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Investigation of the thermodynamic drivers of the interaction between the high mobility group box domain of Sox2 and bacterial lipopolysaccharide
Journal Pre-proof Investigation of the thermodynamic drivers of the interaction between the high mobility group box domain of Sox2 and bacterial lipopolysaccharide
Patrick H. Hewitt, Ernest D. Pianim, Nicholas A. DiCesare, Casey Gray, Trung T. Leong, Kuriko Sakai, Jan V. Bernal, Shweta S. Shetty, Christopher S. Malarkey PII:
S0005-2736(19)30252-4
DOI:
https://doi.org/10.1016/j.bbamem.2019.183106
Reference:
BBAMEM 183106
To appear in:
BBA - Biomembranes
Received date:
23 May 2019
Revised date:
5 September 2019
Accepted date:
8 October 2019
Please cite this article as: P.H. Hewitt, E.D. Pianim, N.A. DiCesare, et al., Investigation of the thermodynamic drivers of the interaction between the high mobility group box domain of Sox2 and bacterial lipopolysaccharide, BBA - Biomembranes(2019), https://doi.org/ 10.1016/j.bbamem.2019.183106
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© 2019 Published by Elsevier.
Journal Pre-proof Investigation of the thermodynamic drivers of the interaction between the high mobility group box domain of Sox2 and bacterial lipopolysaccharide Patrick H. Hewitt1, Ernest D. Pianim1, Nicholas A. DiCesare1, Casey Gray1, Trung T. Leong1, Kuriko Sakai1, Jan V. Bernal, Shweta S. Shetty, and Christopher S. Malarkey1# #
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Corresponding author Christopher S. Malarkey School of Pharmacy, Department of Pharmaceutical Sciences, Rueckert-Hartman College for Health Professions, Regis University 3333 Regis Boulevard, H-28, Denver, CO 80221, USA [email protected] Phone: 001-303-625-1244 Fax: 001-303-625-1305 1 School of Pharmacy, Department of Pharmaceutical Sciences, Rueckert-Hartman College for Health Professions, Regis University 3333 Regis Boulevard, H-28, Denver, CO 80221, USA
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Abstract
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Highlights Sox2 can bind lipopolysaccharide with micromolar affinity. This is the first demonstration of a single domain high mobility group box protein binding to lipopolysaccharide molecules. Sox2 binds lipopolysaccharide in a 2:1 ratio with endothermic and exothermic drivers of the interaction.
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Gastric cancer is associated with high mortality and is preceded by an infection with Helicobacter pylori (H. pylori). H. pylori stimulates inflammation which involves the activation of Toll-like receptor 4 by lipopolysaccharide molecules from the H. pylori. This leads to chronic inflammation that can eventually lead to gastric cancer. Sox2 is a member of the high mobility group (HMG) box family of proteins, and recent studies have shown that HMG box proteins can modulate immune response by altering signaling to Toll-like receptors. Sox2 is overexpressed in most types of cancer with the exception of gastric cancer where expression of Sox2 is decreased. Here, we demonstrate that Sox2 can bind LPS and we investigated the thermodynamic drivers of the Sox2/LPS interaction.
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Graphical Abstract
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Key Words Sox2, high-mobility group box, lipopolysaccharide, isothermal titration calorimetry, tryptophan fluorescence, gastric cancer
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Abbreviations
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Helicobacter pylori (H. pylori), High mobility group (HMG), chronic atrophic gastritis (CAG), lipopolysaccharide (LPS), Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), Toll-like receptor 9 (TLR9), Interleukin-4 (IL-4), bone morphogenetic protein 2 (BMP-2), Caudal-relate homeobox 2 (CDX-2), receptor for advanced glyaction end products (RAGE), mitochondrial transcription factor A (TFAM), neutrophil extracellular traps (NETs) 1. Introduction Gastric (stomach) cancer is the second leading cause of cancer-related death in the world [1, 2]. The progression of gastric cancer is slow, and its pathological presentation is often preceded by a chronic infection in the stomach with the bacterium Helicobacter pylori (H. pylori) [3]. If the H. pylori infection is left untreated, it remains for the lifetime of the patient and causes inflammatory responses leading to chronic atrophic gastritis (CAG) [4, 5]. CAG will then progress to intestinal metaplasia, at which point the progression towards gastric cancer is irreversible. Intestinal metaplasia then proceeds to dysplasia, which in turn progresses to gastric carcinoma with high mortality rates [4].
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H. Pylori induces inflammatory responses in the gastric endothelium by stimulating the innate immune system. The cell membranes of Gram-negative bacteria, such as H. Pylori, contain lipopolysaccharide (LPS) molecules that stimulate the innate immune system by binding to Toll-like receptor 4 (TLR4) [5]. Additionally, there is evidence that H. Pylori LPS can activate Toll-like receptor 2 and induce inflammation [6, 7]. The binding of LPS to TLR4 triggers an immune response cascade causing the release of proinflammatory chemokines and cytokines [8]. The chemokines and cytokines then recruit immune response cells such as macrophages to the site of the chronic H. Pylori infection where they promote chronic inflammation [3]. Since LPS stimulation of the innate immune system in the gastric endothelium can lead to chronic inflammation and the progression to gastric carcinoma, blocking LPS stimulation of TLR4 in the gastric endothelium is of immense clinical interest in the long-term. Sox2 is a member of the high mobility group (HMG) box family of DNA binding proteins and a transcription factor that plays essential roles during many phases of embryonic development [9, 10]. It is also a critical player in the generation of induced pluripotent stem cells [11]. Sox2 is overexpressed in nearly every type of cancer with the unique exception of gastric cancer wherein Sox2 expression decreases during the progression from normal gastric tissue to gastric cancer [2]. Chronic H. pylori infection of the gastric epithelium leads to increased expression of the proinflamatory cytokine Interleukin-4 (IL-4) and bone morphogenetic protein 2 (BMP-2). This in turn leads to decreased Sox2 expression and an increase in expression of Caudal-relate homeobox 2 (CDX-2) a protein shown to be essential for the progression from intestinal metaplasia to gastric cancer [2, 12-14]. The Sox2 protein contains structurally disordered N-, and C-terminal domains flanking the ordered HMG box [9, 15] (Figure 1A). HMG box proteins act as DNA architectural factors and transcription factors by binding to and bending DNA [16-18]. The HMG box domain of this family of proteins consists of three alpha helices rich in basic amino acids that form an “L” shape, that binds to the minor grove of DNA to induce DNA bending [16, 18] (Figure 1B). In addition to their roles as architectural and transcription factors, HMG box proteins have been shown to alter the TLR-mediated immune response when they are shuttled out of the cell nucleus [19-23]. Notably, the HMG box protein HMGB1 has been extensively studied with regards to its role in stimulating inflammatory responses through Toll-like receptor 2 (TLR2), TLR4, the receptor for advanced glyaction end products (RAGE) [24], and its ability to bind LPS molecules [25, 26]. Recent work from our lab and others has shown that the HMG box protein mitochondrial transcription factor A (TFAM) can block Toll-like receptor 9 (TLR9) mediated immune response by binding to CpG rich DNA and preventing it from stimulating TLR9 [22, 23, 27]. Sox2 has been shown to reside outside of the cell nucleus as well [28, 29], and has been shown to bind to bacterial DNA in the cytoplasm of neutrophils altering TLR-mediated immune responses [30]. Additionally, when neutrophils are stimulated with LPS or H. pylori, this can lead to the formation of neutrophil extracellular traps (NETs) where the DNA and DNA binding proteins of the neutrophil are ejected extracellularly to trap microbes [31, 32]. The formation of NETs provides a mechanism by where Sox2 can interact with LPS molecules during an H. pylori infection.
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Journal Pre-proof Since HMG box proteins have been shown to bind to and alter the effect of immune-stimulating molecules, we examined the ability of the HMG domain of Sox2 to bind LPS molecules. Using both fluorescence spectroscopy and isothermal titration calorimetry (ITC), we were able to show that the HMG domain of Sox2 does indeed have the ability to bind LPS. We measured the affinity as well as the stoichiometry and thermodynamic drivers of the LPS/Sox2 HMG interaction. The results of this study have the potential to shed light on a previously unrecognized mechanism for the progression of gastric cancer. 2. Materials and Methods
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2.1. Sox2 HMG Box Expression and Purification
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The plasmid encoding the N-terminal 6xHis tagged HMG box domain of Sox2 (residues 38-124) was a gift from the laboratory of Marius Clore at the National Institute of Health. Our lab expressed and purified the 6xHis tagged HMG box domain of Sox2 using a modified version of previously published protocols [33, 34]. Briefly, the 6xHis tagged HMG box domain of Sox2 was expressed in Rosetta II pLysS cells. When the OD600 of the culture reached 0.6, 1 mM IPTG was added to induce expression of the protein. Cells were harvested by centrifugation and frozen at -80ºC to aid in cell lysis. Cells were lysed by sonication and the lysate was cleared by centrifugation. Purification of the protein was achieved using nickel-nitrilotriacetic acid (Ni-NTA) resin (G Biosciences) in batch purification and the protein was eluted with 300 mM imidazole. The 6xHis tag was removed using thrombin. For pull-down assay experiments, the 6xHis tag was not cleaved from the protein. The Sox2 HMG domain was further purified using ion exchange chromatography with a Hi-Trap S FF column, and then purified by size exclusion using (HiLoad 16/60 Superdex 75 column; GE Healthcare). The purity of the protein was assessed using 4-20% polyacrylamide SDS gels (Bio-Rad) stained with Coomassie blue (Figure 1C). The measured mass of the protein was 10,689.65 Da using electrospray ionization mass spectrometry (data not shown), which is in agreement with the theoretical mass of the Sox2 HMG domain. Purified fractions of Sox2 were stored at -80ºC. 2.2. LPS preparation
Purified LPS isolated from e.coli strain O111:B4 was purchased from EMD Millipore Corporation Billerica, MA, USA. LPS was resuspended in the indicated buffers for the experiments described below. 2.3 Gel Filtration A HiLoad 16/600 Superdex 75 prep grade gel filtration column (GE Healtcare Life Sciences) was equilibrated with 50 mM Hepes, pH 7.4, and 150 mM NaCl on an Äkta Prime FPLC system at a flow rate of 1 ml/min. This same buffer was used for all experiments when running biological molecules through the column. A 2 ml injection
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Journal Pre-proof loop was used for all experiments. The total column volume was 120 ml. All experiments were run at 1 ml/min, and protein elution was monitored at 280 nm. 2.4 6xHis-tagged Sox2 HMG/LPS Pull-down Assays
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Pull-down assays were performed by equilibrating 1 ml of Ni-NTA resin (Gold Bio) in buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, and 20 mM imidazole (buffer A). In the experiment, 20 µM 6xHis Sox2 HMG and 0.35 mg of LPS in 3 ml of buffer A were incubated with 1 ml of Ni-NTA resin for 1 hour at room temperature in a column. The flow through (FT) was eluted, and the column was washed with 3 ml of buffer A, followed by elution with 3 ml of buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, and 300 mM imidazole (buffer B). The collected FT, wash, and elution fractions were digested with 0.25 µg/ml proteinase K for 1 hour at room temperature. After digestion, 10 µl aliquots of the FT, wash, and elution fractions were mixed with 10 µl 2x Laemmli buffer and loaded on a 4-20% polyacrylamide SDS gel (Bio-Rad). As a positive control, 0.25 µg of LPS was loaded on the gel. As a negative control to see if LPS could bind to the Ni-NTA resin in the absence of 6xHis Sox2 HMG, 0.7 mg of LPS were incubated with 1 ml of Ni-NTA resin in 3 ml of buffer A for 1 hour at room temperature. The flow through (FT) was eluted, and the column was washed with 3 ml of buffer A, followed by elution with 3 ml of buffer B. Aliquotes of 10 µl of the FT, wash, and elution were mixed with 10 µl of 2x Laemmli buffer and loaded on the same gel described above. The gel was run at 200 V, and then stained with a silver staining kit (Bio Rad) for LPS visualization.
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2.5. Intrinsic Tryptophan Fluorescence Assays
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Tryptophan fluorescence assays were performed using an AMINCO Bowman Series 2 fluorescence spectrophotometer. In the experiments, 2 ml of 0.5 µM Sox2 HMG domain in 50 mM Hepes, pH 7.4, 150 mM NaCl at 20ºC was titrated with increasing concentrations of LPS and excited at 280 nm. Fluorescence emission was measured between 320 nm and 370 nm. The excitation bandpass was 4 nm and the emission bandpass was 16 nm. A step size of 1 nm and an integration time of 1 second were used during data collection. The buffer was filter sterilized and degassed one hour before experiments were conducted. Three independent experiments were conducted to obtain the measured dissociation constant. 2.6. Isothermal Titration Calorimetry Isothermal titration calorimetry (ITC) experiments were conducted using a General Electric Microcal iTC200 instrument. All buffers for ITC experiments were filter sterilized and degassed before conducting experiments. In the ITC experiments, 20 injections of 2 µl aliquots of 1.25 mg/ml LPS were added to 300 µl of 10 µM Sox2 HMG domain in 50 mM Hepes, pH 7.4, 150 mM NaCl. The same buffer was used for LPS solution. The cell was stirred at 750 RPM at 25ºC. The system was allowed to equilibrate for 10 minutes before injections were initiated and injections were added every two minutes after equilibration. Dilution heats of injection obtained from injecting
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Journal Pre-proof LPS into buffer only were subtracted from the experiments involving titration of LPS into Sox2 HMG. 3. Results 3.1 Identification of a Sox2 HMG LPS complex using gel filtration
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To determine if Sox2 HMG has the ability to bind to LPS molecules and form a complex, we used gel filtration and identified that Sox2 HMG does indeed form a complex with LPS that is identifiable by gel filtration (Figure 2). In gel filtration chromatography, higher molecular weight proteins and biological complexes elute before lower molecular weight proteins and biological complexes. From Figure 2, we see that Sox HMG alone (magenta) elutes between 70 and 80 ml on the column used in this study by measuring the absorbance at 280 nm from the tryptophan residues in the Sox2 HMG protein. When LPS alone (black) is run on the column, we do not see an elution profile since the LPS molecule lacks the conjugated double bond system needed to absorb ultra violet light at 280 nm. When LPS is run on the gel filtration column with an excess of Sox2 HMG (dark green), we see a peak eluting between 45 and 50 ml corresponding to a higher molecular weight complex of Sox2 HMG bound to LPS followed by the excess Sox2 HMG eluting alone. It should be noted that non-globular HMG proteins such as Sox2 exhibit a myriad of odd elution profiles on gel filtration [35, 36], and determination of the stoichiometry HMG proteins alone and in complex with biological molecules should not be over interpreted using gel filtration [35]. Nonetheless, the data from Figure 2 provide initial evidence that Sox2 HMG domains can chelate LPS molecules.
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3.2 Confirmation of the Sox2 HMG/LPS complex using a 6xHis-tagged Sox2 HMG pull-down assay
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To confirm that Sox2 HMG binds LPS molecules, we used LPS pull-down assays where the concentration of Sox2 HMG was in excess of the LPS molecule. As a positive control, we ran 0.25 µg of LPS on the gel and we see faint bands in the 10-25 kDa range corresponding to LPS. We exploited the ability of the 6xHis-tagged Sox2 HMG to bind to Ni-NTA resin with high affinity in this experiment. We incubated 20 µM 6xHis Sox2 HMG and 0.35 mg of LPS in 3 ml of buffer A with Ni-NTA resin in the presence of low concentrations of imidazole (20 mM). Under these conditions, 6xHis-tagged Sox2 HMG can bind the Ni-NTA resin with high-affinity, and if LPS can bind to the HMG region of Sox2, when we elute the buffer from the column containing the Ni-NTA/6xHistagged Sox2 HMG/LPS mixture with imidazole, we would not expect to see much LPS in the elution since it will be bound to the Sox2 HMG which is bound to the Ni-NTA resin. Indeed, after eluting and digesting the elution with proteinase K, we do not see strong LPS bands in the gel (Figure 3 6xHis Sox2 HMG/LPS FT). We then washed the column with 3 ml of buffer A containing 20 mM imidazole and we see the presence of LPS bands suggesting that some of the LPS bound 6xHis-tagged Sox2 HMG was eluted under these conditions (Figure 3 6xHis Sox2 HMG/LPS wash). From our purification of 6xHis-tagged Sox2 HMG, we know that 300 mM imidazole can fully elute
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6xHis-tagged Sox2 HMG (Figure 1C). We therefore would predict the presence of strong LPS bands in this lane when we elute the 6xHis-tagged Sox2 HMG/LPS complex with 3 ml of buffer B, and that is indeed the case (Figure 3 6xHis Sox2 HMG/LPS elute). These results clearly show that Sox2 HMG does in fact bind to LPS molecules. In order to rule out the possibility that LPS could be binding the Ni-NTA resin, we incubated 0.35 mg of LPS in 3 ml of buffer A with Ni-NTA resin. If the LPS does not bind to the Ni-NTA resin, we expect to see strong LPS bands on the gel in this lane and this is what occurred in the experiment (Figure 3 LPS FT). The bands become weaker in intensity with the wash (Figure 3 LPS wash) and finally almost no intensity with the elution step (Figure 3 LPS elute) demonstrating that LPS cannot bind to the Ni-NTA resin on its own and confirming that LPS can bind to the HMG region of Sox2. We then were interested in investigating the affinity and stoichiometry of the Sox2 HMG/LPS interaction.
3.3. Probing the Sox2 HMG/LPS interaction using tryptophan fluorescence assays
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In order to determine the binding affinity of the Sox2 HMG domain with LPS, we used intrinsic tryptophan fluorescence of the two tryptophan residues that reside in the HMG domain of Sox2 to probe LPS binding to the protein (Figure 4). When tryptophan residues are excited with light in the 280 nm-300 nm wavelength range, they emit light in the 320-350 nm wavelength range with the maximum emission wavelength being dependent on the environment of the tryptophan residue. If binding of a ligand to the protein causes the tryptophan residue to be exposed to a more aqueous environment, typically, the intensity of the fluorescence emission decreases. Conversely, if the binding of a ligand to a protein causes the tryptophan to reside in a more hydrophobic environment, the fluorescence emission typically increases [37]. Since LPS contains non-polar regions, we predicted that binding of Sox2 HMG domain to LPS would result in an increase in tryptophan fluorescence emission if binding did occur. Indeed, upon titration of increasing concentrations of LPS into Sox2 HMG, we observed an increase in fluorescence emission at 330 nm, when the Sox2 protein was excited at 280 nm using an AMINCO Bowman Series 2 fluorescence spectrophotometer. We also observed a red shift in the emission maximum suggesting that the Sox2 protein was interacting with a more non-polar environment such as LPS [37] (Figure 4A). We also performed control experiments where LPS was titrated into buffer only, and the minimal fluorescence due to protein contamination from these titrations were subtracted from their corresponding titrations into the HMG domain of Sox2 so that only changes in Sox2 fluorescence were measured. We further analyzed our tryptophan emission data to estimate the binding affinity for the Sox2 HMG domain to LPS (Figure 4B). We normalized the change in fluorescence emission at 330 nm upon titration of LPS into the Sox2 HMG domain, and plotted these changes versus the concentration LPS to obtain a binding isotherm that was fit to the Hill equation. From this binding isotherm, we obtained a dissociation constant (Kd) of 0.90 µM ± 0.05 showing tight binding between Sox2 HMG and LPS. It should be noted that LPS is not a homogeneous molecule, but rather a heterogeneous molecule of varying size (Figure 5) [38] for the outer core polysaccharide unit. The
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Journal Pre-proof manufacturer of the LPS used in this study estimates the size of the LPS to be in the 10-kDa range, and other investigators have used this 10 kDa estimate as well [39]. We also measured a Hill coefficient of ~1.5 suggesting cooperative binding of Sox2 to LPS. Given that there are two regions on the lipid A region of LPS that resemble the backbone of DNA (Figure 5), we propose that there are two Sox2 binding sites on LPS and that sequential binding to LPS is cooperative. Since Sox2 HMG and LPS appear to show cooperative binding in the concentration ranges used in this assay, we further investigated this interaction using ITC. Nonetheless, these initial studies suggest that the Sox2 HMG domain can bind to LPS.
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3.4. Determining the thermodynamic drivers of the Sox2 HMG/LPS interaction using ITC
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To further investigate the stoichiometry of binding and to determine the thermodynamic drivers of the Sox2/LPS interaction, we investigated this interaction using ITC (Figure 4). After allowing the ITC instrument to equilibrate for 10 minutes, we began titrating LPS into the Sox2 HMG domain (Figure 6A). We observed an initial negative change in enthalpy (H) of binding, which contributes to a negative change in Gibbs free energy (G) leading to favorable binding. Interestingly, during the 7th injection of LPS into Sox2 HMG domain, we began to observe a positive H component of binding corresponding to heat being absorbed. When the raw heats were integrated with respect to time and plotted vs. the LPS:Sox2 HMG domain molar ratio (Figure 6B), we were able to see that LPS does indeed have two Sox2 binding sites. The data were fit using a two-site cooperative binding model, and we see that as the first exothermic binding event is halfcomplete, a second endothermic binding event begins to occur in a manner similar to that of HMG box proteins binding to DNA [16, 40, 41]. We were able to directly measure a negative H of binding of -16.1 kJ/mol for the first binding event, and a positive H of binding of 41.1 kJ/mol for the second event. We were also able to measure a K d of 0.46 µM ± 0.1 for the first binding site and a Kd of 1.6 µM ± 0.1 for the second binding site. Interestingly, the dissociation constant of 0.90 µM measured from Figure 4B in the tryptophan fluorescence assays likely represents an average of the two dissociation constants seen in the ITC experiments. From the ITC Kd values, we were able to determine the G of binding for each site from the equation G=-RT ln Ka where R = 8.314 J/molK, T is the temperature in Kelvin, and Ka=1/Kd. Once we determined G and H values for each site, we can determine the change in entropy (S) associated with each binding event from the equation G=H-TS where T is the temperature in Kelvin. This allowed us to determine that binding of the first site is enthalpically and entropically driven, whereas binding to the second site is entirely entropically driven, which is also observed for HMG box proteins binding to DNA [40, 41]. The second entropically driven interaction could be due to the displacement of organized water around the phosphate region in the lipid A region of LPS as is the case with DNA binding [40, 41], or it could be due to Sox2 disrupting hydrophobic interactions between LPS molecules. Nonetheless, sing two different techniques, we have shown that Sox2 can directly bind to LPS. This interaction could alter the immunostimulatory effects of LPS that lead to the progression of gastric cancer.
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Journal Pre-proof 4. Discussion
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Previous studies investigating the binding of the antimicrobial protein rBPI 21 to LPS using ITC showed that the rBPI21/LPS interaction was endothermic and driven entropically [42]. The rBPI21 protein is positively charged like the HMG domain of Sox2, however LPS was shown to only have one binding site for rBPI 21 while LPS has two Sox2 HMG binding sites. This difference could be due to the two proteins binding to different areas of LPS. The HMG domain of Sox2 likely binds to the two sugarphosphate moieties on LPS, whereas the authors of the rBPI21 study suggest that rBPI21 binds to the surface or outer core region of LPS [42]. They suggest that this binding event may cause the release of Ca2+ ions from the bacterial surface and expose the negative charge of LPS on the phosphate regions explaining the entropy driven binding event. On the other hand, the exothermic Sox2 HMG/LPS binding event could be explained by an ionic interaction between the lysine and arginine rich binding face of Sox2 HMG with a negatively charged phosphate on LPS. Since LPS is a lipid and will form membrane layers or micelles with neighboring LPS molecules, the second endothermic binding event driven by entropy could be due to a Sox2 HMG molecule binding to the second phosphate region on LPS and separating this LPS molecule from neighboring LPS molecules, which would be entropically favorable for binding. Work investigating the binding of HMGB1, a protein with two HMG domains, to LPS showed that the box A HMG domain of HMGB1 bound primarily inner/outer core region of LPS while box B HMG domain bound to the lipid A region of LPS [25]. We conducted a sequence alignment analysis of Sox2 HMG with the box A and box B HMG domains of HMGB1 and found that Sox2 HMG has greater sequence similarity with box B (25.6% identical, 50.0% similar) compared to box A (25.0% identical, 36.9% similar)(Figure 7). The high sequence similarity between box B and Sox2 HMG lends credence to the idea that the lipid A portion of LPS is the primary LPS binding site for Sox2 HMG. Interestingly, the putative lipid A binding region of box B in HMGB1 is located on a different alpha helix than the primary and secondary DNA intercalation sites of HMGB1 (Figure 7) suggesting a binding mechanism for LPS that is vastly different from the binding mechanism for DNA, which is not surprising. We identified two putative lipid A in regions Sox2 HMG. The proposed lipid A binging motif of LPS binding proteins is similar to that of heparin binding proteins where the motif BBXB is present and B is any basic residue while X is any residue [43, 44]. Another potential lipid A binding motif is BAB where B is a basic residue and A is an aromatic residue [45]. The identified BBXB and BAB lipid A binding region of KKFK in HMGB1 occurs distal to box B in the disordered linker region of HMGB1 at amino acids 87-90 [25]. We identified a KYR BAB potential lipid A binding motif at amino acids 109111 in a disordered region of Sox2 as well as a KRLR BBXB motif at residues 95-98 in helix III of Sox2 HMG (Figure 7). X-ray crystallography studies have shown that tyrosine residue in the KYR motif makes key hydrogen bonds with base pairs to stabilize the Sox2 HMG/DNA complex, while the KRLR motif in helix III of Sox2 does not make any bonds with the DNA molecule [46] and could be a likely candidate for the lipid A binding region of Sox2 HMG. The LPS dissociation constants for Sox2 HMG measured in this study were found to be on the micromolar scale (Figures 4 and 6) and these values are consistent
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with other proteins that have been shown to bind LPS such as HP0902, an H. pylori protein, which belongs to the cupin superfamily of proteins [39]. In the HP0902 study, the interaction between HP0902 and LPS was shown to be driven entirely by enthalpy [39] while this study demonstrates the interaction between Sox2 HMG and LPS has both entropic and enthalpic components leading towards a favorable binding interaction. This finding suggests that the LPS binding site for Sox2 HMG is different than that of HP0902. Indeed, the cupin superfamily has been shown to include sugar-binding proteins [47] therefore the likely binding LPS binding site for this protein is on the Oantigen, inner core, or outer core region of LPS (Figure 5), whereas the HMG box of Sox2 likely binds the sugar phosphate moiety of the lipid A region of LPS that resembles the backbone of DNA. The results of this study demonstrate that the HMG box region of Sox2 has the ability to bind bacterial LPS molecules in vitro. Sox2 HMG likely binds two regions of LPS at the two sugar phosphate regions of the lipid A region of LPS and Sox2 therefore has the potential to modulate LPS activation of TLR-4 and this will be the subject of a future study. Since Sox2 expression is decreased as an H. pylori infection leads to gastric cancer, it is possible that Sox2 could bind LPS and prevent activation of TLR-4 in the early stages of an H. pylori infection. As the infection progresses to gastric carcinoma and the expression of Sox2 is decreased, this could allow for more LPS molecules to activate TLR-4 and lead to increased inflammation in the gastric endothelium.
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5. Conclusions
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This is the first report showing that a single HMG box containing protein can bind to LPS molecules, however, this is not surprising considering that HMG box proteins have been shown to have a wide array of functions outside of their DNA binding and gene expression activities [15]. Future studies on the interplay between Sox2 and LPS in the progression of gastric cancer should provide interesting results that could have clinical significance in the understanding of gastric cancer pathogenesis. Future work on this project will investigate the ability of Sox2 to bind LPS from different bacterial species and identify key LPS binding regions in the HMG domain of Sox2 through mutagenic studies.
Author Contributions CSM conceived and supervised the study and wrote the manuscript. PH and EDP purified protein, conducted ITC experiments, conducted tryptophan fluorescence experiments, created the sequence alignment, and edited the manuscript. NAD, CG, TL, and KS purified protein, conducted tryptophan fluorescence experiments, and edited the manuscript. JVB and SSS conducted the gel filtration and LPS pull-down experiments.
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Acknowledgements We would like to thank Shaun Bevers and the Biophysics Core at the University of Colorado Anschutz Medical Campus for access to their General Electric Microcal iTC200 instrument. References
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[30] P. Xia, S. Wang, B. Ye, Y. Du, G. Huang, P. Zhu, Z. Fan, Sox2 functions as a sequence-specific DNA sensor in neutrophils to initiate innate immunity against microbial infection, Nat Immunol. 16 (2015) 366-375. [31] A. Hakkim, T.A. Fuchs, N.E. Martinez, S. Hess, H. Prinz, A. Zychlinsky, H. Waldmann, Activation of the Raf-MEK-ERK pathway is required for neutrophil extracellular trap formation, Nat Chem Biol. 7 (2011) 75-77. [32] O. Goldmann, E. Medina, The expanding world of extracellular traps: not only neutrophils but much more, Front Immunol. 3 (2012) 420. [33] D.C. Williams, Jr., M. Cai, G.M. Clore, Molecular basis for synergistic transcriptional activation by Oct1 and Sox2 revealed from the solution structure of the 42-kDa Oct1.Sox2.Hoxb1-DNA ternary transcription factor complex, J. Biol. Chem. 279 (2004) 1449-1457. [34] A. Remenyi, E. Pohl, H.R. Scholer, M. Wilmanns, Crystallization of redoxinsensitive Oct1 POU domain with different DNA-response elements, Acta crystallographica. Section D, Biological crystallography. 57 (2001) 1634-1638. [35] T.A. Gangelhoff, P.S. Mungalachetty, J.C. Nix, M.E. Churchill, Structural analysis and DNA binding of the HMG domains of the human mitochondrial transcription factor A, Nucleic Acids Res. 37 (2009) 3153-3164. [36] R.C. Tarvers, F.C. Church, Use of high-performance size-exclusion chromatography to measure protein molecular weight and hydrodynamic radius. An investigation of the properties of the TSK 3000 SW column, Int. J. Pept. Protein Res. 26 (1985) 539-549. [37] A.B. Ghisaidoobe, S.J. Chung, Intrinsic tryptophan fluorescence in the detection and analysis of proteins: a focus on Forster resonance energy transfer techniques, Int J Mol Sci. 15 (2014) 22518-22538. [38] S. Qiao, Q. Luo, Y. Zhao, X.C. Zhang, Y. Huang, Structural basis for lipopolysaccharide insertion in the bacterial outer membrane, Nature. 511 (2014) 108111. [39] D.W. Sim, J.H. Kim, H.Y. Kim, J.H. Jang, W.C. Lee, E.H. Kim, P.J. Park, K.H. Lee, H.S. Won, Structural identification of the lipopolysaccharide-binding capability of a cupin-family protein from Helicobacter pylori, FEBS Lett. 590 (2016) 2997-3004. [40] A.I. Dragan, J. Klass, C. Read, M.E. Churchill, C. Crane-Robinson, P.L. Privalov, DNA binding of a non-sequence-specific HMG-D protein is entropy driven with a substantial non-electrostatic contribution, J. Mol. Biol. 331 (2003) 795-813. [41] A.I. Dragan, C.M. Read, E.N. Makeyeva, E.I. Milgotina, M.E. Churchill, C. CraneRobinson, P.L. Privalov, DNA binding and bending by HMG boxes: energetic determinants of specificity, J. Mol. Biol. 343 (2004) 371-393. [42] M.M. Domingues, M.L. Bianconi, L.R. Barbosa, P.S. Santiago, M. Tabak, M.A. Castanho, R. Itri, N.C. Santos, rBPI21 interacts with negative membranes endothermically promoting the formation of rigid multilamellar structures, Biochim. Biophys. Acta. 1828 (2013) 2419-2427. [43] D.M. Mann, E. Romm, M. Migliorini, Delineation of the glycosaminoglycan-binding site in the human inflammatory response protein lactoferrin, J. Biol. Chem. 269 (1994) 23661-23667.
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Journal Pre-proof Figure Legends Figure 1. Schematic representation of full-length Sox2 (A). The X-ray crystal structure of the HMG box domain of Sox2 (residues 38-124) bound to DNA modeled from [46] (PDB ID: 1GT0) using pymol. The N and C-termini of the protein are indicated as well as the respective helix numbering, I, II, and III (B). 4-20% polyacrylamide SDS gel with 2 mg/ml of the Sox2 HMG domain (residues 38124), and a protein molecular weight ladder. The theoretical molecular weight of the Sox2 HMG domain after thrombin cleavage of the 6xHis tag is 10.689 kDa (C).
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Figure 2. Gel filtration of Sox2 HMG and the Sox2 HMG/LPS complex. Gel filtration elution profiles of 0.5 mg/ml of LPS alone (black curve), 2 mg/ml of Sox2 HMG alone (magenta curve), and 0.5 mg/ml of LPS and 2 mg/ml Sox2 HMG as a complex (dark green curve). Protein elution was monitored at 280 nm.
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Figure 3. LPS pull-down assay. 4-20% polyacrylamide SDS gel silver stained with a protein molecular weight ladder (ladder) and a positive control of LPS alone (0.25 µg LPS). After equilibrating the column with 20 mM imidazole and Sox2/HMG LPS, the flow through was collected (6xHis Sox2 HMG/LPS FT), the column was then washed with 20 mM imidazole again (6xHis Sox2 HMG/LPS wash), followed by elution of the Sox2/LPS complex with 300 mM imidazole (6xHis Sox2 HMG/LPS elute). For a negative control, LPS alone was allowed to equilibrate with the column with 20 mM imidazole, and the flow through was collected (LPS FT), followed by washing the column with 20 mM imidazole again (LPS wash), and finally eluting the column with 300 mM imidazole (LPS elute). LPS is visualized as the dark bands of varying molecular weights. Samples containing 6xHis Sox2 were digested with proteinase K before being loaded onto the gel.
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Figure 4. Binding of LPS to Sox2 HMG. Representative fluorescence emission spectra of 0.5 µM Sox2 HMG domain in 50 mM Hepes, pH 7.4, 150 mM NaCl at 20ºC titrated with increasing concentrations of LPS and excited at 280 nm. Reducing agents were not used in the experiment since the HMG domain of Sox2 lacks cysteine residues (A). Binding isotherm of LPS bound to the HMG domain of Sox2 (n=3). The change in fluorescence emission at 330 nm was normalized, and plotted versus the concentration of LPS. The resulting binding isotherm was fit with the Hill equation: Y=Bmax*x^n/(Kd^n+x^n) where Bmax is maximal change in fluorescence, Kd is the dissociation constant, and n is the Hill coefficient of cooperativity. Error bars represent s.d. Figure 5. Representative structure of LPS. LPS contains three distinctive units, the lipid A component (black), inner core (blue), and outer core (orange), which contains varying sizes of O-antigen polysaccharides. Saccharides are depicted as hexagons. Figure 6. Thermodynamics of the Sox2/LPS interaction. Representative raw heats emitted and absorbed after injecting 2 µl aliquots of 125 µM LPS into 10 µM Sox2 HMG domain. The buffer was 50 mM Hepes, pH 7.4, 150 mM NaCl and was stirred at 750
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Journal Pre-proof RPM at 25ºC (n=3) (A). Binding and enthalpy (H) analysis of the integrated heats with heats of mixing were subtracted. The molar ratio is LPS to Sox2 HMG domain. The data were fitted to a two-two site cooperative binding model of Y=((DH1*x)/(Kd1+x))+((DH2*x^n)/(Kd2^n+x^n)), where DH1 and DH2 are the change in enthalpy of the first and second binding events, respectively and Kd1 and Kd2 are the dissociation constants of the first and second binding events, respectively. The n parameter is a measure of cooperativity (B). Data were collected on a General Electric MicroCal iTC200 instrument.
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Figure 7. Alignments of Sox2 HMG with the HMG domains of HMGB1. Sequence alignment of Sox2 HMG with box A of HMGB1 and box B of HMGB1. Green cylinders indicate where helicies in Sox2 HMG form. Numbers to the left of the sequences indicate the starting amino acid number in the respective proteins. HMGB1 box residues shown in blue are identical at that position to Sox2 HMG while residues shown in red are similar to Sox2 at that position. The putative lipid A binding regions of Sox2 HMG are underlined in cyan. Primary and secondary DNA intercalating residues are indicated by a black 1 and 2, respectively.
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The authors have nothing to disclose.
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Patrick H. Hewitt, Ernest D. Pianim, Nicholas A. DiCesare, Casey Gray, Trung T. Leong, Kuriko Sakai, Jan V. Bernal, Shweta S. Shetty, Christopher S. Malarkey PII:
S0005-2736(19)30252-4
DOI:
https://doi.org/10.1016/j.bbamem.2019.183106
Reference:
BBAMEM 183106
To appear in:
BBA - Biomembranes
Received date:
23 May 2019
Revised date:
5 September 2019
Accepted date:
8 October 2019
Please cite this article as: P.H. Hewitt, E.D. Pianim, N.A. DiCesare, et al., Investigation of the thermodynamic drivers of the interaction between the high mobility group box domain of Sox2 and bacterial lipopolysaccharide, BBA - Biomembranes(2019), https://doi.org/ 10.1016/j.bbamem.2019.183106
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© 2019 Published by Elsevier.
Journal Pre-proof Investigation of the thermodynamic drivers of the interaction between the high mobility group box domain of Sox2 and bacterial lipopolysaccharide Patrick H. Hewitt1, Ernest D. Pianim1, Nicholas A. DiCesare1, Casey Gray1, Trung T. Leong1, Kuriko Sakai1, Jan V. Bernal, Shweta S. Shetty, and Christopher S. Malarkey1# #
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Corresponding author Christopher S. Malarkey School of Pharmacy, Department of Pharmaceutical Sciences, Rueckert-Hartman College for Health Professions, Regis University 3333 Regis Boulevard, H-28, Denver, CO 80221, USA [email protected] Phone: 001-303-625-1244 Fax: 001-303-625-1305 1 School of Pharmacy, Department of Pharmaceutical Sciences, Rueckert-Hartman College for Health Professions, Regis University 3333 Regis Boulevard, H-28, Denver, CO 80221, USA
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Abstract
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Highlights Sox2 can bind lipopolysaccharide with micromolar affinity. This is the first demonstration of a single domain high mobility group box protein binding to lipopolysaccharide molecules. Sox2 binds lipopolysaccharide in a 2:1 ratio with endothermic and exothermic drivers of the interaction.
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Gastric cancer is associated with high mortality and is preceded by an infection with Helicobacter pylori (H. pylori). H. pylori stimulates inflammation which involves the activation of Toll-like receptor 4 by lipopolysaccharide molecules from the H. pylori. This leads to chronic inflammation that can eventually lead to gastric cancer. Sox2 is a member of the high mobility group (HMG) box family of proteins, and recent studies have shown that HMG box proteins can modulate immune response by altering signaling to Toll-like receptors. Sox2 is overexpressed in most types of cancer with the exception of gastric cancer where expression of Sox2 is decreased. Here, we demonstrate that Sox2 can bind LPS and we investigated the thermodynamic drivers of the Sox2/LPS interaction.
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Graphical Abstract
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Key Words Sox2, high-mobility group box, lipopolysaccharide, isothermal titration calorimetry, tryptophan fluorescence, gastric cancer
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Abbreviations
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Helicobacter pylori (H. pylori), High mobility group (HMG), chronic atrophic gastritis (CAG), lipopolysaccharide (LPS), Toll-like receptor 2 (TLR2), Toll-like receptor 4 (TLR4), Toll-like receptor 9 (TLR9), Interleukin-4 (IL-4), bone morphogenetic protein 2 (BMP-2), Caudal-relate homeobox 2 (CDX-2), receptor for advanced glyaction end products (RAGE), mitochondrial transcription factor A (TFAM), neutrophil extracellular traps (NETs) 1. Introduction Gastric (stomach) cancer is the second leading cause of cancer-related death in the world [1, 2]. The progression of gastric cancer is slow, and its pathological presentation is often preceded by a chronic infection in the stomach with the bacterium Helicobacter pylori (H. pylori) [3]. If the H. pylori infection is left untreated, it remains for the lifetime of the patient and causes inflammatory responses leading to chronic atrophic gastritis (CAG) [4, 5]. CAG will then progress to intestinal metaplasia, at which point the progression towards gastric cancer is irreversible. Intestinal metaplasia then proceeds to dysplasia, which in turn progresses to gastric carcinoma with high mortality rates [4].
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H. Pylori induces inflammatory responses in the gastric endothelium by stimulating the innate immune system. The cell membranes of Gram-negative bacteria, such as H. Pylori, contain lipopolysaccharide (LPS) molecules that stimulate the innate immune system by binding to Toll-like receptor 4 (TLR4) [5]. Additionally, there is evidence that H. Pylori LPS can activate Toll-like receptor 2 and induce inflammation [6, 7]. The binding of LPS to TLR4 triggers an immune response cascade causing the release of proinflammatory chemokines and cytokines [8]. The chemokines and cytokines then recruit immune response cells such as macrophages to the site of the chronic H. Pylori infection where they promote chronic inflammation [3]. Since LPS stimulation of the innate immune system in the gastric endothelium can lead to chronic inflammation and the progression to gastric carcinoma, blocking LPS stimulation of TLR4 in the gastric endothelium is of immense clinical interest in the long-term. Sox2 is a member of the high mobility group (HMG) box family of DNA binding proteins and a transcription factor that plays essential roles during many phases of embryonic development [9, 10]. It is also a critical player in the generation of induced pluripotent stem cells [11]. Sox2 is overexpressed in nearly every type of cancer with the unique exception of gastric cancer wherein Sox2 expression decreases during the progression from normal gastric tissue to gastric cancer [2]. Chronic H. pylori infection of the gastric epithelium leads to increased expression of the proinflamatory cytokine Interleukin-4 (IL-4) and bone morphogenetic protein 2 (BMP-2). This in turn leads to decreased Sox2 expression and an increase in expression of Caudal-relate homeobox 2 (CDX-2) a protein shown to be essential for the progression from intestinal metaplasia to gastric cancer [2, 12-14]. The Sox2 protein contains structurally disordered N-, and C-terminal domains flanking the ordered HMG box [9, 15] (Figure 1A). HMG box proteins act as DNA architectural factors and transcription factors by binding to and bending DNA [16-18]. The HMG box domain of this family of proteins consists of three alpha helices rich in basic amino acids that form an “L” shape, that binds to the minor grove of DNA to induce DNA bending [16, 18] (Figure 1B). In addition to their roles as architectural and transcription factors, HMG box proteins have been shown to alter the TLR-mediated immune response when they are shuttled out of the cell nucleus [19-23]. Notably, the HMG box protein HMGB1 has been extensively studied with regards to its role in stimulating inflammatory responses through Toll-like receptor 2 (TLR2), TLR4, the receptor for advanced glyaction end products (RAGE) [24], and its ability to bind LPS molecules [25, 26]. Recent work from our lab and others has shown that the HMG box protein mitochondrial transcription factor A (TFAM) can block Toll-like receptor 9 (TLR9) mediated immune response by binding to CpG rich DNA and preventing it from stimulating TLR9 [22, 23, 27]. Sox2 has been shown to reside outside of the cell nucleus as well [28, 29], and has been shown to bind to bacterial DNA in the cytoplasm of neutrophils altering TLR-mediated immune responses [30]. Additionally, when neutrophils are stimulated with LPS or H. pylori, this can lead to the formation of neutrophil extracellular traps (NETs) where the DNA and DNA binding proteins of the neutrophil are ejected extracellularly to trap microbes [31, 32]. The formation of NETs provides a mechanism by where Sox2 can interact with LPS molecules during an H. pylori infection.
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Journal Pre-proof Since HMG box proteins have been shown to bind to and alter the effect of immune-stimulating molecules, we examined the ability of the HMG domain of Sox2 to bind LPS molecules. Using both fluorescence spectroscopy and isothermal titration calorimetry (ITC), we were able to show that the HMG domain of Sox2 does indeed have the ability to bind LPS. We measured the affinity as well as the stoichiometry and thermodynamic drivers of the LPS/Sox2 HMG interaction. The results of this study have the potential to shed light on a previously unrecognized mechanism for the progression of gastric cancer. 2. Materials and Methods
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2.1. Sox2 HMG Box Expression and Purification
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The plasmid encoding the N-terminal 6xHis tagged HMG box domain of Sox2 (residues 38-124) was a gift from the laboratory of Marius Clore at the National Institute of Health. Our lab expressed and purified the 6xHis tagged HMG box domain of Sox2 using a modified version of previously published protocols [33, 34]. Briefly, the 6xHis tagged HMG box domain of Sox2 was expressed in Rosetta II pLysS cells. When the OD600 of the culture reached 0.6, 1 mM IPTG was added to induce expression of the protein. Cells were harvested by centrifugation and frozen at -80ºC to aid in cell lysis. Cells were lysed by sonication and the lysate was cleared by centrifugation. Purification of the protein was achieved using nickel-nitrilotriacetic acid (Ni-NTA) resin (G Biosciences) in batch purification and the protein was eluted with 300 mM imidazole. The 6xHis tag was removed using thrombin. For pull-down assay experiments, the 6xHis tag was not cleaved from the protein. The Sox2 HMG domain was further purified using ion exchange chromatography with a Hi-Trap S FF column, and then purified by size exclusion using (HiLoad 16/60 Superdex 75 column; GE Healthcare). The purity of the protein was assessed using 4-20% polyacrylamide SDS gels (Bio-Rad) stained with Coomassie blue (Figure 1C). The measured mass of the protein was 10,689.65 Da using electrospray ionization mass spectrometry (data not shown), which is in agreement with the theoretical mass of the Sox2 HMG domain. Purified fractions of Sox2 were stored at -80ºC. 2.2. LPS preparation
Purified LPS isolated from e.coli strain O111:B4 was purchased from EMD Millipore Corporation Billerica, MA, USA. LPS was resuspended in the indicated buffers for the experiments described below. 2.3 Gel Filtration A HiLoad 16/600 Superdex 75 prep grade gel filtration column (GE Healtcare Life Sciences) was equilibrated with 50 mM Hepes, pH 7.4, and 150 mM NaCl on an Äkta Prime FPLC system at a flow rate of 1 ml/min. This same buffer was used for all experiments when running biological molecules through the column. A 2 ml injection
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Journal Pre-proof loop was used for all experiments. The total column volume was 120 ml. All experiments were run at 1 ml/min, and protein elution was monitored at 280 nm. 2.4 6xHis-tagged Sox2 HMG/LPS Pull-down Assays
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Pull-down assays were performed by equilibrating 1 ml of Ni-NTA resin (Gold Bio) in buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, and 20 mM imidazole (buffer A). In the experiment, 20 µM 6xHis Sox2 HMG and 0.35 mg of LPS in 3 ml of buffer A were incubated with 1 ml of Ni-NTA resin for 1 hour at room temperature in a column. The flow through (FT) was eluted, and the column was washed with 3 ml of buffer A, followed by elution with 3 ml of buffer containing 50 mM Tris, pH 8.0, 150 mM NaCl, and 300 mM imidazole (buffer B). The collected FT, wash, and elution fractions were digested with 0.25 µg/ml proteinase K for 1 hour at room temperature. After digestion, 10 µl aliquots of the FT, wash, and elution fractions were mixed with 10 µl 2x Laemmli buffer and loaded on a 4-20% polyacrylamide SDS gel (Bio-Rad). As a positive control, 0.25 µg of LPS was loaded on the gel. As a negative control to see if LPS could bind to the Ni-NTA resin in the absence of 6xHis Sox2 HMG, 0.7 mg of LPS were incubated with 1 ml of Ni-NTA resin in 3 ml of buffer A for 1 hour at room temperature. The flow through (FT) was eluted, and the column was washed with 3 ml of buffer A, followed by elution with 3 ml of buffer B. Aliquotes of 10 µl of the FT, wash, and elution were mixed with 10 µl of 2x Laemmli buffer and loaded on the same gel described above. The gel was run at 200 V, and then stained with a silver staining kit (Bio Rad) for LPS visualization.
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2.5. Intrinsic Tryptophan Fluorescence Assays
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Tryptophan fluorescence assays were performed using an AMINCO Bowman Series 2 fluorescence spectrophotometer. In the experiments, 2 ml of 0.5 µM Sox2 HMG domain in 50 mM Hepes, pH 7.4, 150 mM NaCl at 20ºC was titrated with increasing concentrations of LPS and excited at 280 nm. Fluorescence emission was measured between 320 nm and 370 nm. The excitation bandpass was 4 nm and the emission bandpass was 16 nm. A step size of 1 nm and an integration time of 1 second were used during data collection. The buffer was filter sterilized and degassed one hour before experiments were conducted. Three independent experiments were conducted to obtain the measured dissociation constant. 2.6. Isothermal Titration Calorimetry Isothermal titration calorimetry (ITC) experiments were conducted using a General Electric Microcal iTC200 instrument. All buffers for ITC experiments were filter sterilized and degassed before conducting experiments. In the ITC experiments, 20 injections of 2 µl aliquots of 1.25 mg/ml LPS were added to 300 µl of 10 µM Sox2 HMG domain in 50 mM Hepes, pH 7.4, 150 mM NaCl. The same buffer was used for LPS solution. The cell was stirred at 750 RPM at 25ºC. The system was allowed to equilibrate for 10 minutes before injections were initiated and injections were added every two minutes after equilibration. Dilution heats of injection obtained from injecting
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Journal Pre-proof LPS into buffer only were subtracted from the experiments involving titration of LPS into Sox2 HMG. 3. Results 3.1 Identification of a Sox2 HMG LPS complex using gel filtration
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To determine if Sox2 HMG has the ability to bind to LPS molecules and form a complex, we used gel filtration and identified that Sox2 HMG does indeed form a complex with LPS that is identifiable by gel filtration (Figure 2). In gel filtration chromatography, higher molecular weight proteins and biological complexes elute before lower molecular weight proteins and biological complexes. From Figure 2, we see that Sox HMG alone (magenta) elutes between 70 and 80 ml on the column used in this study by measuring the absorbance at 280 nm from the tryptophan residues in the Sox2 HMG protein. When LPS alone (black) is run on the column, we do not see an elution profile since the LPS molecule lacks the conjugated double bond system needed to absorb ultra violet light at 280 nm. When LPS is run on the gel filtration column with an excess of Sox2 HMG (dark green), we see a peak eluting between 45 and 50 ml corresponding to a higher molecular weight complex of Sox2 HMG bound to LPS followed by the excess Sox2 HMG eluting alone. It should be noted that non-globular HMG proteins such as Sox2 exhibit a myriad of odd elution profiles on gel filtration [35, 36], and determination of the stoichiometry HMG proteins alone and in complex with biological molecules should not be over interpreted using gel filtration [35]. Nonetheless, the data from Figure 2 provide initial evidence that Sox2 HMG domains can chelate LPS molecules.
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3.2 Confirmation of the Sox2 HMG/LPS complex using a 6xHis-tagged Sox2 HMG pull-down assay
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To confirm that Sox2 HMG binds LPS molecules, we used LPS pull-down assays where the concentration of Sox2 HMG was in excess of the LPS molecule. As a positive control, we ran 0.25 µg of LPS on the gel and we see faint bands in the 10-25 kDa range corresponding to LPS. We exploited the ability of the 6xHis-tagged Sox2 HMG to bind to Ni-NTA resin with high affinity in this experiment. We incubated 20 µM 6xHis Sox2 HMG and 0.35 mg of LPS in 3 ml of buffer A with Ni-NTA resin in the presence of low concentrations of imidazole (20 mM). Under these conditions, 6xHis-tagged Sox2 HMG can bind the Ni-NTA resin with high-affinity, and if LPS can bind to the HMG region of Sox2, when we elute the buffer from the column containing the Ni-NTA/6xHistagged Sox2 HMG/LPS mixture with imidazole, we would not expect to see much LPS in the elution since it will be bound to the Sox2 HMG which is bound to the Ni-NTA resin. Indeed, after eluting and digesting the elution with proteinase K, we do not see strong LPS bands in the gel (Figure 3 6xHis Sox2 HMG/LPS FT). We then washed the column with 3 ml of buffer A containing 20 mM imidazole and we see the presence of LPS bands suggesting that some of the LPS bound 6xHis-tagged Sox2 HMG was eluted under these conditions (Figure 3 6xHis Sox2 HMG/LPS wash). From our purification of 6xHis-tagged Sox2 HMG, we know that 300 mM imidazole can fully elute
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6xHis-tagged Sox2 HMG (Figure 1C). We therefore would predict the presence of strong LPS bands in this lane when we elute the 6xHis-tagged Sox2 HMG/LPS complex with 3 ml of buffer B, and that is indeed the case (Figure 3 6xHis Sox2 HMG/LPS elute). These results clearly show that Sox2 HMG does in fact bind to LPS molecules. In order to rule out the possibility that LPS could be binding the Ni-NTA resin, we incubated 0.35 mg of LPS in 3 ml of buffer A with Ni-NTA resin. If the LPS does not bind to the Ni-NTA resin, we expect to see strong LPS bands on the gel in this lane and this is what occurred in the experiment (Figure 3 LPS FT). The bands become weaker in intensity with the wash (Figure 3 LPS wash) and finally almost no intensity with the elution step (Figure 3 LPS elute) demonstrating that LPS cannot bind to the Ni-NTA resin on its own and confirming that LPS can bind to the HMG region of Sox2. We then were interested in investigating the affinity and stoichiometry of the Sox2 HMG/LPS interaction.
3.3. Probing the Sox2 HMG/LPS interaction using tryptophan fluorescence assays
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In order to determine the binding affinity of the Sox2 HMG domain with LPS, we used intrinsic tryptophan fluorescence of the two tryptophan residues that reside in the HMG domain of Sox2 to probe LPS binding to the protein (Figure 4). When tryptophan residues are excited with light in the 280 nm-300 nm wavelength range, they emit light in the 320-350 nm wavelength range with the maximum emission wavelength being dependent on the environment of the tryptophan residue. If binding of a ligand to the protein causes the tryptophan residue to be exposed to a more aqueous environment, typically, the intensity of the fluorescence emission decreases. Conversely, if the binding of a ligand to a protein causes the tryptophan to reside in a more hydrophobic environment, the fluorescence emission typically increases [37]. Since LPS contains non-polar regions, we predicted that binding of Sox2 HMG domain to LPS would result in an increase in tryptophan fluorescence emission if binding did occur. Indeed, upon titration of increasing concentrations of LPS into Sox2 HMG, we observed an increase in fluorescence emission at 330 nm, when the Sox2 protein was excited at 280 nm using an AMINCO Bowman Series 2 fluorescence spectrophotometer. We also observed a red shift in the emission maximum suggesting that the Sox2 protein was interacting with a more non-polar environment such as LPS [37] (Figure 4A). We also performed control experiments where LPS was titrated into buffer only, and the minimal fluorescence due to protein contamination from these titrations were subtracted from their corresponding titrations into the HMG domain of Sox2 so that only changes in Sox2 fluorescence were measured. We further analyzed our tryptophan emission data to estimate the binding affinity for the Sox2 HMG domain to LPS (Figure 4B). We normalized the change in fluorescence emission at 330 nm upon titration of LPS into the Sox2 HMG domain, and plotted these changes versus the concentration LPS to obtain a binding isotherm that was fit to the Hill equation. From this binding isotherm, we obtained a dissociation constant (Kd) of 0.90 µM ± 0.05 showing tight binding between Sox2 HMG and LPS. It should be noted that LPS is not a homogeneous molecule, but rather a heterogeneous molecule of varying size (Figure 5) [38] for the outer core polysaccharide unit. The
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Journal Pre-proof manufacturer of the LPS used in this study estimates the size of the LPS to be in the 10-kDa range, and other investigators have used this 10 kDa estimate as well [39]. We also measured a Hill coefficient of ~1.5 suggesting cooperative binding of Sox2 to LPS. Given that there are two regions on the lipid A region of LPS that resemble the backbone of DNA (Figure 5), we propose that there are two Sox2 binding sites on LPS and that sequential binding to LPS is cooperative. Since Sox2 HMG and LPS appear to show cooperative binding in the concentration ranges used in this assay, we further investigated this interaction using ITC. Nonetheless, these initial studies suggest that the Sox2 HMG domain can bind to LPS.
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3.4. Determining the thermodynamic drivers of the Sox2 HMG/LPS interaction using ITC
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To further investigate the stoichiometry of binding and to determine the thermodynamic drivers of the Sox2/LPS interaction, we investigated this interaction using ITC (Figure 4). After allowing the ITC instrument to equilibrate for 10 minutes, we began titrating LPS into the Sox2 HMG domain (Figure 6A). We observed an initial negative change in enthalpy (H) of binding, which contributes to a negative change in Gibbs free energy (G) leading to favorable binding. Interestingly, during the 7th injection of LPS into Sox2 HMG domain, we began to observe a positive H component of binding corresponding to heat being absorbed. When the raw heats were integrated with respect to time and plotted vs. the LPS:Sox2 HMG domain molar ratio (Figure 6B), we were able to see that LPS does indeed have two Sox2 binding sites. The data were fit using a two-site cooperative binding model, and we see that as the first exothermic binding event is halfcomplete, a second endothermic binding event begins to occur in a manner similar to that of HMG box proteins binding to DNA [16, 40, 41]. We were able to directly measure a negative H of binding of -16.1 kJ/mol for the first binding event, and a positive H of binding of 41.1 kJ/mol for the second event. We were also able to measure a K d of 0.46 µM ± 0.1 for the first binding site and a Kd of 1.6 µM ± 0.1 for the second binding site. Interestingly, the dissociation constant of 0.90 µM measured from Figure 4B in the tryptophan fluorescence assays likely represents an average of the two dissociation constants seen in the ITC experiments. From the ITC Kd values, we were able to determine the G of binding for each site from the equation G=-RT ln Ka where R = 8.314 J/molK, T is the temperature in Kelvin, and Ka=1/Kd. Once we determined G and H values for each site, we can determine the change in entropy (S) associated with each binding event from the equation G=H-TS where T is the temperature in Kelvin. This allowed us to determine that binding of the first site is enthalpically and entropically driven, whereas binding to the second site is entirely entropically driven, which is also observed for HMG box proteins binding to DNA [40, 41]. The second entropically driven interaction could be due to the displacement of organized water around the phosphate region in the lipid A region of LPS as is the case with DNA binding [40, 41], or it could be due to Sox2 disrupting hydrophobic interactions between LPS molecules. Nonetheless, sing two different techniques, we have shown that Sox2 can directly bind to LPS. This interaction could alter the immunostimulatory effects of LPS that lead to the progression of gastric cancer.
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Journal Pre-proof 4. Discussion
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Previous studies investigating the binding of the antimicrobial protein rBPI 21 to LPS using ITC showed that the rBPI21/LPS interaction was endothermic and driven entropically [42]. The rBPI21 protein is positively charged like the HMG domain of Sox2, however LPS was shown to only have one binding site for rBPI 21 while LPS has two Sox2 HMG binding sites. This difference could be due to the two proteins binding to different areas of LPS. The HMG domain of Sox2 likely binds to the two sugarphosphate moieties on LPS, whereas the authors of the rBPI21 study suggest that rBPI21 binds to the surface or outer core region of LPS [42]. They suggest that this binding event may cause the release of Ca2+ ions from the bacterial surface and expose the negative charge of LPS on the phosphate regions explaining the entropy driven binding event. On the other hand, the exothermic Sox2 HMG/LPS binding event could be explained by an ionic interaction between the lysine and arginine rich binding face of Sox2 HMG with a negatively charged phosphate on LPS. Since LPS is a lipid and will form membrane layers or micelles with neighboring LPS molecules, the second endothermic binding event driven by entropy could be due to a Sox2 HMG molecule binding to the second phosphate region on LPS and separating this LPS molecule from neighboring LPS molecules, which would be entropically favorable for binding. Work investigating the binding of HMGB1, a protein with two HMG domains, to LPS showed that the box A HMG domain of HMGB1 bound primarily inner/outer core region of LPS while box B HMG domain bound to the lipid A region of LPS [25]. We conducted a sequence alignment analysis of Sox2 HMG with the box A and box B HMG domains of HMGB1 and found that Sox2 HMG has greater sequence similarity with box B (25.6% identical, 50.0% similar) compared to box A (25.0% identical, 36.9% similar)(Figure 7). The high sequence similarity between box B and Sox2 HMG lends credence to the idea that the lipid A portion of LPS is the primary LPS binding site for Sox2 HMG. Interestingly, the putative lipid A binding region of box B in HMGB1 is located on a different alpha helix than the primary and secondary DNA intercalation sites of HMGB1 (Figure 7) suggesting a binding mechanism for LPS that is vastly different from the binding mechanism for DNA, which is not surprising. We identified two putative lipid A in regions Sox2 HMG. The proposed lipid A binging motif of LPS binding proteins is similar to that of heparin binding proteins where the motif BBXB is present and B is any basic residue while X is any residue [43, 44]. Another potential lipid A binding motif is BAB where B is a basic residue and A is an aromatic residue [45]. The identified BBXB and BAB lipid A binding region of KKFK in HMGB1 occurs distal to box B in the disordered linker region of HMGB1 at amino acids 87-90 [25]. We identified a KYR BAB potential lipid A binding motif at amino acids 109111 in a disordered region of Sox2 as well as a KRLR BBXB motif at residues 95-98 in helix III of Sox2 HMG (Figure 7). X-ray crystallography studies have shown that tyrosine residue in the KYR motif makes key hydrogen bonds with base pairs to stabilize the Sox2 HMG/DNA complex, while the KRLR motif in helix III of Sox2 does not make any bonds with the DNA molecule [46] and could be a likely candidate for the lipid A binding region of Sox2 HMG. The LPS dissociation constants for Sox2 HMG measured in this study were found to be on the micromolar scale (Figures 4 and 6) and these values are consistent
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with other proteins that have been shown to bind LPS such as HP0902, an H. pylori protein, which belongs to the cupin superfamily of proteins [39]. In the HP0902 study, the interaction between HP0902 and LPS was shown to be driven entirely by enthalpy [39] while this study demonstrates the interaction between Sox2 HMG and LPS has both entropic and enthalpic components leading towards a favorable binding interaction. This finding suggests that the LPS binding site for Sox2 HMG is different than that of HP0902. Indeed, the cupin superfamily has been shown to include sugar-binding proteins [47] therefore the likely binding LPS binding site for this protein is on the Oantigen, inner core, or outer core region of LPS (Figure 5), whereas the HMG box of Sox2 likely binds the sugar phosphate moiety of the lipid A region of LPS that resembles the backbone of DNA. The results of this study demonstrate that the HMG box region of Sox2 has the ability to bind bacterial LPS molecules in vitro. Sox2 HMG likely binds two regions of LPS at the two sugar phosphate regions of the lipid A region of LPS and Sox2 therefore has the potential to modulate LPS activation of TLR-4 and this will be the subject of a future study. Since Sox2 expression is decreased as an H. pylori infection leads to gastric cancer, it is possible that Sox2 could bind LPS and prevent activation of TLR-4 in the early stages of an H. pylori infection. As the infection progresses to gastric carcinoma and the expression of Sox2 is decreased, this could allow for more LPS molecules to activate TLR-4 and lead to increased inflammation in the gastric endothelium.
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5. Conclusions
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This is the first report showing that a single HMG box containing protein can bind to LPS molecules, however, this is not surprising considering that HMG box proteins have been shown to have a wide array of functions outside of their DNA binding and gene expression activities [15]. Future studies on the interplay between Sox2 and LPS in the progression of gastric cancer should provide interesting results that could have clinical significance in the understanding of gastric cancer pathogenesis. Future work on this project will investigate the ability of Sox2 to bind LPS from different bacterial species and identify key LPS binding regions in the HMG domain of Sox2 through mutagenic studies.
Author Contributions CSM conceived and supervised the study and wrote the manuscript. PH and EDP purified protein, conducted ITC experiments, conducted tryptophan fluorescence experiments, created the sequence alignment, and edited the manuscript. NAD, CG, TL, and KS purified protein, conducted tryptophan fluorescence experiments, and edited the manuscript. JVB and SSS conducted the gel filtration and LPS pull-down experiments.
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Acknowledgements We would like to thank Shaun Bevers and the Biophysics Core at the University of Colorado Anschutz Medical Campus for access to their General Electric Microcal iTC200 instrument. References
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Journal Pre-proof Figure Legends Figure 1. Schematic representation of full-length Sox2 (A). The X-ray crystal structure of the HMG box domain of Sox2 (residues 38-124) bound to DNA modeled from [46] (PDB ID: 1GT0) using pymol. The N and C-termini of the protein are indicated as well as the respective helix numbering, I, II, and III (B). 4-20% polyacrylamide SDS gel with 2 mg/ml of the Sox2 HMG domain (residues 38124), and a protein molecular weight ladder. The theoretical molecular weight of the Sox2 HMG domain after thrombin cleavage of the 6xHis tag is 10.689 kDa (C).
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Figure 2. Gel filtration of Sox2 HMG and the Sox2 HMG/LPS complex. Gel filtration elution profiles of 0.5 mg/ml of LPS alone (black curve), 2 mg/ml of Sox2 HMG alone (magenta curve), and 0.5 mg/ml of LPS and 2 mg/ml Sox2 HMG as a complex (dark green curve). Protein elution was monitored at 280 nm.
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Figure 3. LPS pull-down assay. 4-20% polyacrylamide SDS gel silver stained with a protein molecular weight ladder (ladder) and a positive control of LPS alone (0.25 µg LPS). After equilibrating the column with 20 mM imidazole and Sox2/HMG LPS, the flow through was collected (6xHis Sox2 HMG/LPS FT), the column was then washed with 20 mM imidazole again (6xHis Sox2 HMG/LPS wash), followed by elution of the Sox2/LPS complex with 300 mM imidazole (6xHis Sox2 HMG/LPS elute). For a negative control, LPS alone was allowed to equilibrate with the column with 20 mM imidazole, and the flow through was collected (LPS FT), followed by washing the column with 20 mM imidazole again (LPS wash), and finally eluting the column with 300 mM imidazole (LPS elute). LPS is visualized as the dark bands of varying molecular weights. Samples containing 6xHis Sox2 were digested with proteinase K before being loaded onto the gel.
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Figure 4. Binding of LPS to Sox2 HMG. Representative fluorescence emission spectra of 0.5 µM Sox2 HMG domain in 50 mM Hepes, pH 7.4, 150 mM NaCl at 20ºC titrated with increasing concentrations of LPS and excited at 280 nm. Reducing agents were not used in the experiment since the HMG domain of Sox2 lacks cysteine residues (A). Binding isotherm of LPS bound to the HMG domain of Sox2 (n=3). The change in fluorescence emission at 330 nm was normalized, and plotted versus the concentration of LPS. The resulting binding isotherm was fit with the Hill equation: Y=Bmax*x^n/(Kd^n+x^n) where Bmax is maximal change in fluorescence, Kd is the dissociation constant, and n is the Hill coefficient of cooperativity. Error bars represent s.d. Figure 5. Representative structure of LPS. LPS contains three distinctive units, the lipid A component (black), inner core (blue), and outer core (orange), which contains varying sizes of O-antigen polysaccharides. Saccharides are depicted as hexagons. Figure 6. Thermodynamics of the Sox2/LPS interaction. Representative raw heats emitted and absorbed after injecting 2 µl aliquots of 125 µM LPS into 10 µM Sox2 HMG domain. The buffer was 50 mM Hepes, pH 7.4, 150 mM NaCl and was stirred at 750
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Journal Pre-proof RPM at 25ºC (n=3) (A). Binding and enthalpy (H) analysis of the integrated heats with heats of mixing were subtracted. The molar ratio is LPS to Sox2 HMG domain. The data were fitted to a two-two site cooperative binding model of Y=((DH1*x)/(Kd1+x))+((DH2*x^n)/(Kd2^n+x^n)), where DH1 and DH2 are the change in enthalpy of the first and second binding events, respectively and Kd1 and Kd2 are the dissociation constants of the first and second binding events, respectively. The n parameter is a measure of cooperativity (B). Data were collected on a General Electric MicroCal iTC200 instrument.
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Figure 7. Alignments of Sox2 HMG with the HMG domains of HMGB1. Sequence alignment of Sox2 HMG with box A of HMGB1 and box B of HMGB1. Green cylinders indicate where helicies in Sox2 HMG form. Numbers to the left of the sequences indicate the starting amino acid number in the respective proteins. HMGB1 box residues shown in blue are identical at that position to Sox2 HMG while residues shown in red are similar to Sox2 at that position. The putative lipid A binding regions of Sox2 HMG are underlined in cyan. Primary and secondary DNA intercalating residues are indicated by a black 1 and 2, respectively.
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Figure 1.
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Figure 2.
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Figure 3.
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Figure 4.
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Figure 5.
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Figure 6.
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Figure 7.
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Journal Pre-proof Conflict of Interest
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The authors have nothing to disclose.
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