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.
Highlights
T-cell immunoglobulin and mucin domain containing protein-3 (TIM-3), known as Hepatitis A virus cellular receptor 2 (HAVCR2), is involved in regulating innate and adaptive immunity and has emerged as a target for numerous therapeutics that are under clinical development
We observed an excellent global fold structure of our purified Ca++-bound hTIM-3 in solution as judged by the large spread of assigned amide resonance peaks in the 15N-heteronuclear single quantum coherence (HSQC) spectrum of 15N/13C-labeled hTIM-3 protein, which consisted mainly of extended beta-strands (Fig. 1a)
To obtain the structural details of hTIM-3 at an atomic level, we determined the crystal structure of the Ca++-bound hTIM-3 IgV domain at 1.7 Å resolution (PDB code 6DHB, Table 1). This structure exhibited features common to the TIM family members[2,9,10,11,21] by possessing a two anti-parallel β-sheet sandwich formed from front AGFCC′C′′ and back BED faces, respectively, which were linked by the B-C, E-F, C′′-D and A-B loops (Fig. 1b,c)
Summary
T-cell immunoglobulin and mucin domain containing protein-3 (TIM-3), known as Hepatitis A virus cellular receptor 2 (HAVCR2), is involved in regulating innate and adaptive immunity and has emerged as a target for numerous therapeutics that are under clinical development. Previous structural and biochemical studies have shown that mouse (m)TIM-32 and mTIM-419 bind phosphatidylserine (PtdSer) at the GFCC′ face of the IgV domain in a calcium (Ca++) dependent manner. The IgV-like domain of hTIM-3 has been demonstrated to bind other ligands such as Immunologic Diseases and Ann Romney Center for Neurologic Diseases, Harvard Medical School and Brigham and Women’s Hospital, 77 Avenue Louis Pasteur, Boston, MA, 02115, USA. (b) Ribbon diagram of the hTIM-3 IgV domain crystal structure with bound Ca++ in red. Carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1)[4], high mobility group protein B1 (HMGB1)[20] and galectin-91,9,18 The study of these interactions suggests that hTIM-3 may exhibit overlapping but distinct structural characteristics that determine specificity for binding of these unique ligands that all coalesce on the IgV-domain. We report the high resolution co-crystal structure of hTIM-3 IgV-domain with bound Ca++ that provides a structural basis for understanding hTIM-3 at an atomic level
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