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In situ cell-surface conformation of the TCR-CD3 signaling complex
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2
Aswin Natarajan1,2, Wenjuan Wang1,2,
Y
, Manuel Becerra Flores3, Tianqi Li1,2, Hye Won
3
Shin1,2, Saikiran Beesam1,2, Timothy Cardozo3 and Michelle Krogsgaard1,2,*
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1Perlmutter Cancer Center, NYU Grossman School of Medicine, New York, NY 10016
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2Department of Pathology, NYU Grossman School of Medicine, New York, NY 10016
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3Department of Biochemistry and Molecular Pharmacology, NYU Grossman School of
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Medicine, New York, NY 10016
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Y
Current address: Tsinghua University, China
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*Corresponding author
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Abstract
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T cells play a vital role in adaptive immune responses to infections, inflammation and cancer
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and are dysregulated in autoimmunity. Antigen recognition by T cells – a key step in adaptive
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immune responses – is performed by the T cell receptor (TCR)-CD3 complex. The extracellular
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molecular organization of the individual CD3 subunits (CD3de and CD3ge) around the abTCR is
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critical for T cell signaling. Here, we incorporated unnatural amino acid (UAA) photo-crosslinkers
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at specific mouse TCRa, TCRb, CD3d and CD3g sites, based on previous mutagenesis, NMR
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spectroscopy and cryo-EM evidence, and crosslinking allowing us to identify nearby interacting
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CD3 or TCR subunits on the mammalian cell surface. Using this approach, we show that CD3g
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and CD3e, belonging to CD3ge heterodimer crosslinks to Cb FG loop and Cb G strand,
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respectively and CD3d crosslinks to Cb CC’ loop and Ca DE loop. Together with computational
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docking, we identify that in in situ cell-surface conformation, the CD3 subunits exists in CD3e’-
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CD3g-CD3e-CD3d arrangement around the abTCR. This unconventional technique, which uses
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the native mammalian cell surface microenvironment, includes the plasma membrane and
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excludes random, artificial crosslinks, captures a dynamic, biologically relevant, cell-surface
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conformation of the TCR-CD3 complex, which is compatible with the reported static cryo-EM
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structure’s overall CD3 subunits arrangement, but with key differences at the TCR-CD3
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interface, which may be critical for experiments in T cell model systems.
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Introduction
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T cell receptors (TCRs), expressed on T cells, recognize antigenic peptides presented by major
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histocompatibility complexes (MHC) expressed on antigen-presenting cells (APCs) and signal
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through associated CD3 subunits, resulting in T cell immune response initiation (Krogsgaard
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and Davis, 2005). The abTCR is a heterodimeric molecule, with each subunit possessing a
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variable (Va, Vb) domain, which recognizes antigen through its complementarity-determining
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regions (CDRs), and a constant domain (Ca, Cb), which facilitates interactions with CD3
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subunits (Davis and Bjorkman, 1988; Natarajan et al., 2016). The TCR-CD3 complex is
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composed of an abTCR heterodimer with membrane embedded C-terminal helices lacking any
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intracellular signaling domains, a CD3ge heterodimer, a CD3de heterodimer and a CD3zz
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homodimer. Each CD3 possesses either 1 or 3 immunoreceptor tyrosine-based activation motifs
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(ITAMs) in their cytoplasmic tails, which can be phosphorylated to propagate signals in the cell
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interior (Kane et al., 2000). This arrangement thus requires that any communication of cognate
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pMHC-TCR interactions into the T cell must occur through the CD3 subunits. Previously, the
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stoichiometry and molecular features of the association of the constituent domains were
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determined by different techniques (Arechaga et al., 2010; Birnbaum et al., 2014; Dong et al.,
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2019; He et al., 2015; Natarajan et al., 2016) but the truncated proteins and experimental
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conditions used raise questions about their physiological relevance.
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The anchoring core of the TCR complex is the bundle of transmembrane helices (TMs) of the
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TCR and the CD3 chains. Highly conserved, charged residues in the TMs and membrane-
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proximal tetracysteine motif are required for clustering all the TCR-CD3 complex components
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(Call et al., 2002; Call and Wucherpfennig, 2005; Xu et al., 2006), and interactions between
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extracellular regions are required for bioactive TCR-CD3ge/de complex formation (Fernandes et
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al., 2012; He et al., 2015) and T cell signaling (Natarajan et al., 2016). NMR chemical shift
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perturbation (CSP) studies involving the extracellular components of abTCR and CD3 subunits
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provided residue-specific information, but suggested different binding modes (single-sided (He
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et al., 2015) and double-sided (Natarajan et al., 2016)) of CD3ge/de to the abTCR. Indeed, the
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peptide linking segment between the CD3 extracellular folded domains and their corresponding
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TMs are long enough to accommodate either a one-sided or two-sided conformation
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(unpublished observation). Our prior two-sided NMR model showed that, in the inactivated T cell
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state, the TCR Cb subunit interacts with CD3ge through its helix 3 and helix 4-F strand regions,
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whereas the TCR Ca subunit interacts with CD3de through its F and C strand regions, thereby
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placing the CD3 subunits on opposite sides of the TCR. This was in general agreement with an
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earlier electron microscopy (EM) structure of the pMHC-TCR-CD3 complex and TCRa-CD3de
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SAXS structure (Birnbaum et al., 2014). While these studies provide important clues about the
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composition and orientation of the TCR-CD3 complex, they did not include the native TCR-CD3
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TMs (Birnbaum et al., 2014; He et al., 2015; Natarajan et al., 2016). A recent 3.7 Å single-
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particle, non-crystalline cryo-EM structure of the human TCR-CD3 complex included the
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connecting peptide linker segments and the 8-TM helix bundle (without the intracellular cytosolic
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regions), revealing an orientation of the CD3 subunits, as well as specific contact details
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between individual subunits (Dong et al., 2019). This acellular structure depicted both CD3ge
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and CD3de binding TCR from the same side in a non-MHC-ligated state. The absence of the
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plasma membrane in this structure and the use of glutaraldehyde to crosslink the TCR-CD3
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subunits, however, leaves open the possibility that some of the observations may not reflect the
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physiologically relevant, native cell surface conformation. In addition, T cell signaling is
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commonly studied in mouse model systems and to what degree this conformation differs
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between mouse and human is not known. Indeed, major aspects of T-cell signaling are known
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to differ between the mouse and human immune systems (Mestas and Hughes, 2004).
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Photo-crosslinking of incorporated unnatural amino acids (UAA) is a powerful tool for studying
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complex protein-protein interactions, molecular mechanisms, and spatiotemporal conformational
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states (Coin, 2018; Coin et al., 2013) and has been used to map ligand-binding sites for multiple
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proteins, including G protein-coupled receptors, neurokinin-1 receptor and a human serotonin
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transporter (Gagnon et al., 2019; Grunbeck et al., 2011; Rannversson et al., 2016; Valentin-
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Hansen et al., 2014). Other photo-crosslinking studies include analysis of histone-histone
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interactions leading to chromatin condensations (Wilkins et al., 2014) and identification of RNA-
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binding sites in riboprotein complexes (Kramer et al., 2014). However, this effective technique
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has not yet been applied extensively to study immune receptors. Here, we report a model of the
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mouse in situ cell-surface conformation of the TCR-CD3 signaling complex using constraints
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obtained from site-specific photo-crosslinkers that reveals a one-sided CD3e’-CD3g-CD3e-CD3d
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subunit arrangement around the abTCR. We compared the model in detail to the previously
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solved acellular glutaraldehyde crosslinked human TCR-CD3 cryo-EM structure (Dong et al.,
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2019) which revealed a similar overall arrangement but with certain differences between them,
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especially in the TCR-CD3 interface residues.
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Results
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The TCR-CD3 complex is amenable to UAA incorporation and photo-crosslinking
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To incorporate photo-crosslinkable UAAs in response to specific codons (e.g., amber stop
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codon) for crosslinking TCR and CD3 subunits, orthogonal tRNA/aminoacyl-tRNA synthetase
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(tRNA-aaRS) were designed that incorporate UAA present in cell culture media into the nascent
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protein within the cell at appropriate sites (Figure 1A). Previously, we co-transfected plasmids
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encoding tRNA-aaRS, TCR and CD3 subunits into human embryonic kidney (HEK) 293T cells
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and successfully incorporated UAA photo-crosslinkers p-azido-phenylalanine (pAzpa) and H-p-
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Bz-Phe-OH (pBpa) site-specifically into the TCR. We demonstrated the effectiveness of this
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approach by probing the interaction between the TCR subunits by photo-crosslinking (Wang et
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al., 2014). Based on this work, in the present study, we aimed to incorporate the unnatural
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amino acid pAzpa into previously identified TCR-CD3 interaction sites (Beddoe et al., 2009;
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Dong et al., 2019; Kim et al., 2009; Kuhns and Davis, 2007; Natarajan et al., 2016) on the
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mouse 2B4 TCR constant regions and crosslink it to neighboring mouse CD3 subunits and vice
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versa by UV (360 nm) activation (Figure 1B). For expression on mammalian cells, the TCR
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subunits (a- and b-) and CD3 subunits (g-, d-, e- and z-) are connected by self-cleavable 2A
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peptides to promote stoichiometric expression of the different subunits in the TCR-CD3 complex
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(Figure 1C). To facilitate detection of crosslinked subunits by Western blot, the following protein
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tags were added to the C-terminal ends of different subunits: TCRa: c-Myc, TCRb: V5, CD3g:
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VSV-G, CD3d: FLAG and CD3e: HA (Figure 1C).
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Figure 1: The TCR-CD3 complex is amenable to UAA incorporation and photo-
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crosslinking: A) Schematic overview of UAA (pAzpa) incorporation into translated protein by
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orthogonal tRNA/tRNA synthetase pair. B) General outline of the steps involved in the
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crosslinking assay in HEK293T cells. C) Illustrations of the 2B4 TCR, CD3 and tRNA-aaRS
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expression plasmids and locations of peptide tags utilized for Western blot identification. D)
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Locations of aS41 (blue), aK65 (red) in the 2B4 TCR crystal structure (PDB: 3QJF). E) TCRb
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and CD3e expression profiles of wildtype 2B4 TCR (green), aS41 (blue), aK65 (red) by flow
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cytometry (stained with APC-conjugated H57-597 antibody – TCRb and PE-conjugated 145-
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2C11 antibody – CD3e) shows successful surface expression after UAA incorporation. The
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percentage of cells positive for both TCRb and CD3e staining is indicated. F) TCR mutant aS41
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(positive control) crosslinks with TCRb with the crosslinking band migrating between 75 to 100
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kDa illustrating the feasibility of the technique to crosslink nearby subunits. The blot was stained
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with rabbit anti-TCRa (cMyc) antibody and mouse anti-TCRb (V5). Anti-rabbit IRDye 680LT-
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and anti-mouse IRDye 800CW were used as secondary antibodies for detection.
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To verify pAzpa incorporation and test photo-crosslinking, we determined the ability of TCRa
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with S41 and K65 mutations to crosslink TCRb (Wang et al., 2014) (Figure 1D). The TCRa S41
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mutant serves as a positive control, as it is proximal to the TCRb subunit, and the K65 TCRa
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mutant serves as a negative control, as it is distal to the TCRb subunit in the CDR region.
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Following transfection, 87.9% of 293T cells transfected with the wild type TCRa stained positive
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for both TCRb and CD3e, compared with 87.6% for the S41 TCRa mutation and 89.6% for the
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K65 TCRa mutation (Figure 1E). After UV excitation, we detected crosslinked TCRa-TCRb for
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the aS41 mutant by Western blot as a band between 75 and 100 kDa, which corresponds to a
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size equaling TCRa+TCRb (Figure 1F). No such band was observed for the negative control
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K65 TCRa mutant, and non-crosslinked TCRa and TCRb subunits were observed at bands
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between 37 and 50 kDa (Figure 1F). Taken together, these results show that we can efficiently
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express TCR-CD3 complexes on the 293T cell surface, incorporate pAzpa into specific
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locations in the TCR a-subunit, and crosslink it to the adjacent TCRb-subunit.
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CD3
subunits crosslinks with specific TCR regions indicating one-sided CD3 subunits
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arrangement around the TCR
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Earlier studies involving mutagenesis (Kuhns and Davis, 2007), docking (Sun et al., 2004),
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molecular dynamics (Martinez-Martin et al., 2009), NMR (He et al., 2015; Natarajan et al.,
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2016), cryo-EM (Dong et al., 2019) and inference from crystal structures (Arnett et al., 2004;
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Kjer-Nielsen et al., 2004) identified multiple CD3 interaction sites on the TCR. We analyzed
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these proposed interaction sites, namely the AB loop, DE loop of TCR Ca and CC’ loop, FG
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loop, G strand, helix 3 and helix 4- F strand of TCR Cb (Figure 2A, Figure 2 – table supplement
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1), by UAA (pAzpa) incorporation and crosslinking, to provide a detailed model of native TCR-
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CD3 complex assembly in situ on mammalian cells.
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Figure 2: CD3
subunits crosslinks with specific TCR regions indicating one-sided CD3
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subunits arrangement around the TCR. A) Location of Ca DE loop (orange), Cb CC’ loop
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(red), Cb FG loop (blue), Cb G strand (yellow), Cb helix 3 (olive) and Cb helix 4 – F strand
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(magenta) on the 2B4 TCR crystal structure (PDB: 3QJF). B) Ca DE loop A172 and D174
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crosslinks with CD3d. The blot was stained with rabbit anti-TCRa (cMyc) antibody and mouse
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anti-CD3d (FLAG). C) Cb CC’ loop S170 and G171 crosslinks with CD3d. The blot was stained
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with anti-TCRb (V5) antibody and anti-CD3d (FLAG). D) Cb FG loop E221 and W225 crosslink
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to the CD3g subunit. The blot was stained with rabbit anti-TCRb (V5) antibody and mouse anti-
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CD3g (VSVG). (E) Summary of the mass spectrometry analysis on the resected Cb E221
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crosslinking band, which reveals the presence of unique CD3g and TCRb peptides. G) Cb G
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strand S238, E240 and W242 crosslink to CD3e. The bE240 blot was stained with mouse anti-
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TCRb (V5) antibody and rabbit anti-CD3e (HA). The bS238 and bW242 blots were stained with
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mouse anti-CD3e (HA) and rabbit anti-TCRb (V5). G) Cb helix 3 E136, K140 and K142 crosslink
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to TCRa. The blots were stained with mouse anti-TCRb (V5) and rabbit anti-TCRa (cMyc). H)
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Cb helix 4 – F strand F202 and R207 crosslink with CD3d and CD3e, respectively. The F202
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blot was stained with mouse anti-CD3d (FLAG) and rabbit anti-TCRb (V5). The R207 blot was
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stained with rabbit anti-CD3e (HA) and mouse anti-TCRb (V5). The crosslinking bands in each
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case is apparent below 75 kDa. Anti-rabbit IRDye 680LT- and anti-mouse IRDye 800CW were
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used for all blots as secondary antibodies for detection.
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Mutagenesis of the TCR demonstrated that the TCR Cb CC’ loop interacts with CD3eg subunits
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and the Ca DE loop interacts with CD3ed (Kuhns and Davis, 2007). To test these interaction
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sites, we transfected TCRs containing the following mutants into 293T cells: A172, D174 in the
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Ca DE loop and N164, K166, V168, S170 and G171 in the Cb CC’ loop (Figure 2A). 87.8% of
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cells transfected with the A172 mutant and 58.5% of cells transfected with the D174 mutation
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stained positive for TCRb and CD3e (Figure 2 - figure supplement 1A). However, the
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percentage of cells that expressed TCRb and CD3e for the Cb CC’ loop mutants ranged from
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20.8% for the N164 mutation to 91.2% for the S170 mutation (Figure 2 - figure supplement 2A),
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suggesting that some of these residues might be important for the stability of the complex. Our
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crosslinking studies revealed that A172, D174 (both Ca DE loop), S170 and G171 (both Cb CC’
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loop), crosslinked with the CD3d subunit of the CD3de heterodimer (Figure 2B, 2C, Figure 2 -
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figure supplement 1B, 2B). We observed a crosslinked TCRa-CD3d band for A172 and D174
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and a crosslinked TCRb-CD3d band for S170 and G171 below the 75 KDa molecular marker
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(Figure 2B, 2C) corresponding to the size equaling the two crosslinked subunits. Ca 172, Ca
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D174, Cb S170 and Cb G171 cluster together in the mouse/human TCR crystal 3D structural
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model (Figure 2A). In the recent cryo-EM structure, the residue corresponding to A172 in the Ca
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DE loop, S186 in the human TCR Ca, contacts CD3d in the complex. (Note: the alternate
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residue numbering in human cryo-EM structure is shown in smaller font size). There is no
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contact between the Cb CC’ loop and CD3d but the other end of the CC’ loop (G182) interacts
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with CD3g in the cryo-EM structure. There is no evidence of the Cb CC’ loop crosslinking with
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CD3g in our crosslinking analysis. Overall, the relative positioning of TCRa to CD3d determined
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from our crosslinking data suggests close correlation to the cryo-EM structure except for the
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lack of interaction between Cb CC’ loop and CD3g.
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High-resolution X-ray structural studies and fluorescence-based experiments have
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demonstrated a large conformational change in the AB loop of the TCR Ca domain upon
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agonist binding leading to T cell activation. Moreover, deletion of the AB loop impaired T cell
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activation indicated by low CD69 upregulation (Beddoe et al., 2009). Allosteric changes upon
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antigen binding were observed in the AB loop in an NMR CSP study (Rangarajan et al., 2018).
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These findings led to a hypothesis that the AB loop was a possible CD3 interaction site. Based
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on this, we transfected and UV-crosslinked the following Ca AB loop mutants: D132
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(transfection efficiency - 54.5%), R134 (23.2%) and Q136 (45.3%) (Figure 2 - figure supplement
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3A, 3B). However, we observed no crosslinks between the Ca AB loop and any of the CD3
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subunits (Figure 2 - figure supplement 3C), suggesting that the CD3 subunits are not near the
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Ca AB loop. Our finding is in agreement with the cryo-EM structure (Dong et al., 2019), which
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showed no interactions between Ca AB loop and CD3 subunits.
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Upon antigen ligation, the TCR behaves as an anisotropic mechanosensor, wherein the Cb FG
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loop interacts with neighboring CD3ge by acting as a lever (Kim et al., 2009; Kim et al., 2010).
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Other studies using single molecule analyses, NMR and molecular dynamics revealed that the
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FG loop allosterically controls TCR CDR catch-bond formation, peptide discrimination and CD3
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communication (Das et al., 2015; Rangarajan et al., 2018). Moreover, the FG loop is also
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theorized to be involved in thymic selection and T cell function and development (Sasada et al.,
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2002; Touma et al., 2006). Based on this and the known importance of the FG loop to T cell
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function, we transfected, and analyzed by crosslinking, the following TCR Cb FG loop mutants
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(transfection efficiency in brackets): L219 (53.2%), E221 (84.5%), D223 (73.9%), W225
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(79.0%), S229 (61.7%) and K231 (37.3%) (Figure 2A, Figure 2 – figure supplement 4A). Of
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these mutants, we found that Cb FG loop residues E221 and W225 crosslink with CD3g of the
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TCR-CD3 complex based on the presence of a band below the 75 kDa molecular marker
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indicating crosslinked TCRb-CD3g in the Western blot analysis (Figure 2D, Figure 2 – figure
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supplement 4B). However, in the cryo-EM structure CD3e’ (belonging to CD3ge heterodimer) is
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in closer proximity to the Cb FG loop (Dong et al., 2019). The crosslinking assay clearly
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indicates that CD3g is closer to the Cb FG loop than CD3e, possibly due to differing surface
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charges between mouse and human species (see computational docking results section). To
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further confirm that the CD3g subunit is closer to the Cb FG loop than CD3e we performed mass
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spectrometry analysis on the resected crosslinked band below 75 kDa from the SDS-PAGE gel
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for the E221 TCR Cb FG loop mutant. More unique peptide fragments belonging to CD3g and
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TCRb were identified in the crosslinked band (15 and 11, respectively) than other TCR-CD3
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complex subunits such as CD3d (6), CD3z (7), TCRa (4) and CD3d (1) (Figure 2E, Figure 2 –
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data supplement 1), further strengthening the possibility that CD3g is closer to the Cb FG loop in
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the cell surface conformation.
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Next, we identified residues belonging to the Cb G strand region that interact with different CD3
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subunits, as it was implicated in CD3e binding in the cryo-EM structure (Dong et al., 2019). We
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transfected and UV-crosslinked the following Cb G strand mutants (transfection efficiency in
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brackets): N236 (57.0%), S238 (48.6%), E240 (60.0%), W242 (66.2%) and R244 (38.1%)
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(Figure 2A, Figure 2 – figure supplement 5A). We found that residues S238, E240 and W242
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crosslinked with CD3e, indicated by the presence of a band below the 75 kDa molecular marker
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in the Western blot analysis (Figure 2F, Figure 2 – figure supplement 5B). Interestingly, the
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residue corresponding to W242 in the cryo-EM structure, W259, interacts with CD3e (Dong et al.,
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2019), indicating consistency in this aspect between our crosslinking and the cryo-EM data
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(Dong et al., 2019). Overall, these crosslinking experiments show that CD3ge binds to the TCR
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region around the Cb FG loop and the Cb G strand with CD3g nearer to the FG loop.
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Our earlier NMR analysis of TCR-CD3 ectodomains indicated interactions between TCR Cb
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helix 3, helix 4 – F strand regions and CD3ge (Natarajan et al., 2016). This is supported by other
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NMR CSP studies that suggested helix 3, helix 4 regions as CD3 binding regions (He et al.,
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2015) and that these regions undergo allosteric changes upon antigen ligation (Natarajan et al.,
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2017; Rangarajan et al., 2018). Another study showed that amino acid changes in the helix 3
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region led to improved TCR expression and CD3 pairing (Sommermeyer and Uckert, 2010).
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Based on these earlier reports, we transfected and UV-crosslinked the following mutants in
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293T cells: In Cb helix 3: E136 (63.4%), I137 (55.1%), A138 (26.8%), K140 (46.8%), Q141
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(72.1%) and K142 (40.9%) (Figure 2A, Figure 2 – figure supplement 6A); in Cb helix 4- F strand
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region: F202 (78.6%), H204 (84.3%), P206 (68.0%), R207 (77.9%), N208 (74.3%) and F210
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(91.8%), (Figure 2A, Figure 2 – figure supplement 7A). We did not identify any crosslinks
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between TCR Cb helix 3 residues and any CD3 subunits, but instead helix 3 residues E136,
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K140 and K142 crosslinked with the neighboring TCR a-subunit (Figure 2G, Figure 2 – figure
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supplement 6B). Likewise, the cryo-EM structure did not show any interactions between helix 3
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residues and CD3 subunits. For the TCR Cb helix 4- F strand mutants, crosslinking bands were
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observed for F202 and R207 below the 75 kDa molecular marker in the Western blot that
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corresponded with TCRb+CD3d and TCRb+CD3e, respectively (Figure 2H, Figure 2 – figure
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supplement 7B). This is in contrast to the cryo-EM structure (Dong et al., 2019) as F strand
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residue H226, which is near R207, stacks against CD3g in the cryo-EM structure (Dong et al.,
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2019), possibly due to differences in mouse-human surface charges. Overall, our crosslinking
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analysis suggests that residues F202 and R207 in the TCR Cb helix 4 – F strand region could
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13
potentially interact with CD3d and CD3e, respectively, and that residues in Cb helix 3 are not
273
directly involved in CD3 interactions.
274
275
Overall, by incorporating UAA into different abTCR sites, we identified that in the TCR-CD3
276
complex, CD3d is nearer in space to the Ca DE loop and Cb CC’ loop; CD3de is nearer in space
277
to the Cb helix 4-F strand regions; CD3g is nearer in space to the Cb FG loop and CD3e is
278
nearer in space to the Cb G strand in the abTCR-CD3 complex.
279
280
Crosslinking identifies the sides of CD3 subunits facing the TCR in the TCR-CD3
281
complex
282
After identifying specific TCR residues that are closer to CD3 subunits, we performed reciprocal
283
experiments to identify CD3 residues that are closer to TCR residues using the same UAA
284
incorporation and photo-crosslinking approach. The CD3g and CD3d residues for UAA
285
incorporation were selected based on their presence in the interface, near the interface or away
286
from the interface in the human TCR-CD3 cryo-EM complex structure (Dong et al., 2019).
287
Mutations were not introduced into the CD3e subunit as it would not be possible to distinguish
288
between CD3e subunit belonging to CD3de or CD3ge. Based on cryo-EM structure, we
289
transfected and UV-crosslinked the following CD3d mutants in 293T cells: In A strand: T5
290
(66.7%); AB loop: E8 (68.5%), D9 (71.7%); BC loop: T17 (76.3%); CD loop: V26 (31%); E
291
strand: T35 (73.6%); and EF loop: K40 (62.8%) (Figure 3A, Figure 3 – figure supplement 1A,
292
Figure 3 – table supplement 1). Of these mutants, we found that T5 crosslinks to TCRb (Figure
293
3B, Figure 3 – figure supplement 1B) and T35 and K40 crosslinks to TCRa (Figure 3C, 3D,
294
Figure 3 – figure supplement 1B). The conserved residue K40 (K62 in the cryo-EM structure)
295
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14
that crosslinks to TCRa is involved in H-bond interaction with TCRa connecting peptide residue
296
K234 in the cryo-EM structure (Dong et al., 2019).
297
298
Figure 3: Crosslinking identifies the sides of CD3 subunits facing the TCR in the TCR-
299
CD3 complex. A) Left, location of CD3d A strand (red), AB loop (green), BC loop (blue), CD
300
loop (yellow), E strand (magenta) and EF loop (cyan) on the CD3de mouse structure. Right,
301
location of CD3g AB loop (blue), CD loop (yellow), DE loop (magenta), EF loop (cyan) and FG
302
loop (orange). B) CD3d A strand T5 crosslinks to TCRb. The blot was stained with rabbit anti-
303
CD3d (FLAG) and mouse anti-TCRb (V5). C) CD3d E stand T35 crosslinks to TCRa. The blot
304
was stained with rabbit anti-CD3d (FLAG) and mouse anti-TCRa (cMyc). D) CD3d EF loop K40
305
crosslinks to TCRa. The blot was stained with rabbit anti-CD3d (FLAG) and mouse anti-TCRa
306
(cMyc). E) CD3g AB loop S14 and R15 crosslink to TCRb. The blot was stained with mouse
307
anti-CD3g (VSV-G) and rabbit anti-TCRb (V5). The crosslinking bands in each case is apparent
308
below 75 kDa. Anti-rabbit IRDye 680LT- and anti-mouse IRDye 800CW were used for all blots
309
as secondary antibodies for detection.
310
311
Residues T5 and T35 (corresponding to E27 and R57 in cryo-EM structure) that crosslink to
312
TCRb and TCRa, respectively, are involved in polar interactions with TCRa residue R185 in the
313
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15
cryo-EM structure. Interestingly, we do not see crosslinks for the conserved AB loop residues
314
E8 and D9, which interact with TCRa in the cryo-EM structure (Dong et al., 2019). These
315
differences could arise due to mouse/human species-specific surface charge differences
316
between crosslinking and cryo-EM experiments.
317
318
For CD3g, we transfected and UV-crosslinked the following mutants in 293T cells: AB loop: S14
319
(57.8%), R15 (66.8%); CD loop: D36 (59.6%); DE loop: T46 (67.6%), K47 (61.5%); EF loop:
320
K57 (64.8%) and FG loop: A68 (63.2%) (Fig. 3A, Figure 3 – figure supplement 2A). From these,
321
we found that AB loop residues S14 and R15 crosslink with TCRb (Fig. 3E, Figure 3 – figure
322
supplement 2B). The corresponding residues in the cryo-EM structure, Y36 and Q37, contact
323
H226 and G182, respectively (Dong et al., 2019). The other mutants tested were on loops away
324
from the AB loop, suggesting that the region around the AB loop is the one facing and nearer to
325
TCRb.
326
327
CD3-crosslinking interacting C
b
G strand residues are important for T cell functionality
328
Based on our crosslinking results, we used site-directed mutational and functional assays in T
329
cell hybridoma 58-/- (Letourneur and Malissen, 1989; Zhong et al., 2010) (expresses CD3
330
subunits but not TCRab) to determine whether the CD3-crosslinking TCR residues are
331
functionally relevant to T cell activation. Multiple alanine mutations were introduced in the 2B4
332
TCR residues that were shown to crosslink with CD3 and mutant T cell clones were obtained by
333
retroviral transduction (Natarajan et al., 2016; Zhong et al., 2010). Specific target sites included
334
Cb CC’ loop, Cb FG loop, Cb G strand and Ca DE loop (Figure 4A, 4B). Cells were co-cultured
335
with MHCII IEk-expressing CHO cells (CHO-IEk) loaded with K5 peptide and assessed for IL-2
336
production via ELISA sandwich assay (Malecek et al., 2014; Natarajan et al., 2016). Activated T
337
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16
cell hybridoma clones containing alanine mutations at NMR identified CD3 interaction sites such
338
as Cb helix 3 (E136A/I137A, N139A/K140A and Q141A/K142A), Cb helix 4-F strand
339
(H204A/N205A, R207A/N208A and N208A/H209A) showed less than 50% IL-2 production of
340
the wild type TCR, indicating their importance in T cell activation (Natarajan et al., 2016).
341
342
Figure 4: CD3-crosslinking C
b
G strand residues enhance T cell functionality. A)
343
Sequence alignment of TCR constant regions from different species to indicate the conserved
344
residues and the location of the TCR mutations based on cross-linking and tested in functional
345
analysis when expressed in 58-/- T cell hybridoma. B) Locations of the mutated residues
346
indicated on the 2B4 crystal structure. C3(S170A/G171A, red) is located in the Cb CC’ loop, F7
347
(E221A/W225A, blue) is located in the Cb FG loop, G1 (S238A, yellow) and G3 (W242A,
348
yellow) is located in Cb G strand and D11 (A172G/G174A, orange) is located in Ca DE loop. C)
349
ELISA assays (plot of IL-2 produced vs concentration of activating peptide) for mutant 2B4 T
350
cell hybridoma clones activated with CHO/I-Ek/K5. D) Percentage change in the area under the
351
curve for IL-2 production between the indicated mutant T cell and wild type 2B4 T cell when
352
activated with CHO cells expressing the cognate pMHC IEk/K5.
353
354
Alanine mutations at Cb G strand residues S238 (G1) and W242 (G3), which crosslink to CD3e,
355
resulted in IL-2 production increase of 74.2 ± 7.4% and 108.6 ± 8.9% that of wild type,
356
-6 -4 -2 0
-10
0
10
20
30
40
50
log K5 (mM)
% change AUC IL-2 production
58-/-
G1
G3
WT
-6 -4 -2 0
-10
0
10
20
30
40
log K5 (mM)
IL-2 production (pg/uL)
IL-2 production (pg/uL)
58-/-
C3
D11
F7
WT
C
D11 (C DE loop)
C3 (C CC’ loop)
B
F7 (C FG loop)
G3 (C G strand)
G1 (C G strand)
GKEVHS
GKEVHS
GKEVHN
GKEIRN
RKQVTT
HUMAN
BOVINE
RABBIT
MOUSE
RAT
CCC’ loop aa164-171
Mutations tested
EH
DQD
QWEEQNR
DDDEWTYSG-
ENDEWTQDR-
EEDKWPEGS-
EEDNWSEDS-
HUMAN
BOVINE
RABBIT
MOUSE
RAT
F7
CFG loop aa219-231
Mutations tested
C DE loop aa170-175
HUMAN
BOVINE
RABBIT
MOUSE
RAT
MKAMDS
MRSMDF
MEILGS
MKSLDS
MKAMDS
Mutations tested
D11
MKA
MS
G
G
G
G
G
G
N
N
N
N
N
C3
NGK
SL PK
SL
T
T
T
L
L
LTK
AK
SK
PK
DE KPEGS-
SL PA A A
V
QISAEAWGR
QNISAEAWGR
V
QNISAEAWGR
Q SAGR
V
NR
T
H
QNISAEAWGR
Mutations tested
CG strand aa235-244
HUMAN
BOVINE
RABBIT
MOUSE
RAT
QNI A A GR
G1
A
A
EW
QNI A A GR
G3
A
E
S
AA
D
WT
C3
D11
F7
G1
G3
0
50
100
150
200
250
Mutants
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respectively, possibly due to mechanistic changes in the complex as a result of alanine
357
substitutions even though S238A and W242A mutants showed surface expression comparable
358
to the wild type (Figure 4 – figure supplement 1). Interestingly, S238R and W242R mutations
359
increased TCR b-subunit surface expression more than 150% compared to wild type in J.RT3-
360
T3.5 cells (Fernandes et al., 2012) indicating S238 and W242 play vital role in complex
361
assembly and possibly T cell signaling. Overall, based on activation assays, Cb G strand
362
residues S238 and W242 play a crucial role in T cell signaling, similar to Cb helix 3 and helix 4-
363
F strand residues (Natarajan et al., 2016). However, the functional effects of the crosslinking
364
mutants were not as intense as the NMR-identified mutants (Natarajan et al., 2016), Cb
365
S170A/G171A (C3, CC’ loop), Cb E221A/W225A (F7, FG loop) and Ca A172G/D174A (D11,
366
DE loop), which led to changes in T cell activation of +37.6 ± 6.6%, +8.5 ± 5.4% and +20 ±
367
5.5%, respectively (Figure 4C, 4D).
368
369
Computational docking reveals a CD3
e
’-CD3
g
-CD3
e
-CD3
d
model for CD3 binding.
370
To generate a crosslink-guided 3D model of the TCR-CD3 complex using crosslinking
371
constraints (Figure 5A), we used computational molecular protein-protein docking (Fernandez-
372
Recio et al., 2003) to generate all possible unclashed, compact conformers of mouse CD3ge
373
and CD3de domains with the mouse 2B4 TCR domains (Figure 5 – table supplement 1). The
374
mouse TCR-CD3 components were modeled from available structures of human proteins
375
(Figure 5 – table supplement 1). Thousands of unclashed, compact TCR-CD3ge-CD3de
376
extracellular conformations were ranked based on the following hierarchy of constraints: 1)
377
geometric and spatial compatibility of the C-termini of the CD3ge and CD3de extracellular folded
378
domains with the N-termini of their corresponding TM helices in the cryo-EM TM bundle, 2)
379
CD3 subunit crosslinks to TCR chains, 3) TCR crosslinks to CD3 subunits, 4) calculated
380
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18
biophysical energy (van der Waals, solvation electrostatics, hydrogen bonding), 5) absence of
381
encroachment on plasma membrane location, 6) absence of clash with 2C11 antibody structure
382
bound CD3e and 7) absence of encroachment on pMHC binding site.
383
384
Figure 5: Computational docking reveals a CD3
e
’-CD3
g
-CD3
e
-CD3
d
model for CD3 binding.
385
A) Left, crosslinking TCR residues indicated as spheres on the crystal structure of 2B4 TCR (with
386
human constant domains, PDB: 3QJF). Residues interacting with CD3g indicated in smudge
387
green, with CD3e indicated in raspberry and with CD3d indicated in light blue. Center, crosslinking
388
CD3d residues indicated as spheres on the mouse CD3de structure. Residues interacting with
389
TCRa indicated in green, and with TCRb indicated in cyan. Right, crosslinking CD3g residues
390
indicated as spheres on the mouse CD3ge structure. Residues interacting with TCRb are indicated
391
in cyan B) Left, docked TCR-CD3 complex structure based on crosslinking derived constraints.
392
CD3ge interact primarily on the TCRb face of the complex. CD3de interacts in a region involving
393
the interface of TCRa-TCRb. The transmembrane bundle is derived from the cryo-EM human
394
TCR-CD3 transmembrane helical bundle (PDB: 6JXR) (Dong et al., 2019). Center, TCR-CD3
395
crosslink-guided model depicted in cartoon representation (60% transparency) with crosslinking
396
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TCR-CD3 residues in spheres. Same color scheme for the residues in spheres as in A). Right,
397
Cryo-EM TCR-CD3 structure. TCRa, TCRb, CD3g, CD3d, CD3e/e’ are indicated in green, cyan,
398
smudge, light blue and raspberry, respectively.
399
400
Based on these criteria, the top scoring TCR-CD3 conformation that satisfies all crosslinking
401
constraints (Figure 5 – table supplement 2) is similar to the cryo-EM structure (Figure 5B, left,
402
right). The TMs of all the subunits from the cryo-EM structure and connecting linkers were
403
modeled into the crosslink-guided model and found to be consistent with the model (e.g. the
404
CD3 domains were docked without constraints and the cross-links were recorded without bias: if
405
the TM helices clashed with the CD3 domains, this would be an indication of either an invalid
406
docking method or invalid cross-links, as would the inability of the linkers to connect the CD3 C-
407
termini to the TM N-termini). This final structural model had an overall contact area of 491.2 Å2
408
between crosslinked TCR residues and CD3ge and a contact area of 342.4 Å2 between
409
crosslinked TCR residues and CD3de, with energies of -23.6 and -14.5, respectively. The CD3e'
410
chain of the CD3ge’ heterodimer is behind the Cb FG loop (Figure 5B, left) rather than directly
411
below the FG loop, as seen in the cryo-EM structure (Figure 5B, right). The crosslink-guided
412
model shows that the CD3g subunit is closer to the Cb FG loop, as we detected crosslinking
413
between the Cb FG loop residues and CD3g (Figure 5B, center, Figure 5 – figure supplement 2).
414
The electrostatic surfaces participating in binding interfaces in the individual components of the
415
human TCR-CD3 complex differed when compared to their modeled mouse counterparts,
416
especially on the CD3 subunits (Figure 5 – figure supplement 1), suggesting that there could be
417
differences between the human and mouse TCR-CD3 complexes, as seen in our model, even
418
though the overall arrangement of the components are similar. The distances between the
419
center of mass of the CD3de and CD3ge relative to the TCR when compared to the cryo-EM
420
structure are 14.7 and 36.05, respectively. The location of CD3de on the crosslink-guided TCR-
421
CD3 structure that was implicated in CD3d binding is nearly identical as the same regions (A172
422
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in crosslinking and S186 in cryo-EM, both belonging to Ca DE loop) in the cryo-EM structure,
423
although the relative orientation of CD3de in crosslink-guided structure is different than the cryo-
424
EM structure (Figure 5 – figure supplement 2). Overall, our crosslinking analysis combined with
425
computational docking identifies a cell-surface native conformation of the abTCR-CD3 complex
426
that is overall similar to the human cryo-EM structure but with differences in interface contacts.
427
Our structure identifies a CD3e’-CD3g-CD3e-CD3d model for CD3 binding with contacts between
428
CD3g (belonging to CD3ge) and CD3e (belonging to CD3de) (Figure 5B, Figure 5 – figure
429
supplement 2).
430
431
Discussion
432
A molecular phenomenon that can confound precise translation of molecular observations to
433
clinical relevance is structural/atomic accuracy of 3D receptor models. X-ray crystallography,
434
NMR and cryo-EM are the leading structural biology techniques that provide detailed and
435
tangible atomistic details about protein structure and interactions. However, some biologically
436
relevant conformations may not be identified via these techniques due to experimental and
437
nonnative conditions used. Site-specific crosslinking using unnatural amino acids combined with
438
computational analysis can provide a robust alternative towards obtaining both atomistic and
439
species-specific information on intermediate or dynamic states with small amounts of protein
440
and, significantly, on in situ states present in complex cellular environment (Coin, 2018;
441
Grunbeck et al., 2011; Valentin-Hansen et al., 2014). As far as we know, our study is the first to
442
successfully photo-crosslink and subsequently provide a native structural model of an immune
443
protein receptor complex. The results from our study indicating overall similarity between cryo-
444
EM structure and crosslinking model validate photo-crosslinking-docking technique as an
445
attractive option for structural/in situ analysis of protein complexes. A similar approach can be
446
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undertaken to study other immune protein complexes in their native states, such as gdTCR-CD3
447
complex, B cell receptor complex, CD19/CD21 coreceptor complex.
448
449
For unnatural amino acid (pAzpa) incorporation and crosslinking, we co-transfected tRNA-aaRS
450
and mouse TCR and CD3 plasmids (with amber mutations in specific sites on TCR or CD3) into
451
293T cells, and expressed and UV crosslinked 47 different mutants. From this, 16 specific TCR-
452
CD3 subunit crosslinks were identified, including residues in the Ca DE loop crosslinking with
453
CD3d, the Cb CC’ loop crosslinking to CD3d, the Cb FG loop crosslinking to CD3g, the Cb G
454
strand crosslinking to CD3e and the Cb helix 4-F strand crosslinking to CD3d and CD3e (Figure
455
5A). Similarly, the CD3d A strand crosslinks to TCRb, CD3d E strand, the EF loop crosslinks to
456
TCRa and the CD3g AB loop crosslinks to TCRb. Utilizing these specific crosslinks as distance
457
restraints in a biophysical search of conformations, we visualized an in situ cell-surface model of
458
the TCR-CD3 complex (Fernandez-Recio et al., 2003).
459
460
Comparing the crosslink-guided model with the recently published cryo-EM model (Dong et al.,
461
2019), we found that the CD3 arrangement from the crosslink-guided model is largely
462
comparable to the CD3 arrangement in the cryo-EM structure (Figure 5B). The gross locations
463
of the CD3-TCR interfaces within the complexes are all similar between our photo-crosslinking
464
and the cryo-EM, however, the Cb FG loop is above and in-between CD3g and CD3e’ in the
465
crosslink-guided model. This is different from the cryo-EM structure, which places FG loop
466
above CD3e’ in the CD3ge heterodimer. One reason for the difference could be the usage of
467
glutaraldehyde crosslinking to fix the complex in one acellular conformation in cryo-EM
468
analysis(Dong et al., 2019). Our crosslink-based conformer could represent the true resting
469
conformation of the complex resulting from the physiological cell surface condition with Cb FG
470
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loop closer to CD3g in the human TCR-CD3 complex as well. However, examining this
471
experimentally through photo-crosslinking-computational docking is beyond the scope of this
472
current research. The other and most probable reason could be because of the species-specific
473
differences in the amino acid composition of the electrostatic surfaces of the mouse proteins
474
used in our study and the human proteins in the cryo-EM study (Figure 5 – figure supplement
475
1). By observing overall consistent locations of the CD3-TCR interfaces, our findings
476
independently validate the mechanism of this aspect of TCR signaling between mice and
477
humans, but, the regions of the CD3-TCR interaction that differ between mouse and human
478
suggest that signaling thresholds, and therefore pharmacology, may be different between the
479
species. Species-specific differences between mice and humans can confound translation of
480
observations in more easily controlled experiments in mice to clinical relevance. Drug
481
candidates targeting TCR-CD3 complex derived from pre-clinical murine models, ‘humanized’
482
murine models with human CD3 subunits (Crespo et al., 2021; Ueda et al., 2017) and CD3
483
copotentiation (Becher et al., 2020; Hoffmann et al., 2015) should take our crosslink-guided
484
model into account before translating the pharmacology to human studies. For investigators
485
using mouse systems to investigate TCR signaling and phenotypes, our crosslink-guided model
486
may serve as a useful reference point for interpreting translatability of findings to the human
487
TCR via comparison with the cryo-EM model.
488
489
Our crosslink-guided model differs substantially from our previously reported NMR-based model
490
(Natarajan et al., 2016), which was based on CSP data that showed peak intensity losses or
491
peak shits in the TCR upon CD3ge and CD3de addition. These sites include Cb helix 3, Cb helix
492
4-F strand, Cb FG loop, Ca F strand, Ca C strand and Ca tail (Natarajan et al., 2016). This
493
spectral change could result from direct CD3 subunit interaction to the particular TCR site or
494
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from conformational changes at the site upon CD3 binding elsewhere on the TCR. A ranking
495
mechanism similar to the one used in the crosslinking study was used for TCR-CD3ge and TCR-
496
CD3de docking. Based on this, our top-scoring TCR-CD3ge and TCR-CD3de docking models
497
showed that the CD3 heterodimers interact on opposite sides of the TCR (Natarajan et al.,
498
2016). One possible reason for this discrepancy could be the absence of membrane, CPs and
499
TMs in the soluble protein domains used in the NMR study, which could allow for orientation of
500
the CD3 molecules away from the membrane, instead of proximal to the membrane. Further, IL-
501
2 production upon activation of NMR-identified Cb helix 3 mutants indicated a loss of >50%
502
compared to the wild type TCR (Natarajan et al., 2016). Moreover, other studies have identified
503
some of the same TCR sites (Cb helix 3 and helix 4) as CD3 interaction regions and are
504
involved in allosteric interactions upon antigen ligation (He et al., 2015; Natarajan et al., 2017;
505
Rangarajan et al., 2018). Thus, this discrepancy remains unresolved for Cb helix 3 and other
506
sites such as the Ca F and C strands. Importantly, in this study, we identified Cb G strand
507
residues S238 and W242 as playing a role in T cell activation. Interestingly, these same
508
residues show increased occupancy in the TCR-CD3 interface during force-based steered
509
molecular dynamics simulations, thereby strengthening TCR-CD3 interactions under force (Z.
510
Yuan, 2021). Thus, the G strand residues- S238 and W242, conserved between mouse and
511
human, are possible candidates for protein engineering to enhance TCR signaling.
512
513
Based on data from earlier NMR and cryo-EM studies (Arechaga et al., 2010; Birnbaum et al.,
514
2014; Dong et al., 2019; He et al., 2015; Natarajan et al., 2016), we inferred that the
515
extracellular part of the TCR-CD3 complex could exist in multiple, biologically-relevant
516
conformations on the T cell surface and, here, we sought to identify them. This kind of
517
conformational switch is not uncommon in the TCR-CD3 transmembrane space, as the TCRa
518
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24
transmembrane helix exists in L- and E- states (Brazin et al., 2018), CD3zz juxtamembrane
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regions exist in open and closed conformations (Lee et al., 2015), and TCRb switches between
520
inactive and active conformations upon cholesterol binding and unbinding (Swamy et al., 2016).
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Using photo-crosslink-guided computational molecular docking we visualized a conformer that is
522
similar overall to the recent 3.7 Å cryo-EM structure (Dong et al., 2019), providing validation of
523
this model of the TCR-CD3 signaling complex. Extending the crosslink-guided model via an
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antigen activation system could reveal the broader mechanism by which pMHC activates the
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TCR-CD3 complex and identify structure-activity relationships that can be exploited to modulate
526
signaling pharmaceutically, with potential benefits for the treatment of cancer, infectious
527
diseases and autoimmune diseases.
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Materials and Methods
530
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Plasmid construction:
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The tRNA synthetase for recognition of pBpa in PU6-pBpa plasmid, a generous gift from Peter
533
G. Schultz, Scripps Research Institute, was replaced with tRNA synthetase for pAzpa to create
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PU6-pAzpa plasmid(Wang et al., 2014). This PU6-pAzpa plasmid contains mutant E.coli tyrosyl-
535
tRNA synthetase (EcTyrRS), one copy of B. stearothermophilus tRNAs (BstRNA) and human
536
U6 small nuclear promoter (U6)(Wang et al., 2014). cDNA encoding mouse TCR 2B4 a (with c-
537
Myc-tag) and b (with V5-tag) sequences with 2A sequence linking each other were cloned into
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pCDNA3.1/Zeo(+) vector (Life Technologies) using Not1 and Xho1 restriction enzymes.
539
Similarly, cDNA encoding mouse CD3d (with FLAG-tag), mouse CD3g (with VSV-G tag), mouse
540
CD3e (with HA-tag) and CD3z interconnected with 2A sequence were cloned in
541
pCDNA3.1/Zeo(+) vector using Not1 and Xho1 restriction enzymes. Amber (TAG) codons were
542
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.07.479368doi: bioRxiv preprint
25
introduced site-specifically in the 2B4 TCR and CD3 plasmid using Quikchange mutagenesis kit
543
(Agilent).
544
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Transfections into HEK293T cells:
546
HEK293T cells (ATCC) were cultured in DMEM media, supplemented with 10% FBS, sodium
547
pyruvate, non-essential amino acids, glutaMAX-1, penicillin-streptomycin and b-
548
mercaptoethanol and grown at 37 °C, 5% CO2 to 80% confluency in a collagen-coated 6-well
549
plate before transfections. To incorporate unnatural amino acids, pAzpa (p-azido-
550
phenylalanine), into predetermined sites on the TCR and CD3 extracellular regions (Table S1,
551
S2), plasmid expressing amber suppressor tRNA-aminoacyl-tRNAsynthetase (tRNA-aaRS) –
552
PU6-pAzpa was co-transfected with plasmids expressing full-lengths mutant 2B4 TCR and CD3
553
subunits using Xfect transfection kit (TaKaRa). For incorporating pAzpa into TCR sites, as
554
optimized previously, 7.5 µg of TCR:CD3 in 8:1 ratio and 2.5 µg of PU6-pAzpa plasmids were
555
co-transfected into HEK293T cells(Wang et al., 2014). For incorporating pAzpa into CD3 sites,
556
7.5 µg of TCR:CD3 in 4:1 ratio and 2.5 µg of PU6-pAzpa plasmids were co-transfected into
557
HEK293T cells. After 4 hours of culture at 37 °C, 5% CO2, the media was replaced with fresh
558
DMEM media containing 1 mM pAzpa (Chem-Implex International) and cultured for 48 hours.
559
560
Flow cytometry analysis:
561
After 48 hours of culture, the cells were harvested and washed in FACS buffer (PBS + 2%
562
FBS). A small portion of the cells were treated with allophycocyanin (APC) anti-TCRb (clone
563
H57-597, eBioscience) and phycoerythrin (PE) anti-CD3e (clone 145-2C11, eBioscience) in
564
FACS buffer for 30 minutes. Subsequently, the samples were analyzed for TCRb and CD3e
565
expression in FACSCalibur (BD Biosciences) and data was analyzed using FlowJo (ver 10.5.3).
566
.CC-BY 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.07.479368doi: bioRxiv preprint
26
567
Photo-crosslinking, Immunoprecipitation and Western blotting:
568
Cells were photo-crosslinked by exposing them to 360 nm UV light source for 45 minutes on ice.
569
Following that, the cells were washed in antibody buffer - Hank’s balanced salt solution/2%
570
FBS/0.05% (m/v) sodium azide. The cells were treated with 25 ug/mL biotinylated mouse anti-
571
CD3e (clone 145-2C11, eBioscience) for 30 minutes and washed in 1X TBS (Tris-buffered
572
saline). The cells were lysed in TBS/1% (v/v) IGEPAL-630 (sigma) containing 1X Complete
573
protease cocktail inhibitors (Roche). The TCR-CD3 complex was purified from the lysate using
574
Dynabeads M-280 streptavidin (Invitrogen). The beads were subsequently washed with 1X TBS
575
and boiled with SDS-PAGE reducing buffer with b-mercaptoethanol. The subunits were resolved
576
by SDS-PAGE electrophoresis and transferred to nitrocellulose membranes (ThermoFisher).
577
For Western blot analysis, the following pairs of primary antibodies were used: 1) TCRa-TCRb
578
crosslinking: rabbit anti-c-Myc (Genscript) and mouse anti-V5 (Genscript); 2) TCRa-CD3d
579
crosslinking: rabbit anti-cMyc and mouse anti-FLAG (Genscript); 3) TCRa-CD3d crosslinking:
580
mouse anti-cMyc (Genscript) and rabbit anti-FLAG (Genscript); 4) TCRb-CD3d crosslinking:
581
mouse anti-V5 and rabbit anti-FLAG (Genscript); 5) TCRb-CD3d crosslinking: rabbit anti-V5 and
582
mouse anti-FLAG; 6) TCRb-CD3g crosslinking: rabbit anti-V5 (Genscript) and mouse anti-VSV-
583
G (Abcam); 7) TCRb-CD3e crosslinking: rabbit anti-V5 and mouse anti-HA (Genscript); 8)
584
TCRb-CD3e crosslinking: mouse anti-V5 and rabbit anti-HA (Genscript); 9) TCRa-CD3g
585
crosslinking: rabbit anti-cMyc and mouse anti-VSV-G. The following secondary antibodies were
586
used for detection: IRDye 680LT-conjugated donkey anti-rabbit IgG (H+L) (LI-COR) and IRDye
587
800CW-conjugated donkey anti-mouse IgG (H+L) (LI-COR). Images were collected using LI-
588
COR Odyssey and analyzed using Image Studio Lite (LI-COR, ver 4.0.21).
589
590
.CC-BY 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.07.479368doi: bioRxiv preprint
27
Functional analysis with mutant T cell hybridoma:
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Mouse 58-/- T cell hybridoma cells (Letourneur and Malissen, 1989), which expresses mouse
592
CD3 but not TCRab, (from David Kranz, University of Illinois) and Chinese hamster ovary
593
(CHO) cells expressing I-Ek (Krogsgaard et al., 2005)(from Mark M. Davis, Stanford University)
594
were cultured in RPMI 1640 medium and DMEM respectively, supplemented with 10% FBS,
595
sodium pyruvate, non-essential amino acids, glutaMAX-1, penicillin-streptomycin and b-
596
mercaptoethanol. The mutant mouse 2B4 TCR constructs were generated by PCR using
597
overlapping primers containing the mutant sequences and cloned into the pCDNA3.1 vector.
598
Retroviral transductions of the hybridoma cells were done as described previously (Zhong et al.,
599
2010). The transduced cells were stained with PE anti-CD3e (clone 145-2C11) and APC anti-
600
TCRb (clone H57-597) antibodies. The transduced cells were sorted, expanded for 6 days,
601
quantified for TCRb/CD3e expression, and prepared for the cytokine assay. 104 CHO-IEk cells
602
were loaded with different concentrations of a variant of moth cytochrome c (K5) (Krogsgaard et
603
al., 2005) peptide and incubated with 104 T cell hybridoma clones (wild type and mutants) in
604
triplicates for 16 hours at 37 °C, 5% CO2. A standard ELISA sandwich was used to quantify
605
cytokine IL-2 production (Malecek et al., 2013). The area under the curve for wildtype and
606
mutant IL-2 production, a cumulative response measure, was calculated after non-linear fitting
607
using Prism (GraphPad software).
608
609
TCR and CD3 subunit structure generation and complex docking:
610
Crosslink-guided Model: 3D models of individual murine CD3 domains (CD3g, CD3d) were built
611
by homology modeling using ICM-Pro software (Molsoft LLC. La Jolla CA)(Fernandez-Recio et
612
al., 2003) applied to different structures as templates found in the PDB database as shown in
613
Table S3, as for the murine CD3e its structure was taken from a published crystal structure of
614
.CC-BY 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.07.479368doi: bioRxiv preprint
28
the monomer binding to the antibody OKT3 (PDB: 1SY6), . The coordinates of the TMs of all
615
subunits relative to the TCR subunits were inherited from the human cryo-EM structure (PDB:
616
6JXR)(Dong et al., 2019). 3D models of the two CD3 hetero-oligomers were docked to the 3D
617
model of the 2B4 murine TCR. Absence of clashes and compact conformations were identified
618
by calculated, estimated free energy of the complex, as previously described (Fernandez-Recio
619
et al., 2002a, b, 2003; Garzon et al., 2009), which includes terms for van der Waals,
620
electrostatics, hydrogen bonding and solvation and the energy units approximate kcals in free
621
energy calculations. All compact (all CD3 and TCR domains contacting at least one other
622
domain) and conformations without clashes were retained and their calculated free energy
623
score was re-weighted by their contact area of the UAA side-chains with the CD3 domains and
624
the distance between these UAA side-chains and the CD3. Docked conformations that were
625
inconsistent with the length of the linking segments that connect the TMs with the CD3 folded
626
domains were discarded. Conformations impinging on the membrane or the 2C11 antibody
627
were discarded, if there existed at least one unclashed, compact, cross-link compatible
628
conformation that did not impinge on the membrane or 2C11.
629
630
Acknowledgements
631
We thank Peter G. Schultz, Scripps Research Institute for the PU6-pBpa plasmid. We also
632
thank David Kranz (University of Illinois) for providing us 58 −/− T-cell hybridoma and Mark M.
633
Davis (Stanford University) for providing us with Chinese hamster ovary (CHO) cells expressing
634
I-Ek. We also thank Thomas P. Sakmar (Rockefeller University) for protocols and advice on
635
photo-crosslinking technique. We thank Duane Moogk (McMaster University) and Yury
636
Patskovsky (NYU Grossman School of Medicine) for helpful discussions and critical reading of
637
the manuscript. We thank Eric Ni (Yale University) for helpful discussions. This work was
638
supported by the NIH grant NIGMS R01 GM124489 (to M.K).
639
.CC-BY 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.07.479368doi: bioRxiv preprint
29
640
Footnotes
641
Author Contributions: Experiments were conceptualized by A.N., W.W., T.C. and M.K. Photo-
642
crosslinking experiments were performed by A.N. Computational docking was performed by
643
M.B.F. T cell activation experiments were performed by A.N. Crosslinking TCR, CD3 mutant
644
constructs and mutant retroviral TCR constructs were generated by W.W., T.L., S.B. and H.S.
645
Data was analyzed and interpreted by A.N., T.C., and M.K. The original draft was written by
646
A.N. and the final draft was reviewed and edited by A.N., T.C., and M.K.
647
648
The authors declare no competing interests.
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E., Schulze, A.K., Blanco, R., et al. (2016). A Cholesterol-Based Allostery Model of T Cell Receptor
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Phosphorylation. Immunity 44, 1091-1101.
771
Touma, M., Chang, H.C., Sasada, T., Handley, M., Clayton, L.K., and Reinherz, E.L. (2006). The TCR C beta
772
FG loop regulates alpha beta T cell development. Journal of immunology 176, 6812-6823.
773
Ueda, O., Wada, N.A., Kinoshita, Y., Hino, H., Kakefuda, M., Ito, T., Fujii, E., Noguchi, M., Sato, K., Morita,
774
M., et al. (2017). Entire CD3epsilon, delta, and gamma humanized mouse to evaluate human CD3-
775
mediated therapeutics. Scientific reports 7, 45839.
776
Valentin-Hansen, L., Park, M., Huber, T., Grunbeck, A., Naganathan, S., Schwartz, T.W., and Sakmar, T.P.
777
(2014). Mapping substance P binding sites on the neurokinin-1 receptor using genetic incorporation of a
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photoreactive amino acid. The Journal of biological chemistry 289, 18045-18054.
779
Wang, W., Li, T., Felsovalyi, K., Chen, C., Cardozo, T., and Krogsgaard, M. (2014). Quantitative analysis of
780
T cell receptor complex interaction sites using genetically encoded photo-cross-linkers. ACS chemical
781
biology 9, 2165-2172.
782
Wilkins, B.J., Rall, N.A., Ostwal, Y., Kruitwagen, T., Hiragami-Hamada, K., Winkler, M., Barral, Y., Fischle,
783
W., and Neumann, H. (2014). A cascade of histone modifications induces chromatin condensation in
784
mitosis. Science 343, 77-80.
785
Xu, C., Call, M.E., and Wucherpfennig, K.W. (2006). A membrane-proximal tetracysteine motif
786
contributes to assembly of CD3deltaepsilon and CD3gammaepsilon dimers with the T cell receptor. The
787
Journal of biological chemistry 281, 36977-36984.
788
Z. Yuan, P.C., A. Natarajan, C. Ge, S. Travaglino, S. Beesam, D. Grazette, M. Krogsgaard, C. Zhu (2021).
789
Cooperative ectodomain interaction among TCRab, CD3ge and CD3de. (in preparation).
790
Zhong, S., Malecek, K., Perez-Garcia, A., and Krogsgaard, M. (2010). Retroviral transduction of T-cell
791
receptors in mouse T-cells. J Vis Exp.
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793
794
795
796
797
798
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800
801
802
803
804
805
806
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808
809
810
811
812
813
814
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.07.479368doi: bioRxiv preprint
33
Supplementary Figures
815
816
817
818
819
Figure 2 – figure supplement 1: CD3
d
interacts with the TCR C
a
DE loop. A) TCRb and
820
CD3e expression histograms of Ca DE loop mutants – aA172 and aD174 by flow cytometry.
821
The percentage of cells positive for both TCRb and CD3e staining is indicated. B) Western blot
822
analysis of Ca DE loop mutants - aA172 and aD174. CD3d crosslinked bands for aA172 and
823
aD174 are apparent around 75 kDa. The blot was stained with rabbit anti-TCRa (cMyc)
824
antibody and mouse anti-CD3d (FLAG). Anti-rabbit IRDye 680LT- and anti-mouse IRDye
825
800CW were used as secondary antibodies for detection.
826
827
828
.CC-BY 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.07.479368doi: bioRxiv preprint
34
829
830
831
Figure 2 – figure supplement 2: CD3
d
interacts with the TCR C
b
CC’ loop. A) TCRb and
832
CD3e expression histograms of Cb CC’ loop mutants - bN164, bK166, bV168, bS170 and
833
bG171 by flow cytometry. The percentage of cells positive for both TCRb and CD3e staining is
834
indicated. B) Western blot analysis of Cb CC’ loop mutants - bN164, bK166, bV168, bS170 and
835
bG171. CD3d crosslinked bands for bS170 and bG171 are apparent around 75 kDa. The blot
836
was stained with rabbit anti-TCRb (V5) antibody and mouse anti-CD3d (FLAG). Anti-rabbit
837
IRDye 680LT- and anti-mouse IRDye 800CW were used as secondary antibodies for detection.
838
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.07.479368doi: bioRxiv preprint
35
839
840
841
842
Figure 2 – figure supplement 3: The TCR C
a
AB loop is not near any CD3 subunits. A)
843
Location of the Ca AB loop (in orange) on the 2B4 TCR crystal structure (PDB: 3QJF). B) TCRb
844
and CD3e expression histograms of Ca AB loop mutants - aD132, aR134 and aQ136 by flow
845
cytometry. The percentage of cells positive for both TCRb and CD3e staining is indicated. C)
846
Western blot analysis of Ca AB loop mutants - aD132, aR134 and aQ136. No crosslinking
847
bands were evident for these mutants. The blots were stained with rabbit anti-TCRa (cMyc)
848
antibody and mouse anti-TCRb (V5). Anti-rabbit IRDye 680LT- and anti-mouse IRDye 800CW
849
were used as secondary antibodies for detection.
850
10
0
10
1
10
2
10
3
10
4
TCRexpression
D132
Q136
R134
NEG
B. TCR/CD3 expressions of C AB loop mutants
10
0
10
1
10
2
10
3
10
4
CD3expression
D132 (54.5%)
Q136 (45.3%)
R134 (23.2%)
NEG
A. Location of C AB loop
R134
R134
WB: anti-TCRcMyc
Q136D132
TCR
Q136D132
WB: anti-TCRV5
TCR
C. Western blot analysis of C AB loop mutants
AB loop
TCRTCR
100 KD
75 KD
50 KD
37 KD
25 KD
100 KD
75 KD
50 KD
37 KD
25 KD
.CC-BY 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.07.479368doi: bioRxiv preprint
36
851
852
853
Figure 2 - figure supplement 4: CD3
g
interacts with the TCR C
b
FG loop. A) TCRb and
854
CD3e expression histograms of Cb FG loop mutants - bL219, bE221, bD223, bW225, bS229
855
and bK231 by flow cytometry. The percentage of cells positive for both TCRb and CD3e staining
856
is indicated. B) Western blot analysis of Cb FG loop mutants - bL219, bE221, bD223, bW225,
857
bS229 and bK231. CD3g crosslinked bands seen for bE221 and bW225 are apparent below 75
858
kDa. The blot was stained with anti-TCRb (V5) antibody and anti-CD3g (VSV-G). Anti-rabbit
859
IRDye 680LT- and anti-mouse IRDye 800CW were used as secondary antibodies for detection.
860
861
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.07.479368doi: bioRxiv preprint
37
862
Figure 2 - figure supplement 5: CD3
e
interacts with the TCR C
b
G strand. A) TCRb and
863
CD3e expression histograms of Cb G strand mutants - bN236, bS238, bE240, bW242 and
864
bR244 by flow cytometry. The percentage of cells positive for both TCRb and CD3e staining is
865
indicated. B) Western blot analysis of Cb G strand mutants - bN236, bS238, bE240, bW242 and
866
bR244. CD3e crosslinked bands seen for bS238, bE240 and bW242 are apparent below 75
867
kDa. bN236, bS238, bW242 and bR244 blots were stained with rabbit anti-TCRb (V5) antibody
868
and mouse anti-CD3e (HA). bE240 blot was stained with mouse anti-TCRb (V5) antibody and
869
rabbit anti-CD3e (HA). Anti-rabbit IRDye 680LT- and anti-mouse IRDye 800CW were used as
870
secondary antibodies for detection.
871
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.07.479368doi: bioRxiv preprint
38
872
873
874
875
Figure 2 - figure supplement 6: C
b
helix 3 residues interact with the TCR
a
subunit. A)
876
TCRb and CD3e expression histograms of Cb helix 3 mutants – bE136, bI137, bA138, bK140,
877
bQ141 and bK142 by flow cytometry. The percentage of cells positive for both TCRb and CD3e
878
staining is indicated. B) Western blot analysis of Cb helix 3 mutants - WT, bE136, bI137, bA138,
879
bK140, bQ141 and bK142. TCRa crosslinked bands seen for bE136, bK140 and bK142 are
880
apparent between 75 and 100 kDa. WT, bE136, bA138, bK140 and bK142 blots were stained
881
with rabbit anti-TCRa (cMyc) antibody and mouse anti-TCRb (V5). bI137 and bQ141 blots were
882
stained with rabbit anti-TCRb (V5) antibody and mouse anti-CD3d (FLAG). Anti-rabbit IRDye
883
680LT- and anti-mouse IRDye 800CW were used as secondary antibodies for detection.
884
885
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.07.479368doi: bioRxiv preprint
39
886
Figure 2 - figure supplement 7: CD3
de
interacts with the TCR C
b
helix 4-F strand region.
887
A) TCRb and CD3e expression histograms of Cb helix 4–F strand mutants – bF202, bH204,
888
bP206, bR207, bN208 and bF210 by flow cytometry. The percentage of cells positive for both
889
TCRb and CD3e staining is indicated. B) Western blot analysis of Cb helix 4-F strand mutants -
890
bF202, bH204, bP206, bR207, bN208 and bF210. CD3d and CD3e crosslinked bands seen for
891
bF202 and bR207, are apparent below 75 kDa. bH204, bP206, bN208 and bF210 blots were
892
stained with rabbit anti-TCRb (V5) antibody and mouse anti-CD3e (HA). bF202 blot was stained
893
with rabbit anti-TCRb (V5) antibody and mouse anti-CD3d (FLAG). bR207 blot was stained with
894
mouse anti-TCRb (V5) antibody and rabbit anti-CD3e (HA). Anti-rabbit IRDye 680LT- and anti-
895
mouse IRDye 800CW were used as secondary antibodies for detection.
896
100 KD
75 KD
50 KD
37 KD
25 KD
H204 F210N208P206
WB: anti-TCR(V5)
TCR
100 KD
75 KD
50 KD
37 KD
25 KD
H204 F210N208P206
WB: anti-CD3(HA)
CD3
A. TCR/CD3 expressions of C helix 4 - F strand mutants
10
0
10
1
10
2
10
3
10
4
TCRexpression
10
0
10
1
10
2
10
3
10
4
CD3expression
F210 (91.8%)
F210 (91.8%)
N208 (74.3%)
N208 (74.3%)
P206 (68.0%)
P206 (68.0%)
H204 (84.3%)
H204 (84.3%)
NEG
NEG
B. Western blot analysis of C helix 4 - F strand mutants
100 KD
75 KD
50 KD
37 KD
25 KD
F202
Crosslinked
CD3
CD3
WB: anti-CD3(FLAG)
100 KD
75 KD
50 KD
37 KD
25 KD
F202
WB: anti-TCR(V5)
R207
WB: anti-TCR(V5)
R207
WB: anti-CD3(HA)
Crosslinked
CD3
CD3
TCRTCR
Crosslinked
TCR
Crosslinked
TCR
F202 (78.6%)
R207 (77.9%)
NEG
10
0
10
1
10
2
10
3
10
4
TCRexpression
10
0
10
1
10
2
10
3
10
4
CD3expression
F202 (78.6%)
R207 (77.9%)
NEG
100 KD
75 KD
50 KD
37 KD
25 KD
100 KD
75 KD
50 KD
37 KD
25 KD
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.07.479368doi: bioRxiv preprint
40
897
898
Figure 3 - figure supplement 1: TCR
a
crosslinks to CD3
d
E strand, CD3
d
EF loop and
899
TCR
b
crosslinks to CD3
d
A strand. A) TCRb and CD3e expression histograms of CD3d
900
mutants – A strand: T5; AB loop: E8, D9; BC loop: T17; CD loop: V26; E strand: T35; and EF
901
loop: K40 by flow cytometry. The percentage of cells positive for both TCRb and CD3e staining
902
is indicated. Western blot analysis revealed CD3d T5 crosslinked with TCRb and CD3d T35 and
903
K40 crosslinked with TCRa with crosslinking bands apparent below 75 kDa. E8, T35 and K40
904
were stained with mouse anti-TCRa (cMyc) antibody and rabbit anti-CD3d (FLAG). T5 and D9
905
were stained with mouse anti-TCRb (V5) and rabbit anti-CD3d (FLAG). T17 and V26 were
906
stained with rabbit anti-TCRa (cMyc) and mouse anti-CD3d (FLAG). Anti-rabbit IRDye 680LT-
907
and anti-mouse IRDye 800CW were used as secondary antibodies for detection.
908
A. TCR/CD3 expressions of CD3 mutants
B. Western blot analysis of CD3 mutants
10
0
10
1
10
2
10
3
10
4
TCRexpression
10
0
10
1
10
2
10
3
10
4
CD3expression
T35 (73.6%)
NEG
T5 (66.7%)
K40 (62.8%)
D9 (71.7%)
T35 (73.6%)
NEG
T5 (66.7%)
K40 (62.8%)
D9 (71.7%)
10
0
10
1
10
2
10
3
10
4
TCRexpression
NEG
E8 (68.5%)
10
0
10
1
10
2
10
3
10
4
CD3expression
NEG
E8 (68.5%)
10
0
10
1
10
2
10
3
10
4
TCRexpression
10
0
10
1
10
2
10
3
10
4
CD3expression
NEG
T17 (76.3%)
NEG
T17 (76.3%)
10
0
10
1
10
2
10
3
10
4
TCRexpression
NEG
V26 (31.0%)
10
0
10
1
10
2
10
3
10
4
CD3expression
NEG
V26 (31.0%)
T5
WB: anti-TCR(V5)
TCR
Crosslinked
TCR
100 KD
75 KD
50 KD
37 KD
25 KD
D9
100 KD
75 KD
50 KD
37 KD
25 KD
WB: anti-CD3(FLAG)
T5 D9
Crosslinked
CD3
CD3
100 KD
75 KD
50 KD
37 KD
25 KD
WB: anti-TCR(cMyc)
T35 K40
TCR
Crosslinked
TCR
100 KD
75 KD
50 KD
37 KD
25 KD
WB: anti-CD3(FLAG)
T35 K40
Crosslinked
CD3
CD3
100 KD
75 KD
50 KD
37 KD
25 KD
WB: anti-CD3(FLAG)
T17 V26
CD3
100 KD
75 KD
50 KD
37 KD
25 KD
TCR
WB: anti-TCR(cMyc)
T17 V26 E8
WB: anti-CD3(FLAG)
CD3
WB: anti-TCR(cMyc)
E8
TCR
100 KD
75 KD
50 KD
37 KD
25 KD
Crosslinked
TCR
100 KD
75 KD
50 KD
37 KD
25 KD
Crosslinked
CD3
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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41
909
Figure 3 - figure supplement 2: TCR
b
crosslinks to CD3
g
AB loop. A) TCRb and CD3e
910
expression histograms of CD3d mutants – AB loop: S14, R15; CD loop: D36; DE loop: T46,
911
K47; EF loop: K57 and FG loop: A68 by flow cytometry. The percentage of cells positive for both
912
TCRb and CD3e staining is indicated. Western blot analysis revealed CD3g S14 and R15
913
crosslinked with TCRb with crosslinking bands apparent below 75 kDa. S14, R15, K57 and A68
914
were stained with mouse anti-CD3g (VSV-G) and rabbit anti-TCRb (V5) antibody. T46 and K47
915
were stained with mouse anti-CD3g (VSV-G) and rabbit anti-CD3e (HA). D36 was stained with
916
mouse anti-CD3g (VSV-G) and rabbit anti-TCRa (cMyc). Anti-rabbit IRDye 680LT- and anti-
917
mouse IRDye 800CW were used as secondary antibodies for detection.
918
919
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.07.479368doi: bioRxiv preprint
42
920
921
Figure 4 - figure supplement 1: TCRa and CD3e expression histograms of T cell hybridoma
922
mutants tested: G3 – Cb W242A, G1 – Cb S238A, F7 – Cb E221A/W225A, D11 – Ca
923
A172G/D174A and C3 – Cb S170A/G171A by flow cytometry.
924
925
926
.CC-BY 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.07.479368doi: bioRxiv preprint
43
927
Figure 5 – figure supplement 1: Comparison of surface charges of TCR-CD3 interface
928
residues between human and mouse species. Positive charges are indicated in red, negative
929
charges in blue. Green residues indicate identical residues. Overall, there is better conservation
930
in the TCR interface between human and mouse than CD3de and CD3ge.
931
932
933
.CC-BY 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.07.479368doi: bioRxiv preprint
44
934
Figure 5 – figure supplement 2: Overlay of crosslink-guided and cryoEM TCR-CD3
935
structures. A) Overlay of TCR-CD3de crosslink (orange) and cryoEM (magenta) structures, B)
936
Overlay of TCR-CD3ge crosslink (red) and cryoEM (blue) structures with TCR indicated in surface
937
(grey) representation. C) The CD3de (orange) and CD3ge (red) interface residues located on the
938
TCR (grey) in the crosslink model. D) The CD3de (magenta) and CD3ge (blue) interface residues
939
located on the TCR (grey) in the cryo-EM structure.
940
941
942
943
944
CD3 crosslink model
CD3 cryo-EM
CD3 crosslink model
CD3 cryo-EM
C FG loop
CD3 crosslink model
CD3 crosslink model
CD3 cryo-EM
CD3 cryo-EM
AB
A
C D
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The copyright holder for this preprintthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.07.479368doi: bioRxiv preprint
45
Supplementary Tables
945
Region
Sequence
Interacts with
Methodology
Reference
Cb CC’
loop
164NGKEVHSG171
CD3ge
Mutagenesis,
EM structure
(Dong et
al., 2019;
Kuhns and
Davis,
2007)
Ca DE
loop
170MKAMDS175
CD3de
Mutagenesis,
EM structure
(Dong et
al., 2019;
Kuhns and
Davis,
2007)
Ca AB
loop
132DPRSQDS138
Conformational
change upon
antigen ligation
NMR,
fluorescence
and
mutagenesis
(Beddoe et
al., 2009;
Rangarajan
et al.,
2018)
Cb FG
loop
219LSEEDKWPEGSPK231
CD3ge
NMR,
antibody
binding and
docking
(Kim et al.,
2009;
Natarajan
et al.,
2016)
.CC-BY 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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46
Cb
Helix 3
136EIANKQK142
CD3ge
NMR,
mutagenesis
and docking
(He et al.,
2015; Kim
et al.,
2010;
Natarajan
et al.,
2016;
Natarajan
et al.,
2017)
Cb
Helix
4-F
strand
204HNPRNHFRC212
CD3ge
NMR, EM
structure,
mutagenesis
and docking
(Dong et
al., 2019;
He et al.,
2015; Kim
et al.,
2010;
Natarajan
et al.,
2016)
Cb G
strand
232PVTQNISAEAWGRADC247
CD3e
EM structure
(Dong et
al., 2019)
946
.CC-BY 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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47
Figure 2 - table supplement 1: The regions of the TCR from which specific residues were
947
tested for crosslinking. The TCR region, sequence, speculated interacting CD3 subunits,
948
methodology used and reference are tabled.
949
950
Region
Sequence
Interacts with
Methodology
Reference
CD3d A
strand
3QVT5
TCRa
EM structure
(Dong et
al., 2019)
CD3d AB
loop
7YEDK10
TCRa
EM structure
(Dong et
al., 2019)
CD3d BC
loop
16NTS18
CD3d CD
loop
25TVE27
CD3d E
strand
35TLN37
TCRa
EM structure
(Dong et
al., 2019)
CD3d EF
loop
40KGVLD44
TCRa
EM structure
(Dong et
al., 2019)
CD3g AB
loop
13GSRGDGSV20
TCRb
EM structure
(Dong et
al., 2019)
CD3g CD
loop
36DG37
CD3g DE
loop
45ATKN48
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
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48
CD3g EF
loop
55NAKDP59
CD3g FG
loop
67GAKET71
Figure 3 - table supplement 1: CD3 regions used for testing. The CD3 region, sequence,
951
speculated interacting TCR subunit, methodology used and reference are tabled.
952
953
954
955
Subunit
Species in vitro
PDB
PDB Template-
Species
TCR (a and b) V & C
domains
Mus musculus
3QJF and 1TCR
Mus musculus and
Homo sapiens
chimera; Mus
musculus
TCR (a and b) TMs
Mus musculus
6JXR
Homo sapiens
CD3ge
Mus musculus
1JBJ
Mus musculus
CD3de
Mus musculus
1XIW and 3R08
Homo sapiens and
Mus musculus
956
Figure 5 - table supplement 1: PDB structures used for the docking crosslink-guided structure.
957
958
959
CL Subunit
CL Residue
Subunit binds to
Distance (Å)
TCR b
S238
CD3 e
7
TCR b
E240
CD3 e
3
TCR b
W242
CD3 e
3
TCR b
F202
CD3 d
2
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(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.07.479368doi: bioRxiv preprint
49
TCR b
S170
CD3 d
3
TCR b
G171
CD3 d
3
TCR b
R207
CD3 e
6.5
TCR b
E221
CD3 g
6
TCR b
W225
CD3 g
7
TCR a
A172
CD3 d
5
TCR a
D174
CD3 d
7
CD3 g
S14
TCR b
5
CD3 g
R15
TCR b
8
CD3 d
T5
TCR b
1
CD3 d
T35
TCR a
2
CD3 d
K40
TCR a
5
960
Figure 5 – table supplement 2: Crosslinking residues to subunit distances
961
962
963
Figure 2 – data supplement 1: Mass spectrometry results indicating the proteins identified in
964
bE221 crosslinking band. The TCR and CD3 subunits are highlighted in yellow.
965
966
Figure 1 – source data 1: Western blot image and files corresponding to Figure 1F.
967
968
Figure 2 – source data 1: Western blot images and files corresponding to Figure 2
969
(crosslinkers attached to the TCR regions).
970
971
Figure 3 – source data 1: Western blot images and files corresponding to Figure 3
972
(crosslinkers attached to the CD3 regions).
973
974
Figure 4 – source data 1: ELISA IL-2 production data for mutant T cell hybridoma and area
975
under the curve (AUC) normalization to the wild type.
976
.CC-BY 4.0 International licenseavailable under a
(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made
The copyright holder for this preprintthis version posted February 11, 2022. ; https://doi.org/10.1101/2022.02.07.479368doi: bioRxiv preprint
