Comparative Analyses of the Conformational Dynamics Between the Soluble and Membrane-Bound Cytokine Receptors

www.nature.com/scientificreports OPEN Comparative Analyses of the Conformational Dynamics Between the Soluble and Membrane-Bound Cytokine Receptors Chao-Yie Yang Cytokine receptors receive extracellular cues by binding with cytokines to transduce a signaling cascade leading to gene transcription in cells. Their soluble isoforms, functioning as decoy receptors, contain only the ectodomain. Whether the ectodomains of cytokine receptors at the membrane exhibit different conformational dynamics from their soluble forms is unknown. Using Stimulation-2 (ST2) as an example, we performed microsecond molecular dynamics (MD) simulations to study the conformational dynamics of the soluble and the membrane-bound ST2 (sST2 and ST2). Combined use of accelerated and conventional MD simulations enabled extensive sampling of the conformational space of sST2 for comparison with ST2. Using the interdomain loop conformation as the reaction coordinate, we built a Markov State Model to determine the slowest implied timescale of the conformational transition in sST2 and ST2. We found that the ectodomain of ST2 undergoes slower conformational relaxation but exhibits a faster rate of conformational transition in a more restricted conformational space than sST2. Analyses of the relaxed conformations of ST2 further suggest important contributions of interdomain salt-bridge interactions to the stabilization of different ST2 conformations. Our study elucidates differential conformational properties between sST2 and ST2 that may be exploited for devising strategies to selectively target each isoform. The interleukin-1 (IL-1) family of cytokines and their receptors are key regulators of innate immunity that can initiate inflammatory response in hosts to fend off foreign antigens1,2. Ten IL-1 receptors (IL-1R) have been identified including the IL-1R1, IL-1R2, IL-1R accessory protein (IL-1RAcP or IL-1R3), IL-1R like 1 (IL-1RL1, ST2 or IL-1R4), IL-18Rα/β (or IL-1R4/7) and IL-1R accessory protein like 1 (IL-1RAPL or IL-1R9)3,4. IL-1R are single pass transmembrane proteins that contain an ectodomain (ECD) and a conserved cytoplasmic Toll-IL-1-Receptor (TIR) domain2. The ectodomain consists of three consecutive immunoglobulin-like C2 type-1,2,3 domains (denoted as D1-D3) connected by short linkers. The current model of the IL-1 pathway activation suggests that the IL-1 cytokine binds to its cognate IL-1R to recruit a second IL-1R member forming a hetero-trimeric protein complex and causing dimerization of TIR domains for downstream signaling5. Activation of the IL-1 pathway by extracellular cytokines can be regulated by negative or decoy receptors. The negative receptors, such as IL-1R2, lack the cytoplasmic domain to induce downstream signaling6. The decoy receptors include circulatory soluble receptors7,8 that sequester cytokines and limit the pool of free cytokines for binding to cytokine receptors on the cell membrane. The interplay of the binding between the cytokines and the membrane and soluble cytokine receptors allows to control the strength and duration of cytokine-mediated inflammatory response after cytokines are secreted to blood circulation. Among the IL-1R members, ST2 is expressed on hematopoietic cells including T helper type 2 (Th2) cells, group 2 innate lymphoid cells (ILC2), regulatory T cells (Tregs) and mast cells9,10. Membrane-bound ST2 binds with the only known ligand IL-33 to recruits IL-1RAcP resulting in TIR domain dimerization between ST2 and IL-1RAcP5,11. Signal transduction via the ST2/IL-33 pathway leads to p38 MAP kinases phosphorylation and nuclear factor (NF)-κB activation11. Activation of the ST2/IL-33 axis in Th2 cells causes secretion of IL-4, IL-5, IL-1312–14 and IL-915 that elicit type 2 immune response16. Dysregulation in the ST2/IL-33 signaling has been associated with several disease progression including excessive induction of ST2/IL-33 in Th2 cells14 found in Department of Pharmaceutical Sciences, College of Pharmacy, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America. e-mail: Scientific Reports | (2020) 10:7399 | https://doi.org/10.1038/s41598-020-64034-z 1 www.nature.com/scientificreports/ www.nature.com/scientificreports Figure 1. The crystal and the model structures of ST2 and sST2. (A) The model structure of mouse IL-1RAcP, mouse ST2, mouse IL-33 and the POPC membrane. (B) Models of human ST21, ST22 and ST23. The lipid membrane built with ST23 is shown and human IL-33 (yellow surface) bound to ST21 is displayed as a reference. All ST21, ST22 and ST23 are aligned to the transmembrane and the cytosolic domains in ST23. (C) The crystal structure of human ST2ECD and human IL-33 (PDBID: 4KC3). ST2ECD is used to construct the sST2 model. The lipid membrane is shown as a quick surface model. Figures are prepared using PyMOL 2.3.0 (www.github.com/ schrodinger/pymol-open-source) and VMD 1.9.4 (www.ks.uiuc.edu/Research/vmd)27,28. asthma patients17. In patients developing graft versus host disease (GVHD) after hematopoietic cells transplantation (HCT), excessive increases of the sST2 level reduce the pool of IL-3318 for activation of the ST2/IL-33 axis in Th2, ILC2, and Tregs cells that leads to unrestrained inflammation in early GVHD progression19–21. Antibodies20,22 and small-molecule inhibitors23 targeting membrane-bound and soluble ST2 have been reported. Both isoforms contain the same cytokine binding domains. This presents a challenge to develop specific inhibitors for use in different disease settings. Although antibodies therapeutics targeting the extracellular domains of cytokine receptors22,24 can recognize specific epitopes, no selectivity between the two forms has been reported. We25 and other groups5,26 have studied the conformations of the ectodomain of ST2 (ST2ECD) using Small Angle X-ray scattering (SAXS) and computational simulations. These data showed that ST2ECD possess high conformational flexibility. A recent study indicated that ST2 undergoes a greater conformational motion than IL-1R1 before binding to the cognate cytokine on the cell membrane5. However, the extent of different conformational flexibilities between sST2 and ST2 remains unknown. Despite that sST2 and ST2 both contain the D1-3 domains, we hypothesized that ST2 may have limited conformational flexibility than sST2 because ST2 is fixated on the membrane via the transmembrane and the cytoplasmic domains. A better understanding of the differences between sST2 and ST2 conformations will provide insights to develop selective inhibitors. In this work, we performed MD simulations of sST2 and ST2 in their glycosylated forms using the physiological salt concentration. A broad conformational space of sST2 was mapped using conformations obtained from conventional MD and accelerated MD (cMD and aMD) simulations. Because aMD was not implemented to sample ST2, we performed cMD simulations using three different orientations of ST2ECD on the mem (...truncated)