The structures of the mTIM-4 IgV domain identified a Metal Ion-dependent Ligand Binding Site (MILIBS) used for binding to phosphatidylserine by the TIM proteins. The ligand binding specificity is mediated by both the constrained size of the cavity and the requirement for an acidic group in the ligand for coordination with the metal ion. Although the most likely ion seen in the structures is sodium, other cations with similar coordination behaviour such as calcium could bind to this site depending on the ligand. The narrow cavity where the PS penetrates is built by two distinctive loops in the IgV domains of TIM proteins, so that the MILIBS is a specific signature of this receptor family. The lack of a ligand binding pocket in mTIM-2 due to its distinct CC’-loop and the absence of cation coordination residues in its FG loop further prove a divergent ligand recognition mode for this TIM family member (Santiago et al., 2007
Comparison of the ligand-free mTIM-1 with the mTIM-4 structure in complex with PS showed certain requirements for residue rearrangement on the CC’FG loop epitope for ligand binding. In the open conformation of the MILIBS revealed by the mTIM-4 structure the hydrophobic Phe and Trp residues on the tip of the loop were primed for penetration into the lipid bilayer upon binding to PS on the cell surface. The hydrophobic tip of the FG loop in TIM-1 and TIM-4 resemble fusion loops described in class II virus membrane proteins (Harrison, 2005
), which are known to penetrate into cellular membranes. The interaction of the hydrophobic residues with the cell membrane would strengthen the monomeric binding of the TIM proteins to PS, while the metal ion coordination and the conserved interactions of the Ser residue with the protein must provide the phospholipid binding specificity Even though the metal ion coordination residues and the conformation of the CC’ loop are conserved in all TIM but mTIM-2, there are certain differences in the FG and CC’ loops building up the binding pocket. These differences could have a significant influence on the binding affinity. Additional interaction of the IgV domain with the membrane could be mediated by residues at the tip of the BC loop, such as the Arg residues found in mTIM-1 and mTIM-4 or the residues that defined a polymorphism in the mTIM-3 BC loop (McIntire et al., 2001
). The interaction model presented here for TIM binding to PS in cellular membranes is analogous to that proposed for binding of annexin-V and protein kinase C (Swairjo et al., 1995
; Verdaguer et al., 1999
). In those models hydrophobic residues near the PS binding site penetrated into the membrane and residues on neighbouring protein loops, such as a Lys in protein kinase C, defined a second interacting site with the bilayer.
Because the expression of TIM-4 is restricted to phagocytic cells, including macrophages and dendritic cells, the binding of PS to TIM-4 suggests that a major function of TIM-4 is the recognition and removal of apoptotic cells by such phagocytic cells (see accompanying manuscript by Kobayashi et al). Indeed, TIM-4 expressing cells rapidly and specifically phagocytized apoptotic cells, and this process was prevented by anti-TIM-4 blocking mAb and mutations in the MILIBS residues. On the other hand, since expression of TIM-1 is restricted to T cells and to ischemic kidney cells, the binding of PS to TIM-1 may mediate resolution of ischemic kidney damage by clearance of dying renal tubular epithelial cells and regulate TIM-1 intracellular trafficking in T cells.
The ability of mTIM-1 to bind phospholipids, its intracellular distribution and the absence of the T1.10 antibody epitope in the membrane bound wild type protein led us to propose two distinct conformations for the protein on the cell surface, that could be shared by TIM proteins binding to PS: One folded molecule with the tip of the IgV domain penetrating into the lipid bilayer and a second extended conformation where the IgV domain would be available for ligand binding on the cell surface. Moreover, TIM binding to PS on its own membrane could sequester the proteins inside of the cell, where PS resides, preventing their exposure on the cell surface for ligand binding and subsequent receptor-mediated intracellular signalling. According to this model, the efficient trafficking of the mTIM-1 protein to the cell surface observed upon cell activation could be related to either its intracellular release from PS or to the flopping of the PS bound TIM toward the outer leaflet of the cell membrane by changes in the lipid membrane polarity. It is well documented that the increase of the intracellular concentration of calcium during cell activation flops PS out of the cell membrane by the simultaneous activation of the enzyme scramblase and the inhibition of the aminophospholipid-specific translocase (Zwaal and Schroit, 1997
). This regulatory mechanism for mTIM-1 trafficking must be specific for TIM proteins that bind PS, so that it might differ from other regulatory processes such as that described for CTLA-4 (Linsley et al., 1996
According to the trafficking regulatory model presented here, the exposure of a functional mTIM-1 and perhaps other TIM proteins for ligand binding would be controlled and restricted to certain cellular conditions. Uncontrolled exposure of TIM molecules on the cell surface could come from changes in the IgV domain affecting its interaction with PS and the lipid bilayer, such as those seen here with the MILIBS mutants. Polymorphisms in mTIM-1 (McIntire et al., 2001
), which modify the signal peptide and extend the mucin domain in the BALBc strain variant, could increase the distance between the tip of the domain and the lipid bilayer prior to the cleavage of the signal peptide, affecting binding of the IgV domain to PS and the amount of molecules on the cell surface. This uncontrolled exposure of TIM proteins could trigger undesired intracellular signals leading to lymphocyte activation upon binding to exogenous ligands.
We have shown here a unique Metal Ion-dependent Ligand Binding Site in the Ig domain of TIM proteins built up by distinctive structural motifs specifically found in the N-terminal domains of this family. We showed this is a phospatidylserine binding site and it could be also involved in binding to an identified mTIM-3 ligand and HAV, whose binding surface has been mapped on the GFC face of the IgV domain where the MILIBS locates (Cao et al., 2007
; Santiago et al., 2007
). Small molecules containing acidic groups could bind also to the MILIBS, while binding to multimeric ligands might crosslink TIM proteins on the cell surface and trigger intracellular signals. On the contrary, the critical contribution of the MILIBS to several TIM functions and its concave and well defined size opens up the rational design of small molecules targeting this site and modulating the functions of the TIM family in the immune system.