Chapter 3 Fragment screen of T cell immunoglobulin mucin receptor 3 (TIM-3)
3.1 Introduction
3.1.2 TIM-3 structure and function
TIM-3 is a single-pass transmembrane protein that consists of a membrane distal variable immunoglobulin (IgV) domain, a mucin domain, a transmembrane domain, and a cytoplasmic tail involved in phosphotyrosine-dependent signaling. The primary receptor interaction domain in TIM-3 is the membrane-distal IgV domain (residues 22-130) which is largely conserved with mTIM-3. The IgV domain is characterized by a two anti-parallel
-sheet sandwich formed from front AFGCC’C” and back BED faces which are linked by
B-C, E-F, C”-D, and A-B loops (Figure 3-2A). The two faces are stabilized by inter-sheet interactions and three disulfide bonds: one internal bond between C38 and C110 which stabilizes the two -sheets and two noncanonical bonds between C52 and C63, and C58 and C109 which stabilize the upward fold of the CC’ loop forming the FG-CC’ cleft. The FG-CC’ cleft is a conserved feature of TIM family proteins and has been reported to possess a conserved metal ion-dependent ligand binding site that is capable of chelating a single calcium cation (Ca2+) (Figure 3-2B).111,112 The TIM-3 IgV domain has three predicted N-linked glycosylation sites (N33, N100, and N124).
Figure 3-2. TIM-3 primary and tertiary structure.
(A) 3D structure of the TIM-3 IgV domain (PDB 6DHB) with labeled -strands and loops. Disulfide bonds and potential N-linked glycosylation sites (purple) are shown as sticks. Calcium (Ca2+) coordinated in the FG-CC’ cleft is shown as a green sphere. (B) Sequence alignment of human and mouse TIM-3 IgV domains.
Conserved residues are red the -strands are labeled above the sequences and underlined in red and black for conserved and no-conserved, respectively.
Previous studies have identified four TIM-3 ligands that interact with the IgV domain: phosphatidylserine (PtdSer), carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), high mobility group box 1 (HMGB1), and galectin-9.113–116 Galectin-9, the first reported natural TIM-3 ligand, was found to bind TIM-3 through the interaction of the two carbohydrate recognition domains of galectin-9 and N-linked oligosaccharides of the IgV domain. The elucidation of the crystal structure of mTIM-3 IgV domain led to the discovery of the conserved FG-CC’ binding cleft shared among TIM
family members as a galectin-9 independent binding site.117 Interestingly, both galectin-9 and CEACAM1 binding events have been separately shown to induce a TIM-3 active state leading to the phosphorylation of Y256 and Y263 despite having independent binding sites.116,118
The Tim-3 cytoplasmic tail adjacent to the trans-membrane domain is devoid of the classical inhibitory switch motifs found in other inhibitory receptors. It does, however, contain a conserved region of five tyrosine residues, two of which have been shown to be critically important for coupling to downstream signaling pathways. Although the precise intracellular signaling mechanism has not been fully elucidated, it has been found that Y256 and Y263 are critical for the binding of HLA-B associated transcript 3 (BAT3), to the C-terminal tail of Tim-3.119 The peptide sequences surrounding these two tyrosine residues are highly conserved and function as SH2 domain-binding motifs, where multiple SH2 domain-containing kinases including Fyn, Lck, PI3K p85, and Itk are found to bind.118,120 Many of these molecules are key components of the T cell receptor (TCR) signaling pathway, indicating a functional relationship between TIM-3 and the TCR pathway. It has been found that Tyr256 and Tyr263 are critical for the binding of HLA-B associated transcript 3 (Bat3), to the C-terminal tail of TIM-3.119 In its inactive state, Bat3 recruits the catalytically active form of Lck and forms an intracellular molecular complex with TIM-3 that preserves and potentially promotes T cell signaling and represses TIM-3- mediated cell death and exhaustion (Figure 3-3A). When ligand bound and in its active state, Y256 and Y263 are phosphorylated and BAT3 is released from the Tim-3 tail, thereby promoting Tim-3-mediated T cell inhibitory function by allowing binding of tyrosine kinase FYN resulting in immunological synapse disruption and phosphatase recruitment
(Figure 3-3B). Consequently, the cell becomes anergic and undergoes apoptosis through the induced intracellular calcium influx.115 Because FYN and BAT3 bind to the same domain in the TIM-3 cytoplasmic tail, a likely molecular switch between TIM-3-Bat3 and TIM-3-FYN might trigger the switch of TIM-3 function from being permissive to TCR signaling to inhibition of proximal TCR signaling.
Figure 3-3. Models for TIM-3 activation states and ligand binding.
TIM-3 inhibits immune cells when in its active, ligand-bound state. (A) In its inactive, unbound state, the cytoplasmic tail of TIM-3 interacts with BAT3 and maintains T cell activation by LCK recruitment. (B) Ligand binding of galectin-9 or CEACAM1 leads to phosphorylation of Y256 and Y263, release of BAT3, and recruitment of FYN. This results in the disruption of immune synapse formation and phosphatase recruitment, and ultimately leading to cell apoptosis. Adapted from Wolf et al., 2020.121
While phosphorylation of Y256 and Y263 is widely accepted as the trigger of TIM- 3 inhibitory function, the structural dynamics that lead to this event are unknown.
Generally, single-pass transmembrane receptor activation mechanisms are not fully understood. However, the study of transmembrane domains of single-pass receptors has
suggested that most transmembrane domains have a tendency to self-associate.122,123 Transmembrane domain self-association contributes to the overall dimerization of the protein but is also balanced by the potential positive or negative interactions of the soluble domains. It is likely that each receptor has a particular set of conditions, including ligand binding, that leads to receptor oligomerization and activation. A working mechanistic hypothesis of receptor activation includes potential contributions from ligand-induced dimerization, ligand-induced rotation, and clustering (Figure 3-4A).124 In each circumstance, ligand binding to the receptor can lead to the reduction of free energy barriers to oligomerization allowing for dimerization, rotation, and/or clustering (Figure 3- 4B).
Figure 3-4. Single pass transmembrane receptor mechanisms of activation.
(A) Lingand-induced dimerization and ligand-induced rotation hypotheses posit that ligand binding to the extracellular domains brings receptor monomers together or brings intracellular domains into active configurations to form a signaling-competent dimer. Clustering occurs when receptors are stabilized as higher order oligomeric signaling complexes. These mechanisms are not mutually exclusive and may be utilized in combination. (B) Receptor activation is energetically unfavorable when not ligand bound.
In the context of TIM-3, both ligands CEACAM-1, which has been shown to interact with TIM-3 in cis and trans, and galectin-9, which is a tandem-repeat protein containing two carbohydrate recognition domains capable of TIM-3 binding through N-linked glycans, would have potential clustering effects which may lead to receptor activation. It is also possible that these events are not mutually exclusive and galectin-9 induced clustering and CEACAM-1 induced conformational changes to the IgV domain and required for complete signal transduction. Both binding events have separately been shown to induce phosphorylation of Y256 and Y263. However, it should be noted that CEACAM1 engagement of the FG-CC’ cleft alone has not been shown to induce apoptosis, but galectin-9 binding of N-linked glycans does. This may suggest multiple functional and/or activation states of TIM-3 depending on specific ligand binding that lead to different functional cellular outcomes. TIM-3 biology is complicated by its non-canonical signaling, a broad expression across different immune cells, and multiple ligands. Further molecular tools may be necessary to fully understand aspects of TIM-3 activation and subsequent biological outcomes.