantly; 3binding of the third set of [OASS]dimer is accompanied by high positive cooperativity. On the contrary, the reverse reaction would be favored if; 1-two free sites within the CRC2 poses steric problem for additional [OASS]dimer binding; 2-negative cooperativity between adjacent free and bound sites. Reprogramming of metabolic pathways in response to changes in the levels of metabolites and metabolite-specific cycling of multi-enzyme assemblies have become widely recognized phenomenon 42, 43.


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Figure 9. A dynamic model for CRC assembly and dissociation. Cartoon representations of OASS (PDB code: 1OAS) and SAT (PDB code: IT3D), generated from structural coordinates of homologous proteins were used to describe CRC assembly and dissociation cycle. Forward (F) and reverse (R) reactions. Step1, F: free enzymes interact to form CRC1 (low dissociators and abundant OASS). Two of six high-affinity binding sites of SAT are occupied. R:-CRC1 dissociates when OAS levels rise. Step2, F: - two [OASS]dimer bind to CRC1 to form CRC2 (low dissociators and abundant OASS). Four of six high-affinity binding sites of SAT are occupied in the CRC2 state. R: - CRC2 dissociates when dissociator levels increase. Complete dissociation to free enzymes occurs when OAS levels increase significantly high (>8 mM) under sulfate limiting conditions (diagonal arrows). Step2, F: - two [OASS]dimer bind to CRC2 to form CRC3 (low dissociators and abundant OASS. All six high-affinity binding sites of SAT are occupied in the CRC3 state (CRC3 may be favored only if: 1-OASS levels rise very high with a significant reduction in dissociator level or high positive cooperativity between adjacent sites in SAT. R: two free sites within the CRC2 poses a steric problem for additional OASS binding or negative cooperativity.

We present here a dynamic and dual assembly state of CRC, regulated by OASS- and two metabolites. Our results support a model that is in sync with up and down cycles of sulfate flux in the cell. Thermodynamic forces that favor CRC assembly are mostly non-temporal therefore do not change significantly over time. Favorable forces are either inherent properties of components or system; high-affinity interactions between SAT and OASS, multiple binding sites of SAT, and relatively high abundance of OASS over SAT. On the contrary, forces that oppose CRC stability/assembly are temporal and amplitudes or strengths of these forces oscillate in sync


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with oscillations of sulfate flux, suggesting that sulfate flux induced changes in opposing forces alone dictate the cycle of CRC. Therefore, physiological assembly state(s) of CRC should, in principle, follow concentration fluctuations of sulfate, sulfur, and OAS. This implies that both stochastic and temporal trajectories of concentration fluctuations guide the dynamic equilibrium between different assembly states and free enzymes. However, the position of equilibrium would be set not only by the net balance of two opposing forces but also by inherent properties of protein-protein interactions, such as step-wise cooperativity and steric hindrance, etc. The selforganizing property of CRC may favor higher-order formation of CRC2 and even the CRC3 (six dimers of OASS bound to one hexameric SAT) due to the high-affinity interaction between OASS and SAT, and the abundant intracellular OASS concentration. However, the fact that we could not observe the CRC3 complex in the absence of dissociators and even when the concentration of OASS was maintained at several fold excess to SAT suggests that binding of fifth and sixth [OASS]dimers are highly unfavorable events. Although two SAT C-terminals are available as free binding sites, CRC3 formation is not observed. These results suggest that under normal physiological conditions where levels of OAS and sulfide are expected to be low, CRC1 will be preferentially stabilized over CRC2. Although CRC2 complex is consistently observed under in vitro conditions, we conclude here that basal levels of OAS and sulfide under physiological conditions should skew the equilibrium towards CRC1. Results of work presented herein could represent general features of the architecture of bacterial CRC cycle and provides a framework for understanding regulatory features that control dynamic cycle of CRC in bacteria.


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SUPPORTING INFORMATION Size-exclusion elution profiles of protein standards (Figure S1), analyses of molecular mass of CRC from sedimentation velocity profiles (Figure S2), Additional data on isothermal titration calorimetry analyses of CRC formation (Figure S3), information on goodness of fit of steadystate kinetic data (Table S1).

AUTHOR CONTRIBUTIONS S.K. wrote the manuscript, A.K. and M.K.E. performed the experiments and analyzed the results. ACKNOWLEDGEMENTS Abhishek Kaushik and Mary K. Ekka received research fellowships from CSIR, India. This work was funded by CSIR-IMTECH and Department of Biotechnology (DBT), India. REFERENCES 1. Leustek, T. (2002) Sulfate metabolism, Arabidopsis Book 1, e0017. 2. Leustek, T., and Saito, K. (1999) Sulfate transport and assimilation in plants, Plant Physiol 120, 637-644. 3.

Takahashi, H. (2010) Regulation of sulfate transport and assimilation in plants, Int Rev Cell Mol Biol 281, 129-159.

4. Takahashi, H., Kopriva, S., Giordano, M., Saito, K., and Hell, R. (2011) Sulfur assimilation in photosynthetic organisms: molecular functions and regulations of transporters and assimilatory enzymes, Annu Rev Plant Biol 62, 157-184. 5.

Kredich, N. M., Becker, M. A., and Tomkins, G. M. (1969) Purification and characterization of cysteine synthetase, a bifunctional protein complex, from Salmonella typhimurium, J Biol Chem 244, 2428-2439.


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10. Bogdanova, N., and Hell, R. (1997) Cysteine synthesis in plants: protein-protein interactions of serine acetyltransferase from Arabidopsis thaliana, Plant J 11, 251-262. 11. Cook, P. F., and Wedding, R. T. (1977) Overall mechanism and rate equation for Oacetylserine sulfhydrylase, J Biol Chem 252, 3459. 12. Jez, J. M., and Dey, S. (2013) The cysteine regulatory complex from plants and microbes: what was old is new again, Curr Opin Struct Biol 23, 302-310. 13. Banerjee, S., Ekka, M. K., and Kumaran, S. (2011) Comparative thermodynamic studies on substrate and product binding of O-acetylserine sulfhydrylase reveals two different ligand recognition modes, BMC Biochem 12, 31. 14. Huang, B., Vetting, M. W., and Roderick, S. L. (2005) The active site of O-acetylserine sulfhydrylase is the anchor point for bienzyme complex formation with serine acetyltransferase, J Bacteriol 187, 3201-3205. 15. Campanini, B., Speroni, F., Salsi, E., Cook, P. F., Roderick, S. L., Huang, B., Bettati, S., and Mozzarelli, A. (2005) Interaction of serine acetyltransferase with O-acetylserine sulfhydrylase active site: evidence from fluorescence spectroscopy, Protein Sci 14, 21152124. 16. Francois, J. A., Kumaran, S., and Jez, J. M. (2006) Structural basis for interaction of Oacetylserine sulfhydrylase and serine acetyltransferase in the Arabidopsis cysteine synthase complex, Plant Cell 18, 3647-3655. 39 ACS Paragon Plus Environment

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17. Kumaran, S., and Jez, J. M. (2007) Thermodynamics of the interaction between Oacetylserine sulfhydrylase and the C-terminus of serine acetyltransferase, Biochemistry 46, 5586-5594. 18. Mazumder, M., and Gourinath, S. (2016) Structure-Based Design of Inhibitors of the Crucial Cysteine Biosynthetic Pathway Enzyme O-Acetyl Serine Sulfhydrylase, Curr Top Med Chem 16, 948-959. 19. Salsi, E., Bayden, A. S., Spyrakis, F., Amadasi, A., Campanini, B., Bettati, S., Dodatko, T., Cozzini, P., Kellogg, G. E., Cook, P. F., Roderick, S. L., and Mozzarelli, A. (2010) Design of O-acetylserine sulfhydrylase inhibitors by mimicking nature, J Med Chem 53, 345-356. 20. Schnell, R., and Schneider, G. (2010) Structural enzymology of sulphur metabolism in Mycobacterium tuberculosis, Biochem Biophys Res Commun 396, 33-38. 21. Kumar, S., Raj, I., Nagpal, I., Subbarao, N., and Gourinath, S. (2011) Structural and biochemical studies of serine acetyltransferase reveal why the parasite Entamoeba histolytica cannot form a cysteine synthase complex, J Biol Chem 286, 12533-12541. 22. Mino, K., Hiraoka, K., Imamura, K., Sakiyama, T., Eisaki, N., Matsuyama, A., and Nakanishi, K. (2000) Characteristics of serine acetyltransferase from Escherichia coli deleting different lengths of amino acid residues from the C-terminus, Biosci Biotechnol Biochem 64, 1874-1880. 23. Berkowitz, O., Wirtz, M., Wolf, A., Kuhlmann, J., and Hell, R. (2002) Use of biomolecular interaction analysis to elucidate the regulatory mechanism of the cysteine synthase complex from Arabidopsis thaliana, J Biol Chem 277, 30629-30634. 24. Feldman-Salit, A., Wirtz, M., Hell, R., and Wade, R. C. (2009) A mechanistic model of the cysteine synthase complex, J Mol Biol 386, 37-59. 25. Wirtz, M., Birke, H., Heeg, C., Muller, C., Hosp, F., Throm, C., Konig, S., FeldmanSalit, A., Rippe, K., Petersen, G., Wade, R. C., Rybin, V., Scheffzek, K., and Hell, R. (2010) Structure and function of the hetero-oligomeric cysteine synthase complex in plants, J Biol Chem 285, 32810-32817. 26. Yi, H., Dey, S., Kumaran, S., Lee, S. G., Krishnan, H. B., and Jez, J. M. (2013) Structure of soybean serine acetyltransferase and formation of the cysteine regulatory complex as a molecular chaperone, J Biol Chem 288, 36463-36472. 40 ACS Paragon Plus Environment

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27. Pye, V. E., Tingey, A. P., Robson, R. L., and Moody, P. C. (2004) The structure and mechanism of serine acetyltransferase from Escherichia coli, J Biol Chem 279, 4072940736. 28. Wang, T., and Leyh, T. S. (2012) Three-stage assembly of the cysteine synthase complex from Escherichia coli, J Biol Chem 287, 4360-4367. 29. Wirtz, M., Berkowitz, O., Droux, M., and Hell, R. (2001) The cysteine synthase complex from plants. Mitochondrial serine acetyltransferase from Arabidopsis thaliana carries a bifunctional domain for catalysis and protein-protein interaction, Eur J Biochem 268, 686-693. 30. Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. (1995) How to measure and predict the molar absorption coefficient of a protein, Protein Sci 4, 2411-2423. 31. Gaitonde, M. K. (1967) A spectrophotometric method for the direct determination of cysteine in the presence of other naturally occurring amino acids, Biochem. J. 104, 627633, 17 32. Banerjee, S., Ekka, M. K. & Kumaran, S. (2011). Comparative thermodynamic studies on substrate and product binding of O-acetylserine sulfhydrylase reveals two different ligand recognition modes. BMC Biochem. 12, 31 33. Tai, C. H., Nalabolu, S. R., Jacobson, T. M., Minter, D. E., and Cook, P. F. (1993). Kinetic mechanisms of the A and B isozymes of O-acetylserine sulfhydrylase from Salmonella typhimurium typhimurium LT-2 using the natural and alternative reactants. Biochemistry. 32, 6433-6442. 34. Johnson, C. M., Huang, B., Roderick, S. L., and Cook, P. F. (2004). Kinetic mechanism of the serine acetyltransferase from Haemophilus influenza. Arch. Biochem. Biophys. 429, 115-122. 35. Laue, T. M., Shah, B. D. Ridgeway, T.M. & Pelletier, S. L. (1992). Analytical Ultracentrifugation in Biochemistry and Polymer Science, Royal Society of Chemistry, pp. 90-125. 36. Schuck, P. (2000). Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling, Biophys. J. 78, 1606-1619. 37. Hindson, V. J., Moody, P. C., Rowe, A. J., & Shaw, W. V. (2000). Serine acetyltransferase from Escherichia coli is a dimer of trimers. J. Biol. Chem. 275, 461466. 41 ACS Paragon Plus Environment

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Two Distinct Assembly States of the Cysteine Regulatory Complex of

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