Biphasic Force-Regulated Phosphorylation Site Exposure and Unligation of ERM Bound with PSGL-1: A Novel Insight into PSGL-1 Signaling via Steered Molecular Dynamics Simulations

Abstract

The PSGL-1-actin cytoskeleton linker proteins ezrin/radixin/moesin (ERM), an adaptor between P-selectin glycoprotein ligand-1 (PSGL-1) and spleen tyrosine kinase (Syk), is a key player in PSGL-1 signal, which mediates the adhesion and recruitment of leukocytes to the activated endothelial cells in flow. Binding of PSGL-1 to ERM initials intracellular signaling through inducing phosphorylation of Syk, but effects of tensile force on unligation and phosphorylation site exposure of ERM bound with PSGL-1 remains unclear. To answer this question, we performed a series of so-called “ramp-clamp” steered molecular dynamics (SMD) simulations on the radixin protein FERM domain of ERM bound with intracellular juxtamembrane PSGL-1 peptide. The results showed that, the rupture force of complex pulled with constant velocity was over 250 pN, which prevented the complex from breaking in front of pull-induced exposure of phosphorylation site on immunoreceptor tyrosine activation motif (ITAM)-like motif of ERM; the stretched complex structure under constant tensile forces <100 pN maintained on a stable quasi-equilibrium state, showing a high mechano-stabilization of the clamped complex; and, in consistent with the force-induced allostery at clamped stage, increasing tensile force (<50 pN) would decrease the complex dissociation probability but facilitate the phosphorylation site exposure, suggesting a force-enhanced biophysical connectivity of PSGL-1 signaling. These force-enhanced characters in both phosphorylation and unligation of ERM bound with PSGL-1 should be mediated by a catch-slip bond transition mechanism, in which four residue interactions on binding site were involved. This study might provide a novel insight into the transmembrane PSGL-1 signal, its biophysical connectivity and molecular structural basis for cellular immune responses in mechano-microenvironment, and showed a rational SMD-based computer strategy for predicting structure-function relation of protein under loads.

Spatiotemporal characteristics of P-selectin-induced β2 integrin activation of human neutrophils under flow

“…Meanwhile, disruption of the actin cytoskeleton is highly suppressive to b 2 -integrin activation through immobilized chemokine signaling (18). Ezrin/radixin/ moesin (ERM) connects the cytoplasmic domains of PSGL-1 and CD44 with the cytoskeleton (25,26,29,30). Talin-1 (a member of a four-point-one, ezrin, radixin, moesin (FERM)domain protein family) binds the cytoplasmic tail of an integrin and recruits cytoskeletal and signaling proteins to activate integrins synergistically (6).…”

Force-enhanced biophysical connectivity of platelet β3 integrin signaling through Talin is predicted by steered molecular dynamics simulations

“…A force threshold of 60pN was shared by E, N HB and f D , as it should be. The catch-slip bond phenomenon had been observed by AFM and BFP as well as flow chamber experiments for various adhesive molecule systems, such as LFA-1 with ICAM-1 24 , vWF with ADMAMTS13 39 , α IIb β 3 with fibrinogen 5 or, and predicted through MD simulations for the molecular systems, such as von Willebrand factor (vWF) with GPIbα 34 and PSGL-1 with ERM 36 as well as β3 integrin with Kindin3 40 .…”

“…MD simulations were performed with periodical boundary condition and 2 fs timestep as well as the CHARMM27 all-atom force field 36 , along with cMAP correction for backbone, particle mesh Ewald (PME) algorithm for electrostatic interaction, a 12 Å cut off for electrostatic and van der Waals interaction. The system was energy-minimized firstly for 15,000 steps with heavy or non-hydrogen protein atoms being fixed, and then for another 15,000 steps with all atoms free.…”

“…1 b). To reveal the mechanical regulation on biophysical connectivity of β3-integrin signaling through Talin and its molecular basis, we herein investigated interaction of β3-integrin CT domain to Talin F3 domain under various mechanical loads through a series of molecular dynamics (MD) simulations, which were performed for modelling molecular interactions successfully in various extracellular and intracellular molecular systems 34 – 36 . The present results revealed a force-enhanced biophysical connectivity of β3 integrin signaling through Talin, its mechano-kinetics regulation mechanism and structural basis, provide not only a novel insight into β3 integrin signaling in platelet activation, hemostasis and thrombosis under mechano- microenvironments but also a new clue for drug design and treatment of thrombotic diseases.…”

MD Simulations on a Well-Built Docking Model Reveal Fine Mechanical Stability and Force-Dependent Dissociation of Mac-1/GPIbα Complex

“…It signed that the computer strategy with "ramp-clamp" SMD simulation was practicable in examining the mechano-regulation on receptor-ligand interactions, as done in our previous work for the interaction of PSGL-1 with ERM (Feng et al, 2020) or Kindlin 2 with β 3 integrin (Zhang et al, 2020) and Model II, a docking model treated with "force-ramp + snapback" SMD simulation, was suitable in studying the structure-function relation for the complex of Mac-1 with GPIbα.…”