The cytosolic domain of T-cell receptor ζ associates with membranes in a dynamic equilibrium and deeply penetrates the bilayer

Abstract

Interactions between lipid bilayers and the membrane-proximal regions of membrane-associated proteins play important roles in regulating membrane protein structure and function. The T-cell antigen receptor is an assembly of eight single-pass membrane-spanning subunits on the surface of T lymphocytes that initiates cytosolic signaling cascades upon binding antigens presented by MHC-family proteins on antigen-presenting cells. Its ζ-subunit contains multiple cytosolic immunoreceptor tyrosine-based activation motifs involved in signal transduction, and this subunit by itself is sufficient to couple extracellular stimuli to intracellular signaling events. Interactions of the cytosolic domain of ζ (ζ) with acidic lipids have been implicated in the initiation and regulation of transmembrane signaling. ζ is unstructured in solution. Interaction with acidic phospholipids induces structure, but its disposition when bound to lipid bilayers is controversial. Here, using surface plasmon resonance and neutron reflection, we characterized the interaction of ζ with planar lipid bilayers containing mixtures of acidic and neutral lipids. We observed two binding modes of ζ to the bilayers in dynamic equilibrium: one in which ζ is peripherally associated with lipid headgroups and one in which it penetrates deeply into the bilayer. Such an equilibrium between the peripherally bound and embedded forms of ζ apparently controls accessibility of the immunoreceptor tyrosine-based activation signal transduction pathway. Our results reconcile conflicting findings of the ζ structure reported in previous studies and provide a framework for understanding how lipid interactions regulate motifs to tyrosine kinases and may regulate the T-cell antigen receptor biological activities for this cell-surface receptor system.

The TCR is an allosterically regulated macromolecular machinery changing its conformation while working

“…This was also true for a CD3ε cytosolic tail peptide . In both cases, binding to the membrane induced folding of the otherwise unstructured peptides and required the presence of a basic rich sequence (BRS) in those peptides . Membrane binding of the ζ and CD3ε peptides shielded the ITAMs from phosphorylation in an in vitro kinase assay .…”

“…It has been reported that these cytoplasmic tails bind to each other, and might thus form some defined structure by protein‐protein interactions . However, NMR and structural analyses only exist of isolated peptides corresponding to these tails, showing that these peptides are unstructured . It is therefore realistic to think that these peptides in isolation do not find their partners to fold into a common structure and therefore they were unstructured.…”

“…It is still possible that all these changes occur simultaneously and that only two states exist. However, it has been shown using biophysical methods that the isolated ζ and CD3ε tails can adopt several distinguishable conformational states when binding to membranes with acidic lipids. Thus, it might be that the TCR can adopt several distinct states.…”

Electrostatic interactions: From immune receptor assembly to signaling

“…How these lipids relate to biological phospholipids is not known, but point to the possibility that other components of the phospholipid, other than the acidic head group, may be involved in regulating immune receptor binding dynamics and function. Remarkably, lipid association to any acidic, but not zwitterionic, phospholipids prevented CD3ζ CT phosphorylation by a recombinant Scr kinase in an in vitro assay and was due to deep embedding of the side chains within the bilayer . This led the authors to postulate for the first time a model in which the phosphorylation of CD3ζ chains of the TCR was prevented by sequestering the ITAM from kinases through electrostatic interaction‐driven dynamic membrane binding.…”

Clustering of CD3ζ is sufficient to initiate T cell receptor signaling

“…However, a previous in vitro study has showed that clustering of CD3ζ did not change its random-coil-like unstructured status (Sigalov et al, 2004). It is likely that LIC-CD3z remains dynamically membrane associated (Zimmermann et al, 2017) regardless of its clustering state. It should be also considered that previous work showed that ITAMs can still be phosphorylated by Lck in membrane-bound state (Zhang et al, 2011).…”