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. 2007 Mar;26(3):299-310.
doi: 10.1016/j.immuni.2007.01.014.

Structures of T Cell immunoglobulin mucin receptors 1 and 2 reveal mechanisms for regulation of immune responses by the TIM receptor family

César Santiago  1 , Angela Ballesteros, Cecilia Tami, Laura Martínez-Muñoz, Gerardo G Kaplan, José M Casasnovas
Affiliations

Structures of T Cell immunoglobulin mucin receptors 1 and 2 reveal mechanisms for regulation of immune responses by the TIM receptor family

César Santiago et al. Immunity. 2007 Mar.

Abstract

The T cell immunoglobulin mucin (TIM) receptors are involved in the regulation of immune responses, autoimmunity, and allergy. Structures of the N-terminal ligand binding domain of the murine mTIM-1 and mTIM-2 receptors revealed an immunoglobulin (Ig) fold, with four Cys residues bridging a distinctive CC' loop to the GFC beta-sheet. The structures showed two ligand-recognition modes in the TIM family. The mTIM-1 structure identified a homophilic TIM-TIM adhesion interaction, whereas the mTIM-2 domain formed a dimer that prevented homophilic binding. Biochemical, mutational, and cell adhesion analyses confirmed the divergent ligand-binding modes revealed by the structures. Structural features characteristic of mTIM-1 appear conserved in human TIM-1, which also mediated homophilic interactions. The extracellular mucin domain enhanced binding through the Ig domain, modulating TIM receptor functions. These results explain the divergent immune functions described for the murine receptors and the role of TIM-1 as a cell adhesion receptor in renal regeneration and cancer.

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Figures

Figure 1
Figure 1
Crystal Structures of the N-Terminal Cys-Rich Domain of TIM Receptors (A and B) Ribbon diagrams of the mTIM-1 and mTIM-2 structures are shown in (A) and (B), respectively. β strands of one face are red and those in the opposite side are magenta, coil is orange, 310 helix is light blue, α helix in the BC loop is blue, and the loop between C and C′ β strands is light green. Cys residues and disulphide bonds are in green. Strands are labeled with uppercase letters and terminal ends (n and c) are in lowercase. (C) Stereo view of superimposed mTIM-1 (red) and mTIM-2 (magenta) structures, with the regions showing structural variability labeled. (D) Structural alignment of the mTIM-1 and mTIM-2 structures with residues closer than 3 Å aligned. β strands and helical regions defined by the program dssp (Wolfgang and Sander, 1983) are colored as in (A) and (B). The other TIM receptor domains were aligned by sequence. Conserved residues in most TIM receptors are colored in yellow and the six Cys residues in green. N-linked glycosylation sites are underlined and sequences of mTIM-1 and mTIM-2 numbered. Green lines join the disulphide-linked Cys residues.
Figure 2
Figure 2
Conformation of the Loops Connecting C and C′ β Strands in the TIM Structures and in Related IgV Domains (A and B) Ribbon diagram of the GFC face of the mTIM-2 (A) and mTIM-1 (B) domain structures. Insets show lateral views. Residues between the two external disulphide bonds in the CC′ loop and interacting residues at the F and G β strands and at the FG loop are yellow. Hydrogen bonds are shown as pink dashed cylinders. Oxygens and nitrogen atoms are in red and blue, respectively. Residues are labeled according to Figure 1D. Blue dots on the C′C″ loop of (B) indicate location of N-linked glycosylations in human and monkey TIM-1 and in mTIM-3. (C) Stereo view of the superimposed mTIM-1 (red) and CAR (green, 1f5w) homologous domains. The CC′ loop region of the CEA IgV domain (1L6Z) is blue.
Figure 3
Figure 3
N-Terminal Domain Interactions in the TIM Receptors (A and B) Ribbon diagrams of the two domains in the asymmetric unit of the mTIM-2 (A) and mTIM-1 (B) crystals. Side view of the dimer is displayed for mTIM-2, whereas a view along the quasi-2-fold axis (2) is shown for mTIM-1. Molecules presented in Figure 1 have the same coloring scheme, and the neighboring molecules are in yellow. Side chains of residues contributing to the dimer interfaces are included and some central residues are labeled. Acetate ligand found in the mTIM-2 structure is black, water molecules are red spheres, and hydrogen bonds are pink dashed cylinders. Asn residues to which glycans link in mTIM-2 are green. Arrows represent the hypothetical interaction of O-linked glycans from the C-terminal mucin domain with residues at the β strand A, BC, and FG loops of the interacting mTIM-1 domains (see also Figure S3). (C) Self-association of the N-terminal IgV domains in solution. SDS-PAGE under reducing conditions of mTIM-1, mTIM-2, and mTIM-4 domains untreated (−) or treated with the indicated BS3 crosslinker concentration (mM). Treated ICAM-1 protein (IC1-2D) known to dimerize at high concentration and a soluble fragment of CD46 are also included. Size and migration of the molecular weight marker is indicated. Crosslinked dimers are labeled with an asterisk. No dimerization of the mTIM-4 IgV domain is seen here or in the protein crystals (not shown). (D) Structural alignment with residues at the dimer interface in yellow and those at the center of the interacting molecules in blue. β strands are represented by lines.
Figure 4
Figure 4
Oligomerization of the mTIM-2 Receptor (A) Size exclusion chromatography of soluble mTIM-1 (open triangles) and mTIM-2 (open squares) molecules with the complete extracellular regions. mTIM2-BCt1 corresponds to a mutant mTIM-2 receptor where BC loop residues (HLG) were replaced by aligned residues in the mTIM-1 BC loop (YR). Percentage of the total optical density (OD) is plotted for each elution fraction. Exclusion volume and size of the molecular weight markers are indicated. A representative experiment is shown. (B) Immunoblot of BS3 treated (+) or untreated (−) cell supernatants lacking (mock) or having the indicated TIM receptor (see Experimental Procedures). The arrows mark the three mTIM-2 oligomeric species. Size and migration of the molecular weight marker are shown.
Figure 5
Figure 5
Homophilic mTIM-1 Receptor Interaction (A) Binding of soluble Fc fusion proteins to plastic-coated mTIM-1 protein with the complete extracellular region. Binding to the isolated IgV domain is shown in Figure S4. TIM-Fc (mTIM-1 and mTIM-2) and control ICAM-1-Fc (IC1-2D) proteins used are included in the legend. Binding at the indicated protein concentration was determined from the optical density (OD) at 492 nm (see Experimental Procedures). Mean ± SD of three experiments is shown. (B) Normalized binding of mTIM-1-Fc protein to plastic-coated mTIM-1 IgV in the absence (mTIM-1) or presence of mTIM-1 (T1.4 and T1.10) and mTIM-2 (T2.1) antibodies, or EDTA (10 mM). Binding of a mutant mTIM-1-Fc protein where His64 was replaced by a glutamic acid (His64Glu) is also included. Mean + SD of 6 different measurements carried with 20 and 10 μg/ml of Fc protein is shown. (C) 293T cells transfected with the indicated TIM protein tagged with a cyan fluorescent protein (CFP) at their C-terminal ends. Fluorescence images of individual (white framed insets) and adhered cells were acquired 2 days after transfection (see Experimental Procedures). DIC image of the cell cluster with three transfected cells (t) and one cell untransfected (ut) or lacking mTIM-1 is shown in the inset. (D) Fluorescence and DIC images of adhered 300.19 preB-cells transfected with CFP-tagged mTIM-1 are on the top. Individual transfected cells untreated or treated with 1 μg/ml of ionomycin for 30 min at 37°C are in the bottom (see also Figure S4).
Figure 6
Figure 6
Homophilic TIM-1 Receptor Interaction in Humans (A) Binding of soluble Fc fusion proteins to plastic-coated TIM-1 proteins with the complete extracellular receptor as in Figure 5A. Binding of the Arg65Glu TIM-1 mutant or a double mutant Asp62His-Arg65Glu (TIM1-DEt1) in the DE loop of the IgV domain are included. (B) Binding of latex beads coated with the indicated protein to cells expressing GFP, the complete TIM-1 receptor, or a mutant lacking the IgV domain (TIM-1ΔIgV).
Figure 7
Figure 7
Ligand-Binding Surfaces in the IgV Domain of TIM-1 Receptors Surface representation of the mTIM-1 domain structure. Surface involved in the homophilic interaction is pink. Residues in a conformational epitope built by the tip of the long CC′ loop and the FG loop onto the GFC β sheet are colored red and orange, respectively. The surface where an mkTIM-1 polymorphism (Lys88Gln) has been mapped is in blue. The mutation identified the side of the domain recognized by a mAb blocking HAV binding to its mkTIM-1 receptor (Feigelstock et al., 1998a). Surface corresponding to the Asn residue to which glycans will be linked in the primate TIM-1 receptors is green.
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