Structural diagram of a Tim4 molecule attached to a lipid membrane. Lipid is shown as a 2-box model, with head group in burgundy and tail in gray. The coordinating Ca2+, providing main binding is shown as yellow sphere. Four peripheral basic residues, denoted by groups of blue tips, provide secondary binding.

Recognition of phosphatidylserine (PS) lipids exposed on the extracellular leaflet of plasma membranes is implicated in both apoptotic cell removal and immune regulation. The PS receptor T cell immunoglobulin and mucin-domain-containing molecule 4 (Tim4) regulates T-cell immunity via phagocytosis of both apoptotic (high PS exposure) and nonapoptotic (intermediate PS exposure) activated T cells. The latter population must be removed at lower efficiency to sensitively control immune tolerance and memory cell population size, but the molecular basis for how Tim4 achieves this sensitivity is unknown. Using a combination of interfacial X-ray scattering, molecular dynamics simulations, and membrane binding assays, we demonstrate how Tim4 recognizes PS in the context of a lipid bilayer. Our data reveal that in addition to the known Ca2+-coordinated, single-PS binding pocket, Tim4 has four weaker sites of potential ionic interactions with PS lipids. This organization makes Tim4 sensitive to PS surface concentration in a manner capable of supporting differential recognition on the basis of PS exposure level. The structurally homologous, but functionally distinct, Tim1 and Tim3 are significantly less sensitive to PS surface density, likely reflecting the differences in immunological function between the Tim proteins. These results establish the potential for lipid membrane parameters, such as PS surface density, to play a critical role in facilitating selective recognition of PS-exposing cells. Furthermore, our multidisciplinary approach overcomes the difficulties associated with characterizing dynamic protein/membrane systems to reveal the molecular mechanisms underlying Tim4’s recognition properties, and thereby provides an approach capable of providing atomic-level detail to uncover the nuances of protein/membrane interactions.

See:  Gregory T. Tietjena, Zhiliang Gongb,c, Chiu-Hao Chend, Ernesto Vargasa, James E. Crooksa, Kathleen D. Caob,c, Charles T. R. Heffernb,c, J. Michael Hendersonb,c, Mati Merone, Binhua Linc,e, Benoît Rouxa,b,f, Mark L. Schlossmand, Theodore L. Steckf, Ka Yee C. Leea,b,c,1, and Erin J. Adamsa,f,g,1, “Molecular mechanism for differential recognition of membrane phosphatidylserine by the immune regulatory receptor Tim4” PNAS 111 (15), 1463-1472 (2014).

Author affiliations: aProgram in Biophysical Sciences, Institute for Biophysical Dynamics, bDepartment of Chemistry, and cJames Franck Institute, The University of Chicago, Chicago, IL; dDepartment of Physics, University of Illinois at Chicago, Chicago, IL; eCenter for Advanced Radiation Sources, fDepartment of Biochemistry and Molecular Biology, and gCommittee on Immunology, The University of Chicago, Chicago, IL