3D structures inferred from cDNA clones identify the CD1D-Restricted γδ T cell receptor in dromedaries

Abstract

The Camelidae species occupy an important immunological niche within the humoral as well as cell mediated immune response. Although recent studies have highlighted that the somatic hypermutation (SHM) shapes the T cell receptor gamma (TRG) and delta (TRD) repertoire in Camelus dromedarius, it is still unclear how γδ T cells use the TRG/TRD receptors and their respective variable V-GAMMA and V-DELTA domains to recognize antigen in an antibody-like fashion. Here we report about 3D structural analyses of the human and dromedary γδ T cell receptor. First, we have estimated the interaction energies at the interface within the human crystallized paired TRG/TRD chains and quantified interaction energies within the same human TRG/TRD chains in complex with the CD1D, an RPI-MH1-LIKE antigen presenting glycoprotein. Then, we used the human TRG/TRD-CD1D complex as template for the 3D structure of the dromedary TRG/TRD-CD1D complex and for guiding the 3D human/dromedary comparative analysis. The choice of mutated TRG alternatively combined with mutated TRD cDNA clones originating from the spleen of one single dromedary was crucial to quantify the strength of the interactions at the protein-protein interface between the paired C. dromedarius TRG and TRD V-domains and between the C. dromedarius TRG/TRD V-domains and CD1D G-domains. Interacting amino acids located in the V-domain Complementarity Determining Regions (CDR) and Framework Regions (FR) according to the IMGT unique numbering for V-domains were identified. The resulting 3D dromedary TRG V-GAMMA combined with TRD V-DELTA protein complexes allowed to deduce the most stable gamma/delta chains pairings and to propose a candidate CD1D-restricted γδ T cell receptor complex.

Keywords: 3D modelization; Camelus dromedarius; IMGT unique numbering; T cell receptor; TRG and TRD loci; multiple sequence alignments (MSA).

Figure 1

(A) Protein-protein interactions between the Homo sapiens FR-IMGT and CDR-IMGT of the TR V-GAMMA and V-DELTA domains and between the TR V-GAMMA or V-DELTA domains and the Homo sapiens CD1D G-ALPHA1-LIKE and G-ALPHA2-LIKE domains. Five types of interactions are shown: protein-protein ionic interactions within 6 Å (solid brown line), protein-protein side chain-side chain hydrogen bonds (broken brown line), aromatic-aromatic interactions within 4.5 and 7- Å (solid blue line), aromatic-sulphur interactions (solid red line), cation-Pi interactions (broken blue line) (11, 12, 14),. On the left, in red the CDR and FR of the TRGV5 V-GAMMA with the respective amino acids and their positions involved in the interactions (black squares). In blue the CDR and FR of the TRDV1 V-DELTA. In green the CD1D G-ALPHA1-LIKE and the G-ALPHA2-LIKE domains, with their respective amino acids and their positions involved in the interactions with the V-GAMMA and V-DELTA domains. In the upper right, interacting amino acids at the interface of the cited TR gd 3D models with the Homo sapiens CD1D G-ALPHA1-LIKE, and G-ALPHA2-LIKE domains are reported in sticks representation (See also Figure 2B and Table S2 ). (B) IMGT Collier de Perles of the Homo sapiens TRGV5-TRGJ1 domain, the TRDV1-(D)-J1 domain (18, 19) and the G-ALPHA1-LIKE and the G-ALPHA2-LIKE domains of the CD1D, available in IMGT/3Dstructure-DB, http://www.imgt.org, accessed on 4 March 2022.

Figure 2

(A) Flowchart of the overall C. dromedarius experimental setting and analyses of TRG and TRD cDNA clones. In correspondence with each combination of the TRG and TRD examined clones (black arrows), the estimated negative energy values (kcal/mol) of the interactions at the protein interfaces between the paired TRG and TRD V-domains, and at the protein interfaces between the paired TRG/TRD V-domains and the CD1D G-domains are shown in succession. Finally, the V-(D)-J-C gene composition of the paired TRG/TRD V-domains corresponding to each combination is indicated. The values in yellow are those deemed most significant because they refer to those closest to the human negative energy value (see Table S3 ). (B) C.dromedarius TR γδ 3D models in complex with CD1D and B2M. Panel (a) Homo sapiens TR γδ 3D model is in orange (TR γ) and gray (TR δ) cartoon, in complex with CD1D (green cartoon) and B2M (red cartoon) as reported in the available crystallized structure 4hlu.pdb. Panel b-g. Different complex poses showing the investigated combination of C. dromedarius TRG (RTS88, magenta cartoon; 5R1S169, blue cartoon) and TRD (RTVD4m9, white cartoon; RTVD4m14, yellow cartoon; JD3.05, dark green cartoon) chains in complex with CD1D (salmon cartoon) and of B2M (cyan cartoon).

Figure 3

Protein-protein interactions between the Camelus dromedarius FR-IMGT and CDR-IMGT of the TR V-GAMMA (clone RTS88) and V-DELTA (clone RTVD4m9) domains (A) and of V-GAMMA (clone 5R1S169) and V-DELTA (clone JD3.05) domains (B) and between the V-GAMMA or V-DELTA domains and the Camelus dromedarius G-ALPHA1-LIKE and G-ALPHA2-LIKE domains of the CD1D. On the left, in red the CDR and FR of the TRGV1 (A) and TRGV2 (B) domains and in blue the CDR and FR of the TRDV4 (A) and TRDV1 (B) domains; the amino acids and their positions involved in the interactions are shown in black squares. In green the CD1D G-ALPHA1-LIKE and the G-ALPHA2-LIKE domains, with their respective amino acids and their positions involved in the interactions with the V-GAMMA and V-DELTA domains. Amino acids that have no interaction lines, for example RTS88/FR2/49A-50P (A) and 5R1S169/FR2/49A-50P (B) present hydrophobic interactions within 5 Å (see positions marked in yellow in Figures S6, S8 ). (A) Down from left to right, IMGT Collier de Perles of the Camelus dromedarius TRGV1-J1 domain, TRDV4-D6-J4 domain (18, 19) and the G-ALPHA1-LIKE and the G-ALPHA2-LIKE domains of the CD1D, obtained using the IMGT/Collier-de-Perles tool (20). (B) Down from left to right, IMGT Collier de Perles of the Camelus dromedarius TRGV2-J2 domain, TRDV1-D1-D2-J4 domain (18, 19) and the Protein-Protein Interactions Legend. (A, B) In the upper right, interacting amino acids at the interface of the cited TR gd 3D models with the Homo sapiens CD1D G-ALPHA1-LIKE, and G-ALPHA2-LIKE domains are reported in sticks representation (See also Figure 2B and Table S2 ).

Figure 4

The scheme presents a possible modality of the TR γδ of a human NKT cell that can establish protein interactions with the domains of the CD1D associated with an antigen presenting cell (APC). TR γδ (orange/white), CD1D (green), a-GalCer (magenta) beta-2-microglobulin B2M (red).