Exploring the biological functional mechanism of the HMGB1/TLR4/MD-2 complex by surface plasmon resonance
High Mobility Group Box 1 (HMGB1) was first identified as a nonhistone chromatin-binding protein that functions as a pro-inflammatory cytokine and a Damage-Associated Molecular Pattern molecule when released from necrotic cells or activated leukocytes. HMGB1 consists of two structurally similar HMG boxes that comprise the pro-inflammatory (B-box) and the anti-inflammatory (A-box) domains. Paradoxically, the A-box also contains the epitope for the well-characterized anti-HMGB1 monoclonal antibody “2G7”, which also potently inhibits HMGB1-mediated inflammation in a wide variety of in vivo models. The molecular mechanisms through which the A-box domain inhibits the inflammatory activity of HMGB1 and 2G7 exerts anti-inflammatory activity after binding the A-box domain have been a mystery. Recently, we demonstrated that: 1) the TLR4/MD-2 receptor is required for HMGB1-mediated cytokine production and 2) the HMGB1–TLR4/MD-2 interaction is controlled by the redox state of HMGB1 isoforms.
We investigated the interactions of HMGB1 isoforms (redox state) or HMGB1 fragments (A- and B-box) with TLR4/MD-2 complex using Surface Plasmon Resonance (SPR) studies.
Our results demonstrate that: 1) intact HMGB1 binds to TLR4 via the A-box domain with high affinity but an appreciable dissociation rate; 2) intact HMGB1 binds to MD-2 via the B-box domain with low affinity but a very slow dissociation rate; and 3) HMGB1 A-box domain alone binds to TLR4 more stably than the intact protein and thereby antagonizes HMGB1 by blocking HMGB1 from interacting with the TLR4/MD-2 complex.
These findings not only suggest a model whereby HMGB1 interacts with TLR4/MD-2 in a two-stage process but also explain how the A-box domain and 2G7 inhibit HMGB1.
KeywordsHMGB1 TLR4/MD-2 complex Surface plasmon resonance (SPR) Antagonist TLR4-signaling
Damage Associated Molecular Pattern
High Mobility Group Box 1
Equilibrium dissociation constant
MYELOID Differentiation factor 2
Receptor for Advanced Glycation Endproducts
Sodium dodecyl sulfate –polyacrylamide gel electrophoresis
Surface plasmon resonance
Total internal reflection fluorescence
Toll-like Receptors (TLRs)
Tumor necrosis factor
As its name implies, High Mobility Group Box 1 (HMGB1) is a small protein that migrates rapidly on SDS-PAGE gels and was first identified as a nonhistone chromatin-binding protein that has important biological activities in human health and diseases (Kang et al. 2014; Andersson and Tracey 2011; VanPatten and Alabed 2017). HMGB1 resides in the nucleus of most eukaryotic cells, where it functions as a transcriptional regulator facilitating the binding of several regulatory protein complexes to DNA (Wang et al. 2007; Stros 2010). Upon cellular activation, injury or death, HMGB1 translocates to the cytoplasm, where it can activate autophagy by interacting with beclin-1 (Kang et al. 2010), and to the extracellular medium, where it acts as a prototypic Damage Associated Molecular Pattern (DAMP) molecule. This DAMP has cytokine, chemokine, and growth factor activities, orchestrating the inflammatory and immune responses (Bianchi et al. 2017). When extracellular HMGB1 is released passively from damaged necrotic cells or actively from immune and/or stressed cells, it promotes inflammatory responses by binding to key pattern recognition receptors such as the Receptor for Advanced Glycation Endproducts (RAGE) and Toll-like Receptors (TLRs) (Schmidt et al. 2000). Upon binding to its receptors, HMGB1 induces nuclear translocation of nuclear factor-κB (NF-κB), leading to secretion of pro-inflammatory cytokines including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and interleukin-1β (IL-1β) (Wu et al. 2016).
HMGB1-TLR4 signaling has been strongly implicated in the pathogenesis of sterile injury (Maroso et al. 2010; Yang et al. 2013b). TLR4-deficient animals are significantly protected from tissue injury during hepatic ischemia. TLR4, a pivotal receptor for activation of innate immunity, including cytokine release and tissue damage, is required for HMGB1-dependent activation of macrophage TNF release, whereas RAGE and TLR2 are dispensable. TLR4 activity and interaction with its ligands depend on a molecular collaboration with the extracellular adaptor protein Myeloid Differentiation factor 2 (MD-2) (Miyake 2004; Visintin et al. 2006). Surface plasmon resonance (SPR) studies indicate that HMGB1 binds specifically to TLR4/MD-2, and that this binding requires cysteine 106 (Yang et al. 2010). Recently, we demonstrated that MD-2 binds specifically to the disulfide isoform of HMGB1 and not to the other isoforms (Yang et al. 2015a).
Structure–function analyses demonstrated that exogenous B-box recapitulates the cytokine activity of full length HMGB1 and stimulates TNF-훼 release from macrophages. TNF-stimulating activity localizes to 20 amino acids within B-box (B1-B20, HMGB1 amino acids 89 to 108) (Li et al. 2003). In contrast, despite a 40% sequence identity with B-box, A-box not only possesses no TNF-stimulating activity but also acts as an antagonist of HMGB1, and can compete with HMGB1 for binding sites on the surface of activated macrophages and suppresses HMGB1-induced pro-inflammatory cytokines release (Yang et al. 2004). Recombinant A-box was found protective in established preclinical inflammatory disease models (Andersson and Tracey 2011; Venereau et al. 2016), including mouse models of sepsis (Yang et al. 2004; Suda et al. 2006), lung injury induced by LPS, hepatitis, severe acute pancreatitis, ischemia-reperfusion injury in the heart, cerebral ischemia, and epilepsy (Maroso et al. 2010; Andrassy et al. 2008; Muhammad et al. 2008; Yuan et al. 2009). The specific mechanism of how A-box antagonizes HMGB1 remains unknown. Adding to this mystery is the fact that the A-box contains the epitope of the anti-HMGB1 monoclonal antibody, 2G7. How 2G7 inhibits HMGB1 activity through binding to the A-box remains unknown.
As it has long been known that A-box acts as a potent HMGB1 antagonist in many experimental models, we sought to address the underlying molecular mechanism using surface plasmon resonance (SPR). We found that A-box binds to TLR4 with a comparable equilibrium dissociation constant (KD) to HMGB1, but with a 10-fold slower dissociation rate than that of HMGB1. We propose that A-box antagonizes HMGB1 by blocking HMGB1-TLR4 binding sites, thus preventing HMGB1/TLR4/MD-2 complex formation.
Human TLR4/MD-2 complex, human MD-2, TLR4 were obtained from R&D Systems.
Recombinant HMGB1 was expressed in E. coli and purified to homogeneity as described previously (Wang et al. 1999; Li et al. 2004). DTT reduced HMGB1 was prepared as previously described (Yang et al. 2012). The non-oxidizable HMGB1 3S mutant, where serines replace cysteines, was provided by HMGBiotech (Milan, Italy). Recombinant GST-A-box, GST-B-box and GST protein were expressed in E. coli and purified to homogeneity as previously described (Li et al. 2003; Yang et al. 2004; Li et al. 2004). Recombinant A-box without GST tag was also provided by HMGBiotech (Milan, Italy). A-box with or without GST tag showed comparable binding activity in our SPR assay (Additional file 1: Figure S4), both types of A-box were utilized in this study. Typically, LPS content in the protein preparations was less than 1 pg LPS/μg protein.
Surface plasmon resonance analysis
Biacore T200 (GE Healthcare, USA) was used for real-time binding interaction studies. Binding reactions were done in HBS-EP buffer from BIAcore, containing 10 mM hepes, 150 mM NaCl, 3 mM EDTA and 0.05% surfactant p20, pH 7.4. At least 3 independent experiments were performed.
TLR4/MD-2 or TLR4 and redox forms of HMGB1 binding analyses
A slow, high-level immobilization of TLR4/MD-2 or rhTLR4 protein was obtained on a CM5 series chip (GE Healthcare). The TLR4/MD-2 complex was diluted to a concentration of 20 μg/mL in 10 mM Acetate buffer (pH 4.5). A 1:1 mixture of N-hydroxysuccinimide and N-ethyl-N-(dimethyaminopropyl)carbodiimide was used to activate 2 flow-cells of the CM5 chip. One flow-cell was used as a reference and thus immediately blocked upon activation with 1 M ethanolamine (pH = 8.5). The sample flow-cell was injected with the diluted TLR4/MD-2 at a flow rate of 10 μL/min. The TLR4/MD-2 injection was stopped when the surface plasmon resonance reached ~ 1200 RU. For TLR4/MD-2 and HMGB1 isoform kinetics assays, HMGB1 isoforms were sequentially injected at a flow rate of 30 μL/min for 60s at 25 °C; the dissociation time was set for 1 min. The concentrations were 31.25, 62.5, 125, 250, 500, 1000 nM for disulfide and reduced HMGB1; 310, 625, 1250, 2500, 5000 nM for 3S mutant HMGB1. The rhTLR4 protein was diluted to a concentration of 20 μg/mL in 10 mM Acetate buffer (pH 5.0) and was immobilized on the CM5 chip according to the same amine coupling method described above. The TLR4 injection was stopped when the surface plasmon resonance reached ~ 800 RU. For TLR4 and HMGB1 isoform kinetics assays, HMGB1 isoforms were sequentially injected at a flow rate of 30 μL/min for 60s at 25 °C, the dissociation time was set for 1–2 min. The concentrations were 31.25, 62.5, 125, 250, 500, 1000 nM for disulfide and reduced HMGB1; 250, 500, 1000, 2000, 4000, 8000 nM for 3S mutant HMGB1. The equilibrium dissociation constant (KD) for individual analytes was obtained to evaluate the binding affinity by using the BIAEvaluation 2.0 software (GE Healthcare) supposing a 1:1 binding ratio.
TLR4/MD-2 and GST-A-box or GST-B-box binding analyses
TLR4/MD-2 protein was immobilized onto a CM5 series chip (GE Healthcare) by amine coupling chemistry. GST protein alone was used as negative control (Additional file 1: Figures S2-S3). GST-A-box or GST-B-box were sequentially injected over immobilized TLR4/MD-2 (1100 RU) at a flow rate of 30 μL/min for 60s at 25 °C; the dissociation time was set for 1 min. The concentrations were 150, 310, 625, 1250, 2500, 5000 nM for GST-A-box and GST-B-box. The association and dissociation phases of GST-A-box were separately fitted to a 1:1 L binding model provided in the BIAevaluation 2.0 software. Binding affinity of GST-B-box was determined by global fitting of data to a steady-state affinity model in the BIAEvaluation 2.0.
TLR4 and GST-A-box or GST-B-box binding analyses
TLR4 protein was immobilized onto a CM5 series chip (GE Healthcare) by amine coupling chemistry. GST-A-box was sequentially injected over the immobilized TLR4 (800 RU) at a flow rate of 30 μL/min for 60s at 25 °C; the dissociation time was set for 2 min. The concentrations were 310, 625, 1250, 2500, 5000, 10,000 nM. The equilibrium dissociation constant (KD) was obtained by using the BIAEvaluation 2.0 software (GE Healthcare) supposing a 1:1 binding ratio. 1 μM and 5 μM GST-B-box was injected over the immobilized TLR4 at a flow rate of 30 μL/min for 60s at 25 °C, however there has no observed binding activity. GST protein alone was used as negative control; no binding was observed (Additional file 1: Figures S2-S3).
MD-2 and GST-A-box or GST-B-box binding analyses
GST-A-box or B-box protein was immobilized onto a CM5 series chip (GE Healthcare) by amine coupling chemistry. MD-2 was sequentially injected over the immobilized GST-A-box (750 RU) or GST-B-box (1000 RU) at a flow rate of 30 μL/min for 60s at 25 °C; the dissociation time was set for 1 min. The concentrations were 17, 31.25, 62.5, 125, 250, 500 nM. Apparent equilibrium dissociation constants (KD) were determined by global fitting of data to a steady-state affinity model in the BIAEvaluation 2.0 software. Binding affinity and kinetics data of HMGB1 isoforms and segments to TLR4/MD-2 receptors are listed in Additional file 1: Tables S1-2S. Data shown in this report are representative of three independent experiments.
Biacore analysis of complex formation among TLR4/MD-2, TLR4, A-box and HMGB1
The assay was performed using the HBS-EP buffer from BIAcore as described above. HMGB1 (100 nM) was injected on a TLR4/MD-2 complex sensor chip surface followed by HBS buffer or HMGB1 (100 nM) or TLR4/MD-2 (100 nM) or mixture of two proteins (1:1 M ratio) using the dual injection command. In competition experiments, A-box (10 μM) was injected on a TLR4 sensor chip surface followed by 2-fold dilutions of HMGB1 (2.5–5 μg/ml) using the dual injection command. In separate experiments, HMGB1 (500 nM) was injected on a TLR4 sensor chip surface, followed by A-box using the dual injection command (Additional file 1: Figure S5).
The TLR4/MD-2 complex is sensitive to the oxidative state of HMGB1, while TLR4 is not
Previously, we reported that HMGB1 was incapable of directly binding to TLR4 in the absence of MD-2 (Yang et al. 2015a). In this study, we revisited this observation and refined the experimental conditions (immobilization levels and/or switching ligand and analyte) for this binding assay. Using these optimized conditions we found that disulfide HMGB1 does bind to TLR4 (coated on the sensor chip) in a concentration-dependent manner with an apparent KD of 0.64 ± 0.01 μM (Fig. 2d). Next, the other isoforms of HMGB1 were injected onto the TLR4 sensor chip to investigate which redox state of HMGB1 was most conducive to binding (Fig. 2e-f). Reduced HMGB1 bound to TLR4 with a comparable equilibrium dissociation constant KD = 0.65 ± 0.01 μM (Fig. 2e), indicating that a disulfide bond between C23 and C45 has a negligible influence on the binding to TLR4. However, the sensorgrams demonstrated a 2-fold slower dissociation rate (kd = 0.128 ± 0.002 s− 1, Additional file 1: Table S2) for disulfide HMGB1 compared with reduced HMGB1, indicating that the complex with TLR4 is more stable once formed. We also found that 3S, where C23, C45 and C106 are mutated to serines, binds to TLR4 with a weaker affinity (apparent KD = 4.20 ± 0.09 μM) (Fig. 2f), suggesting that three cysteine residues within HMGB1 are essential for binding to TLR4.
A-box binds to TLR4/MD-2 complex and major binding sites are located on TLR4
B-box binds to MD-2 but not TLR4
Plausible mechanism of HMGB1-TLR4 signaling and the role of A-box as antagonist
Binding of HMGB1 to TLR4 has been shown to activate the MyD88 signaling pathway, thus resulting in the release of pro-inflammatory cytokines (Ugrinova and Pasheva 2017). It is unknown whether HMGB1 recognition by the TLR4/MD-2 complex shares similarities with other prototypical activators of TLR4 signaling, such as lipopolysaccharide (LPS). In the last decade, the crystal structures of the LPS receptors and accessory proteins have been characterized. Recently, Kim and coworkers reconstituted the entire cascade of LPS transfer to TLR4/MD-2 in a total internal reflection fluorescence (TIRF) microscope in a single-molecule analysis, reveling that a single LPS molecule bound to CD14 is transferred to TLR4/MD-2 in a TLR4-dependent manner (Kim and Kim 2017). Upon binding of LPS to a hydrophobic pocket in MD-2, two LPS-bound TLR4/MD-2 complexes form an M-shaped dimer, followed by activation of the signaling pathway (Kim and Kim 2017).
We hypothesized that HMGB1 may act in a manner similar to LPS, binding to TLR4/MD-2 complex and then inducing the dimerization of HMGB1/TLR4/MD-2 complexes, which brings together the cytosolic toll/interleukin-1 receptor (TIR) domains of TLR4 to recruit downstream adaptor molecules. To examine the details of the binding of HMGB1 to the TLR4/MD-2 complex, we immobilized one ligand on a Biacore CM5 sensor chip and used a sequential dual-injection method where the second analyte is injected after the first analyte with zero dissociation time. If a two-step increase in the bound mass is observed, we infer that the second analyte can bind to the complex of the ligand and first analyte; conversely, a decrease or unchanged signal after the second injection suggests that the first and second analytes compete for binding to the ligand, and then a ternary complex is not formed.
Previously, SPR studies reported by Yang et al. (Yang et al. 2010) showed that anti-HMGB1 monoclonal antibody (mAb) 2G7 also inhibited the binding of HMGB1 to TLR4/MD-2. This antibody has been extensively used by researchers to block HMGB1 activity both in vitro and in vivo (Venereau et al. 2016; Ugrinova and Pasheva 2017). The epitope in HMGB1 that binds to 2G7 has been identified within amino acids 53–63 of the A-box subunit (Qin et al. 2006). However, the molecular mechanisms whereby binding of 2G7 to A-box has anti-inflammatory activity remained elusive. Based upon the finding of A-box as an HMGB1 antagonist, we speculate that mAb 2G7 would share similar mechanism to inhibit HMGB1 activity (Scheme 1c) by blocking HMGB1-TLR4 interactions.
In summary, SPR studies showed that HMGB1 and its fragments (A-box and B-box) individually interact with the TLR4/MD-2 receptor with different binding and kinetic parameters. Our study also reveals that HMGB1 likely activates TLR4 signaling through inducing TLR4/MD-2 dimerization. The HMGB1 recognition cascade can be disrupted by the antagonistic A-box fragment due to its higher binding affinity to TLR4 or by blocking A-box with anti-HMGB1 mAb 2G7. Ongoing studies further detailing HMGB1/TLR4/MD-2 interactions will facilitate the design and development of therapeutics to inhibit HMGB1-mediated inflammation.
We acknowledge Dr. Sonya VanPatten with help in manuscript editing.
This work was supported by funding from the Feinstein Institute for Medical Research and National Institutes of Health (NIH) Grant S10 RR033072–01 to Y.A-A, and by Associazione Italiana Ricerca sul Cancro (AIRC) Grant IG 18623 to M.E.B.
Availability of data and materials
All data generated or analyzed during this study are included in this published article and its supplementary information files.
Conception and design of experiments, and development of assays: MH, KJT, YAA. Acquisition of data, and/or analysis and interpretation of data: MH, MEB, YAA. Drafting the manuscript: MH, MEB, TRC, YAA. Revising the manuscript: MH, MEB, TRC, KJT, YAA. Final approval prior to submission: MH, MEB, TRC, KJT, YAA.
Ethics approval and consent to participate
YAA is on patents related to HMGB1 antagonists, and MEB on patents related to the HMGB1 3S mutant.
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