Structures of T Cell Immunoglobulin Mucin Receptors 1 and 2 Reveal Mechanisms for Regulation of Immune Responses by the TIM Receptor Family (1)
mTIM-1 is preferentially expressed in Th2 cells and delivers a signal that enhances T cell activation and proliferation, increasing airway inflammation and allergy. mTIM-3 is mainly expressed in Th1 cells and provides a negative costimulatory signal that leads to immune tolerance. Polymorphisms in mTIM-1 and mTIM-3 confer susceptibility to the development of asthma and allergy. The N-terminal Cys-rich domain is critical for binding of the TIM receptors to their ligands.
The structures revealed an Ig domain belonging to the V set (IgV), related to the N-terminal domains of the CD4 and CAR (coxsackievirus and adenovirus receptor) cellular receptors (highest Z score in DALI search). The IgV domains have two antiparallel β sheets with particularly short β strands B, E, and D in one face (BED β sheet) and the A, G, F, C, C′, and C″ β strands in the opposite one (GFC β sheet). A Pro residue found prior to the first Cys in all TIM receptor domains was responsible for the short length of the β strand B. The first and last Cys residues in the N-terminal domain of the TIM receptors bridged the β sandwich as in most Ig domains, whereas the other four Cys residues characteristic of the TIM family formed two disulphide bonds that link a long CC′ loop to the GFC β sheet.
In mTIM-2, the tip of the loop folds down and had a helical conformation, whereas in mTIM-1 it extended up onto the GFC β sheet. These differences arise from distinct contacts between residues in the loop and the β sheet. In mTIM-1, the conformation of the CC′ loop tip was fixed by interactions with the Arg88 and Lys99 residues at the β strands F and G, respectively. Their side chains hydrogen bonded to main-chain oxygen atoms of Pro35, Ser36, and Ala38 in the two molecules of the asymmetric unit. So, the disulphide-bridged CC′ loop was additionally linked to the upper half of the β sheet by the conserved Arg88 and Lys99 residues in the mTIM-1 structure. These basic residues are conserved in all primate and murine TIM receptors that has a distinct CC′ loop conformation from mTIM-1. The FG loop of mTIM-1 is extended by two additional aromatic residues, Trp94 and Phe95. The side chain of Phe95 came close (∼4Å) to the tip of the CC′ loop in the mTIM-1 structure. Interestingly, the mTIM-1 FG loop is conserved in TIM-1 and in the human and mouse TIM-4 receptors, and the length of the loop in human TIM-3 and its mouse ortholog mTIM-3 is as in mTIM-1.
The association of the two IgV domains in the asymmetric unit of the mTIM-1 crystals was remarkably different from mTIM-2. The mTIM-1 domains were related by a rotation angle of about 180° and had their C-terminal ends extending toward opposite directions. The domains interact through the upper half of the BED face, including residues following the short β strand B and at the DE loop. Two Thr17 residues from opposite molecules were hydrogen bonded at the center of the dimer interface. Hydrophobic contacts included Thr13 and Pro15 with the bulky Tyr21 side chain at the BC loop of a neighboring domain. Additional interacting sites between the two mTIM-1 domains engaged several residues at the long DE coil. Asp69 bound to Ser19 on the BC loop of the opposite domain. Also, His64 and Glu67 were involved in interdomain interactions. Although these two residues at the helical DE loop are not conserved, some charged residues alternate at the aligned positions in the TIM receptors. In the native mTIM-1 receptor, O-linked glycans from the contiguous mucin domain could penetrate in the interdomain cavity and bridge interacting receptors from opposite cells. Potential glycan-interacting residues such as the basic Lys5 and Arg22, and the conserved Pro92, Trp94, and Asn96 on the FG loop lay on the upper edge of the cavity.
Homophilic mTIM-1 binding was specifically blocked by the T1.10 mAb that recognizes the IgV domain and by the addition of EDTA, which indicated requirement of divalent cations for high-affinity binding and suggested involvement of carbohydrates from the mucin domain.
The crystal structure of mTIM-1 identified a new homophilic TIM-TIM receptor interaction in mice and humans that could be relevant for the regulation of immune functions by these receptors. the homophilic mTIM-1-binding interaction described here could have important implications in the regulation of immune processes both in mice and humans and could be responsible of the hyperproliferation of T cells observed in mice treated with soluble mTIM-1 molecules. mTIM-1 is expressed on the surface of B cells and activated T cells, so it is feasible that the homophilic mTIM-1 interaction mediates B-T cell adhesion interactions and has important implications in the regulation of immune responses.