Activation of Toll-Like Receptor 5 Immune Signaling by HMGB1

Abstract

Human High Mobility Group Box-1 (HMGB-1) is an ubiquitously expressed nuclear protein which primarily functions as an architectural chromatin binding factor that binds to DNA in a structure specific manner. Recently, it has been linked to the pathogenesis of several inflammatory diseases regulating the innate immune system by two specific toll-like receptors (TLRs) such as TLR2 and TLR4. Here we present human HMGB-1 as an inflammatory regulator for TLR5 in immune signaling. The binding modes between these two proteins were demonstrated by a variety of cellular, biophysical and structural studies. The cellular assays revealed that HMGB1/TLR5 interaction invokes an elevation of downstream pro-inflammatory cytokines such as tumor necrosis factor-α, internuclein-8 (IL-8) which are critical in MyD88 dependant inflammatory pathways. Flourescence anisotropy assays revealed the binding constant (Kd) of 2.2uM between these two proteins with a further validation of interaction sites by solution NMR spectroscopy. For NMR structural studies 15N, 13C uniform labeled full length and tailless HMGB1 proteins was expressed and purified from E.coli BL21(DE3)pLysS cells and the 2D HSQC spectra were used for backbone and side chain residue chemical shift mapping from previously published spectra. Change in chemical shifts showed the direct molecular interactions between the acidic tail of HMGB1with the extracellular domain of TLR5 in physiological conditions. Thus, our studies not only provide compelling new insights into the TLR5 signal transduction by HMGB1 but also open the avenue of future therapeutic development.