High resolution X-ray and NMR structural study of human T-cell immunoglobulin and mucin domain containing protein-3

Abstract

T-cell immunoglobulin and mucin domain containing protein-3 (TIM-3) is an important immune regulator. Here, we describe a novel high resolution (1.7 Å) crystal structure of the human (h)TIM-3 N-terminal variable immunoglobulin (IgV) domain with bound calcium (Ca++) that was confirmed by nuclear magnetic resonance (NMR) spectroscopy. Significant conformational differences were observed in the B-C, C′-C″ and C′-D loops of hTIM-3 compared to mouse (m)TIM-3, hTIM-1 and hTIM-4. Further, the conformation of the C-C′ loop of hTIM-3 was notably different from hTIM-4. Consistent with the known metal ion-dependent binding of phosphatidylserine (PtdSer) to mTIM-3 and mTIM-4, the NMR spectral analysis and crystal structure of Ca++-bound hTIM-3 revealed that residues in the hTIM-3 F-G loop coordinate binding to Ca++. In addition, we established a novel biochemical assay to define hTIM-3 functionality as determined by binding to human carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1). These studies provide new insights useful for understanding and targeting hTIM-3.

CEACAM1 structure and function in immunity and its therapeutic implications

“…The N-terminal IgV domain functions as the extracellular binding element that is responsible for determining CEACAM1's unique homophilic and heterophilic binding properties. The hCEACAM1 IgV domain contains 108 amino acids arranged in 9 beta strands (ABCC'C"DEFG) that fold into the conserved IgV anti-parallel beta-sandwich tertiary structure [15,21] adopted by other IgV-containing proteins including CD2 [22], T cell receptor (TCR) [23], T cell inhibitory and mucin domain containing protein 3 (TIM-3) [24,25], programmed cell death protein 1 (PD-1) and programmed death-ligand 1 (PD-L1) [26] and its murine ortholog mCEACAM1 [21]. The opposing ABED and GFCC'C" faces of the CEACAM1 beta-sandwich are tethered by an internal salt bridge (R64 : D82) that mimics a stabilizing covalent disulfide linkage found in most Ig domains [15,27].…”

“…TIM-3 is also a member of the immunoglobulin superfamily and is expressed as a type I membrane protein consisting of an N-terminal IgV domain, heavily O-linked glycosylated mucin domain stalk, transmembrane sequence and SH2binding motif-containing cytoplasmic domain. The CEACAM1 and TIM-3 IgV domains share a similar beta-sandwich fold [24] and exhibit a calculated main chain r.m.s.d. of 1.31Å indicative of high structural homology.…”

“…That said, the TIM-3 IgV domain, in contrast to CEACAM1, is heavily stabilized by three interchain disulfide linkages in mouse and human. Both biochemical [15,24,27] and NMR studies [27] demonstrated calcium-dependent binding of CEACAM1 IgV to the TIM-3 IgV and biochemically localized the interaction to the GFCC'C" surface where CEACAM1 binds homophilically. CEACAM1 has also been demonstrated to interact with a v and β1-integrins [13,38] and dendritic cell (DC)-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN) [39].…”

“…This raises the possibility that CEACAM1 associates with TIM-3 as it does other activating receptors and serves to inhibit its function or that CEACAM1 association with TIM-3 enables other novel inhibitory signaling mechanisms that remain to be determined. CEACAM1-TIM-3 interactions have been biochemically mapped to the GFCC'C" interface of both molecules that overlaps with those involved in homophilic binding of CEACAM1 and the phosphatidylserine binding sites on TIM-3 [15,24,142]. Interestingly, the binding epitope of anti-TIM-3 antibodies that function as effective anti-cancer agents map to the CEACAM1 binding site on TIM-3 suggesting that targeting TIM-3 requires disabling TIM-3 interactions with CEACAM1 and/or phosphatidylserine [143].…”

Immunological Targets for Immunotherapy: Inhibitory T Cell Receptors

“…Polymorphisms in the TIM family of genes (specifically TIM-1) are associated with airway hyperreactivity [100], suggesting that the TIM-3 family of molecules are broadly involved in Th1/2 differentiation and allergic diseases. Human TIM-3 consists of a distal variable immunoglobulin (IgV)-like domain bound to a proximal mucin domain both of which are extracellular and connected to an intracellular cytoplasmic tail that is involved in phosphotyrosine-dependent signaling [101][102][103][104][105]. The IgV-like domain of TIM-3 binds multiple ligands including carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) [106], high mobility group protein B1 (HMGB1) [107], galectin-9 [101,102,108]and phosphatidylserine (PS) [109,110].…”