T cell/transmembrane, Ig, and mucin (TIM) proteins, identified using a congenic mouse model of asthma, critically regulate innate and adaptive immunity. TIM-1 and TIM-4 are receptors for phosphatidylserine (PtdSer), exposed on the surfaces of apoptotic cells. Herein, we show with structural and biological studies that TIM-3 is also a receptor for PtdSer that binds in a pocket on the N-terminal IgV domain in coordination with a calcium ion. The TIM-3/PtdSer structure is similar to that of TIM-4/PtdSer, reflecting a conserved PtdSer binding mode by TIM family members. Fibroblastic cells expressing mouse or human TIM-3 bound and phagocytosed apoptotic cells, with the BALB/c allelic variant of mouse TIM-3 showing a higher capacity than the congenic C.D2 Es-Hba–allelic variant. These functional differences were due to structural differences in the BC loop of the IgV domain of the TIM-3 polymorphic variants. In contrast to fibroblastic cells, T or B cells expressing TIM-3 formed conjugates with but failed to engulf apoptotic cells. Together these findings indicate that TIM-3–expressing cells can respond to apoptotic cells, but the consequence of TIM-3 engagement of PtdSer depends on the polymorphic variants of and type of cell expressing TIM-3. These findings establish a new paradigm for TIM proteins as PtdSer receptors and unify the function of the TIM gene family, which has been associated with asthma and autoimmunity and shown to modulate peripheral tolerance.

The T cell immunoglobulin and mucin domain (TIM) proteins are important regulators of T cell responses. Crystal structures of the murine TIM-4 identified a metal-ion-dependent ligand binding site (MILIBS) in the immunoglobulin (Ig) domain of the TIM family. The characteristic CC' loop of the TIM domain and the hydrophobic FG loop shaped a narrow cavity where acidic compounds penetrate and coordinate to a metal ion bound to conserved residues in the TIM proteins. The structure of phosphatidylserine bound to the Ig domain showed that the hydrophilic head penetrates into the MILIBS and coordinates with the metal ion, whereas the aromatic residues on the tip of the FG loop interacted with the fatty acid chains and could insert into the lipid bilayer. Our results also revealed an important role of the MILIBS in the trafficking of TIM-1 to the cell surface.

The coronaviruses (CoVs) are enveloped viruses of animals and humans associated mostly with enteric and respiratory diseases, such as the severe acute respiratory syndrome and 10–20% of all common colds. A subset of CoVs uses the cell surface aminopeptidase N (APN), a membrane-bound metalloprotease, as a cell entry receptor. In these viruses, the envelope spike glycoprotein (S) mediates the attachment of the virus particles to APN and subsequent cell entry, which can be blocked by neutralizing antibodies. Here we describe the crystal structures of the receptor-binding domains (RBDs) of two closely related CoV strains, transmissible gastroenteritis virus (TGEV) and porcine respiratory CoV (PRCV), in complex with their receptor, porcine APN (pAPN), or with a neutralizing antibody. The data provide detailed information on the architecture of the dimeric pAPN ectodomain and its interaction with the CoV S. We show that a protruding receptor-binding edge in the S determines virus-binding specificity for recessed glycan-containing surfaces in the membrane-distal region of the pAPN ectodomain. Comparison of the RBDs of TGEV and PRCV to those of other related CoVs, suggests that the conformation of the S receptor-binding region determines cell entry receptor specificity. Moreover, the receptor-binding edge is a major antigenic determinant in the TGEV envelope S that is targeted by neutralizing antibodies. Our results provide a compelling view on CoV cell entry and immune neutralization, and may aid the design of antivirals or CoV vaccines. APN is also considered a target for cancer therapy and its structure, reported here, could facilitate the development of anti-cancer drugs.

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.