title: Quantitative analysis of T cell receptor complex interaction sites using genetically encoded photo-cross-linkers.
abstract: The T cell receptor (TCR)-cluster of differentiation 3 (CD3) signaling complex plays an important role in initiation of adaptive immune responses, but weak interactions have obstructed delineation of the individual TCR-CD3 subunit interactions during T cell signaling. Here, we demonstrate that unnatural amino acids (UAA) can be used to photo-cross-link subunits of TCR-CD3 on the cell surface. Incorporating UAA in mammalian cells is usually a low efficiency process. In addition, TCR-CD3 is composed of eight subunits and both TCR and CD3 chains are required for expression on the cell surface. Photo-cross-linking of UAAs for studying protein complexes such as TCR-CD3 is challenging due to the difficulty of transfecting and expressing multisubunit protein complexes in cells combined with the low efficiency of UAA incorporation. Here, we demonstrate that by systematic optimization, we can incorporate UAA in TCR-CD3 with high efficiency. Accordingly, the incorporated UAA can be used for site-specific photo-cross-linking experiments to pinpoint protein interaction sites, as well as to confirm interaction sites identified by X-ray crystallography. We systemically compared two different photo-cross-linkers--p-azido-phenylalanine (pAzpa) and H-p-Bz-Phe-OH (pBpa)--for their ability to map protein subunit interactions in the 2B4 TCR. pAzpa was found to have higher cross-linking efficiency, indicating that optimization of the selection of the most optimal cross-linker is important for correct identification of protein-protein interactions. This method is therefore suitable for studying interaction sites of large, dynamic heteromeric protein complexes associated with various cellular membrane systems.
authors: Department of Pathology, ‡Laura and Isaac Perlmutter Cancer Center, and §Department of Biochemistry and Molecular Pharmacology New York University School of Medicine , New York, United States.
authors: Wenjuan
authors: W
authors: Wang
authors: Tianqi
authors: T
authors: Li
authors: Klara
authors: K
authors: Felsovalyi
authors: Chunlai
authors: C
authors: Chen
authors: Timothy
authors: T
authors: Cardozo
authors: Michelle
authors: M
authors: Krogsgaard
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism; D017252:CD3 Complex / Q000032:analysis / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000737:chemistry; D018360:Crystallography, X-Ray; D057809:HEK293 Cells; D006801:Humans; D046912:Multiprotein Complexes / Q000737:chemistry; D011401:Promoter Regions, Genetic; D054730:Protein Interaction Domains and Motifs; D011948:Receptors, Antigen, T-Cell / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism*; D016693:Receptors, Antigen, T-Cell, alpha-beta / Q000032:analysis / Q000737:chemistry / Q000378:metabolism
chemical_list: D000596:Amino Acids; D017252:CD3 Complex; D003432:Cross-Linking Reagents; D046912:Multiprotein Complexes; D011948:Receptors, Antigen, T-Cell; D016693:Receptors, Antigen, T-Cell, alpha-beta; D000604:Amino Acyl-tRNA Synthetases