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Mol Med. 2018 May 10;24(1):21. doi: 10.1186/s10020-018-0023-8.

Exploring the biological functional mechanism of the HMGB1/TLR4/MD-2 complex by surface plasmon resonance.

Author information

1
Center for Molecular Innovation, The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, New York, 11030, USA. mhe@northwell.edu.
2
Chromatin Dynamics Unit, Division of Genetics and Cell Biology, San Raffaele University and San Raffaele Scientific Institute IRCCS, Via Olgettina 58, 20132, Milan, Italy.
3
Center for Molecular Innovation, The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, New York, 11030, USA.
4
Center for Biomedical Science, and Center for Bioelectronic Medicine, The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, New York, 11030, USA.
5
Center for Molecular Innovation, The Feinstein Institute for Medical Research, 350 Community Drive, Manhasset, New York, 11030, USA. yalabed@northwell.edu.

Abstract

BACKGROUND:

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.

METHODS:

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.

RESULTS:

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.

CONCLUSIONS:

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.

KEYWORDS:

Antagonist; HMGB1; Surface plasmon resonance (SPR); TLR4-signaling; TLR4/MD-2 complex

PMID:
30134799
PMCID:
PMC6085627
DOI:
10.1186/s10020-018-0023-8
[Indexed for MEDLINE]
Free PMC Article

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