Lipid Membrane Association of the T Cell Antigen Receptor ζ Subunit: Affinities and Structure

The T cell antigen receptor (TCR) is a cell surface protein complex that binds peptide antigen fragments presented by the Major Histocompatibility Complex (MHC). The interaction of TCR with MHC leads to T cell activation and initiation of an immune response,1 but the molecular details of the transmembrane signaling process remain unknown. TCRζ is the 115 amino acid long cytosolic signaling domain of the CD3-T cell receptor complex that plays a central role in this process. TCRζ is predominantly unstructured2 and carries several copies of the immunoreceptor tyrosine-based activation motif (ITAM). ITAMs become phosphorylated upon receptor engagement, constituting an early and obligatory event in the signaling cascade that blocks lipid association and lipid-associated conformational rearrangements3 of TCRζ. We present a characterization of the affinity of TCRζ to tethered lipid bilayers by SPR and a structural study of its association with such membranes using neutron reflectometry (NR). We studied the binding of unphosphorylated TCRζ to highly charged stBLMs rich in anionic phosphatidylserine (PS) or phosphatidylglycerol (PG) and observed affinity constants of Kd ≈ 10 μM. Minute amounts of phosphoinositides (PI(4,5)P2) increase the membrane affinity of the protein by more than 10-fold. An NR characterization of TCRζ in the membrane-associated state shows the major portion of the protein interfacially associated with the bilayer surface and a minor protein penetrating the bilayer deeply. This is consistent with a model in which an α-helical structure is aligned on the membrane surface that may be connected via a hinge with another α-helical segment inserted into the bilayer.[1] Call, M.E., and Wucherpfennig, K.W., Annu. Rev. Immunol. 23:101 (2005).[2] Sigalov, A.B., et al., Biochemistry 45:15731 (2006).[3] Aivazian, D.A., et al., Nat. Struct. Biol. 7:1023 (2000).