Molecular mechanism for differential recognition of membrane phosphatidylserine by the immune regulatory receptor Tim4

Abstract

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 Ca 2+ -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 PSexposing 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. differential membrane recognition | PS recognition

Characterization of Human and Murine T-Cell Immunoglobulin Mucin Domain 4 (TIM-4) IgV Domain Residues Critical for Ebola Virus Entry

“…The four basic residues that were identified were proposed to bind directly to lipid head groups of the membrane bilayer and mediate cooperative binding to PtdSer independently of the PtdSer binding pocket residues (41). We demonstrate that two of these residues (R49 and K63), but not R70 or K124, modestly reduce EBOV entry when mutated to alanine.…”

“…Not all of the mTIM-4 residues found by Tietjen et al to enhance mTIM-4 cooperative binding to PtdSer-containing membranes also affected virus entry (41). The four basic residues that were identified were proposed to bind directly to lipid head groups of the membrane bilayer and mediate cooperative binding to PtdSer independently of the PtdSer binding pocket residues (41).…”

“…Interestingly, mTIM-1 and mTIM-4 have similar binding affinities for PtdSer (27) but mTIM-4 binding to PtdSer-containing artificial membranes displays significant cooperativity compared to the other TIM proteins (27,41). Tietjen et al recently proposed that mTIM-4 binds in a cooperative manner to PtdSer in membranes through ionic interactions with additional IgV domain residues (41). Four mTIM-4 basic IgV domain residues were found (R49, K63, R70, and K124) in that study to mediate direct interactions with membrane-bound PtdSer.…”

“…Four mTIM-4 basic IgV domain residues were found (R49, K63, R70, and K124) in that study to mediate direct interactions with membrane-bound PtdSer. These residues are thought to bind independently of the central residues within the PtdSer binding pocket that most robustly bind PtdSer in a Ca 2ϩ -dependent manner (41). Therefore, the impact of these basic residues on viral entry was assessed.…”

Neurodegenerative disease mutations in TREM2 reveal a functional surface and distinct loss-of-function mechanisms

“…In contrast to highly selective phospholipid binding by other Ig domains (Kobayashi et al, 2007; Miyanishi et al, 2007; Santiago et al, 2007; Simhadri et al, 2012), purified TREM2 shows only broad discrimination for anionic (PA, PG, PI, PS) over more neutral (PC, PE) lipids (Figure 4—figure supplement 1a,b and c). Structurally, Ig domains with selective lipid binding feature a canonical FG loop motif that contains large hydrophobic residues, absent in TREM2, which mediate membrane insertion and acyl chain binding (Tietjen et al, 2014) (Figure 4—figure supplement 1d,e). Moreover, while TREM2 does share a conserved aspartic acid residue from this motif, which chelates a required Ca 2+ ion for the TIM and CD300a proteins, TREM2-lipid binding is not sensitive to EDTA, unlike TIM and CD300 (Figure 4—figure supplement 1e and f).…”