
==== Front
Biochem Biophys Rep
Biochem Biophys Rep
Biochemistry and Biophysics Reports
2405-5808
Elsevier

S2405-5808(24)00177-8
10.1016/j.bbrep.2024.101813
101813
Short Communication
The constant domain of CRTAM is essential for high-affinity interaction with Nectin-like 2
Barragan-Galvez Juan Carlos ab
Hernandez-Flores Araceli c
Lopez-Ortega Orestes d
Rodriguez-Alvarez Adriana A. e
Maravillas-Montero Jose Luis f
Ortiz-Navarrete Vianney vortiz@cinvestav.mx
a⁎
a Department of Molecular Biomedicine, Center for Research and Advanced Studies (CINVESTAV), Mexico City, Mexico
b Departamento de Farmacia, División de Ciencias Naturales y Exactas, Universidad de Guanajuato, Guanajuato, 36200, Mexico
c Universidad de la Sierra Sur, Miahuatlán de Porfirio Díaz, Oaxaca, Mexico
d Université Paris Cité, INSERM UMR-S1151, CNRS UMR-S8253, Institut Necker Enfants Malades, 75015, Paris, France
e Boston University School of Medicine, 72 E Concord St, Boston, MA, 02118, United States
f Research Support Network, Universidad Nacional Autónoma de México and Instituto Nacional de Ciencias Médicas y Nutrición “Salvador Zubirán”, Mexico City, Mexico
⁎ Corresponding author. Av. IPN 2508, Col. San Pedro Zacatenco, Gustavo A Madero, 07360, Mexico City, Department of Molecular Biomedicine, CINVESTAV, Mexico. vortiz@cinvestav.mx
25 8 2024
9 2024
25 8 2024
39 1018133 6 2024
16 8 2024
19 8 2024
© 2024 The Authors. Published by Elsevier B.V.
2024

https://creativecommons.org/licenses/by-nc/4.0/ This is an open access article under the CC BY-NC license (http://creativecommons.org/licenses/by-nc/4.0/).
CRTAM (Class-I MHC restricted T cell-associated molecule) is a member of the Nectin-like family, composed of two extracellular domains, one constant domain (IgC) and another variable domain (IgV), expressed in activated CD8 T cells, epithelial cells, natural killer (NK) cells, and in a subpopulation of CD4 T cells. CRTAM recognizes the ligand Nectin-like 2 (Necl2) through the IgV domain. However, the role of the IgC domain during this ligand recognition has yet to be understood. In this study, we show the purification of soluble-folded Ig domains of CRTAM, and we demonstrate that the IgC domain forms a homodimer in solution via hydrophobic interactions. By surface plasmon resonance (SPR) analysis, we also demonstrate that CRTAM binds to Necl2 with an affinity of 2.16 nM. In conclusion, CRTAM's IgC is essential for a high-affinity interaction with Necl-2.

Highlights

• The constant domain of CRTAM forms a dimer in solution.

• The constant domain of CRTAM interacts with the ligand Necl-2 with low affinity.

• The variable and constant domains of CRTAM are required for high-affinity interaction with Necl-2.

Keywords

CRTAM
Nectin-like 2
Constant domain
CADM1
Affinity
Dimer
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pmc1 Introduction

Cell–cell adhesion is essential for the ontogenesis and maintenance of tissues and organs [1]. Cell adhesion molecules (CAM) mediate cell-cell adhesion through homophilic or heterophilic interactions. Also, the binding of CAMs, among them, involves cis or trans interactions. There are four families of CAMs: cadherins, selectins, integrins, and the immunoglobulin superfamily (IgSF) [1,2].

IgSF established Ca + -independent homophilic and heterophilic interactions through its extracellular domains similar to immunoglobulin that result in effector and cell signaling functions [3,4]. Nectin and Nectin-like molecules (Necls) belong to the IgSF family [5,6]. The Nectin family has four members: nectin-1, -2, -3, and -4, while the Necls have five: Necl-1, -2, -3, -4, and -5 [1,5,7]. Each member of the family forms homophilic and heterophilic interactions that contribute to cell-cell adhesion. During cell-cell adhesion, the IgV domains establish interactions in trans with the opposite molecule expressed in the front cell, while constant domains form interactions in cis (with the lateral molecules) on the cell surface [5,7,8].

CRTAM is another member of the Nectin-like family and is expressed after activation in CD8 + T cells, natural killer T cells (NKT), NK cells and a subpopulation of CD4 + T cells [[9], [10], [11], [12]]. As an adhesion molecule, CRTAM is induced and expressed on the cell surface in activated CD8+ T cells, establishing a heterotypic trans-interaction with Necl2, which is expressed in CD8+ dendritic cells of lymph nodes. The interaction between CRTAM and Nectin-like2 participates in CD8+T cell and NKT cell activation, IFNγ, IL-17, and IL-22 production. It also improves the cytotoxic activity of NK cells against tumors [9,10,12].

[13,14]. For this in vivo interaction, the extracellular domains of both molecules are involved [9,10]. CRTAM has two extracellular immunoglobulin-like domains: IgV (V-set) and IgC (C1-set); exhibiting the class I postsynaptic density (PSD95), large disc (Dlg), and zonula occludens (ZO-1) (PDZ) binding motif ESIV at its carboxyl-terminal end. The IgV domain is responsible for interacting with its ligand Necl2 [10,11,15], but the role of the IgC domain of CRTAM is unknown.

The dissociation constant between the IgV of CRTAM and the IgV domain of Necl2 was determined to be 12.5 μM [15]. This affinity is much lower compared to other nectin molecules, for example 2 nM for nectin 1 and nectin 3 and 360 nM for nectin 2 and nectin 3 [5,7,8,16].

We postulate that the low affinity of CRTAM for Necl2 could increase if the IgC domain is included for binding determination. Therefore, in this study, we describe how the IgC of CRTAM forms a dimer in solution. Additionally, the affinity between CRTAM and Necl2 increased to 2.16 nM when both extracellular domains were included in the SPR analysis.

2 Materials and method

2.1 Reagents

Reagents Tris-Hydrocloride (HCl; cat. 648317), sodium dodecyl sulphate (SDS; cat. 817034), NaCl (cat. 71836), glycine (cat 104201), coomasie blue R-250 (cat. 112553), Bovine serum albumin (BSA, cat A3294) and 2x Laemmli sample buffer (cat S3401) were acquired from the Merck company.

2.2 Cloning constructions of CRTAM and Necl2

Design constructions are schematized in Fig. 1: CRTAMEC, CRTAMIgV-Stalk, CRTAMIgC-Stalk, and Necl2EC. These constructions were cloned into a pET28a (+) expression vector (Novagen) as previously described [17]. The transmembrane and intracellular regions were removed for all cloning, whereas the CRTAMIgV-stalk construction does not contain a linker between the Variable domain (IgV) and the stalk region, only the IgC domain was removed (Fig. 1).Fig. 1 Schematic representation of the cloning constructions. a) scheme of CRTAM constructions and b) scheme of the Necl2EC construction cloned into pET28a (+) vector. The theoretical molecular weight was calculated by amino acid sequence and is denoted in kDa in each construction. EC, extracellular region; IgV, variable Ig-like domain; IgC, constant Ig-like domain; TM: transmembrane region; ESIV, intracellular motif of CRTAM; EYFI, intracellular motif of Necl2. Empty boxes with dashed line borders mean that regions and domains were removed. No linker was added between IgV domain and stalk region for the CRTAMIgV-stalk construction.

Fig. 1

2.3 Protein expression and purification

The expression and purification of recombinant proteins (schematized in Fig. 1) from bacterial lysates were performed as previously described [17] purified by immobilized metal affinity chromatography (IMAC). The purified proteins were subjected to a chemical refolding as described by Zhang [18]. Folded proteins were purified by gel filtration chromatography using a Sephacryl S-200 HR (GE) column with a buffer composed of 30 mM Tris and 100 mM NaCl at pH 8.0.

2.4 Gel filtration assay

The precollected fractions by S-200 (HR) with the refolded CRTAM and Necl2 proteins were concentrated using an Amicon ultra-4 centrifugal filter unit with a 3K molecular weight cutoff membrane. The proteins were then loaded onto a high resolution Superdex 75 10/300 GL column (Cytiva) at a flow rate of 0.5 ml/min in a buffer composed of 30 mM Tris-HCl, 100 mM NaCl at pH 8.0, using an AKTA FPLC chromatography system (GE). The main fractions of each elution peak were dissolved in 2x Laemmli sample buffer (in denaturing and reducing conditions) and boiled for 10 min. The samples were loaded in 10 % gels and resolved on sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE), using running buffer with 25 mM Tris-HCl, 1.92 M glycine and 1 % (w/v) of SDS, at pH 8.3) at 110 V/90 min. The gels were stained with 0.2 % coomasie blue R-250 solution. A Bio-Rad gel filtration standard (#1511901) was used to equilibrate the Superdex 75 10/300 column (Supplementary Fig. 1).

2.5 Surface plasmon resonance analysis

NECL2EC was captured on a Biacore CM5 sensor chip (GE Healthcare) with a final immobilisation density of ∼1600 resonance units (RU). All kinetic experiments were carried out at 25 °C with a flow rate of 30 μl/min. Two-fold serial dilutions of CRTAMEC, CRTAMIgV-Stalk and CRTAMIgC-Stalk were injected into HBS-P+ buffer (GE Healthcare) was injected over the CM5 chip. Sensograms were fit with BIAcore T200 software using 1: 1 Langmuir binding (GE Healthcare). Binding analyses were performed previously to kinetic experiments, using 1 μM of BSA (as irrelevant protein) and 1 μM of the recombinant proteins injected onto the CM5 chip with immobilized NECL2EC under the same conditions described above (Supplementary Fig. 2).

2.6 Structural protein modelling

Homology modelling for the constant domain of CRTAM was performed using the I-TASSER server [19], entering the primary sequence of 114–218 residues of human CRTAM (number accession UniProt: O95727). The protein model was submitted to the CLUS-PRO server [20] for molecular docking (dimer prediction), and the Ramachandran plot and the QMEANDisCo Scort were generated for quality assessment using the ProCheck and SWISS-MODEL server, respectively [21,22]. The 3D images of the model were obtained by PyMOL [23].

3 Results

3.1 The constant domain of CRTAM forms a dimer in solution

We previously produced the IgV domain (CRTAMIgV-stalk), the IgC domain (CRTAMIgC-stalk), and the extracellular region of CRTAM (CRTAMEC) as recombinant proteins (Fig. 1) [17]. However, we noticed that these recombinant proteins formed in aggregates with molecular weights greater than 2000 kDa (Supplementary Fig. 3). To overcome this inconvenience, we refolded the proteins and then purified them by gel filtration. As shown in Fig. 2a, CRTAMEC eluted as a single peak at 7.7 ml with a molecular weight slightly higher than the BSA marker (∼66 kDa). CRTAMIgC-stalk eluted at 9.12 ml with a molecular weight between BSA (66 kDa) and OVA (44 kDa). CRTAMIgV-stalk eluted with two peaks at 8.94 ml and 10.20 ml. The result suggests that, in solution, there are two protein populations for CRTAMIgV-stalk. The first peak might contain the dimer, and the second might correspond to the monomer. However, these dimers and monomers peaks were not fully separated during chromatography. SDS-PAGE shows that each eluted peak contains monomeric proteins: ∼37 kDa for CRTAMEC, ∼26 kDa for CRTAMIgC-stalk, and ∼27 kDa for CRTAMIgV-stalk. It is important to note that in SDS-PAGE, the two peaks for CRTAMIgV-stalk were resolved as monomers. (Fig. 2b). These results demonstrate that the extracellular region of CRTAM forms a dimer in solution. In contrast, the IgV domain was found as a dimer and monomer, as others found [15]. As previously reported, we also identified that Necl2EC forms a dimer in solution (Supplementary Fig. 4) [24].Fig. 2 The oligomerization of CRTAM. a) Elution profile (280-nm absorbance curves) by gel-filtration chromatography of CRTAMEC (black line), CRTAMIgC-stalk (dark grey), and CRTAMIgV-Stalk (light grey) from the preequilibrated Superdex 75 (10/300) column. b) SDS-PAGE (10 % under reducing conditions) analysis shows the profile analysis of each peak elution from a. P1(peak 1) and P2 (peak 2) of the CRTAMIgV-stalk profile elution are denoted on the gel. c) 3D homology modelling of the IgC dimer of CRTAM. A hydrophobic site (shown in red) in the dimer is marked by a grey circle (left) and is zoomed (right), where the residues involved are denoted by a one-letter code. The hydrophobicity (red) and hydrophilic (white) scales were calculated by PyMOL. The yellow region shows a disulphide bond. Mw, protein markers; BSA, bovine serum albumin marker; OVA, ovalbumin (chicken) marker; Myo, myoglobin (horse) marker; V–B12, vitamin B-12. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

3.2 Homology modeling of CRTAM IgC

The homology modelling method was performed to predict the 3D structure of a homodimer of the IgC domain of CRTAM using the I-TASSER server to build the model. Molecular modelling showed a classical beta-sandwich topology of the immunoglobulin domain composed of six antiparallel beta sheets bonded by a disulphide bridge (Fig. 2c, left panel). This dimer prediction model displayed 71.9 % residues in the most favored region of the Ramachandran analysis (Supplementary Fig. 5a) and a global QMEAN score of 0.68 (scale from 0 to 1.0; Supplementary Fig. 5b), showing a good prediction model. The homodimer model shows an interface site of interaction between monomers with a visible cleft that is predominantly composed of a hydrophobic region (Fig. 2c). Hydrophobic and neutral residues, such as Phe170, Leu167, Thr166, and V162 of the first monomer and the residues Phe119, Ile122, Leu123, Leu208, Val209, Ala210, and Phe212 residues of the second monomer, form the main cleft with van der Waals interaction (Fig. 2c, right panel). This model suggests that the homodimer of the constant domain of CRTAM is bound mainly by hydrophobic interactions.

3.3 The constant domain is involved in the high affinity of the CRTAM-Necl2 interaction

SPR technology was used to evaluate the participation of the constant domain of CRTAM in the interaction with Necl2. The refolded NECL2EC was immobilized on a CM5 chip and CRTAMEC, CRTAMIgV-stalk or CRTAMIgC-stalk, was injected with a gradient of two-fold serial dilutions. The kinetic constant (KD)was calculated as 2.16 nM, with Kon = 6.04x104 M−1s−1 and a Koff = 1.30x10−4s−1 (Fig. 3a). This high affinity was decreased without the constant domain with a KD = 971 nM, Kon = 5.76x102 M−1s−1, and Koff = 5.6x10−4s−1 (Fig. 3b). A weaker affinity (KD = 479 nM) and faster binding of Kon = 9.16x104 M−1s−1 Koff = 9.90x10−4s−1 was observed between CRTAMIgC-Stalk and Necl2EC (Fig. 3c). No response units were detected when 1 μM of BSA and HBS-P+ buffer was injected into the CM5 chip (Supplementary Fig. 2). These results show that the IgC domain of CRTAM is essential for the high affinity interaction with Necl2.Fig. 3 High affinity in the CRTAM-Necl2 interaction. Binding kinetics of the extracellular region (CRTAMEC) (a), variable domain (CRTAMIgV-stalk) (b) and constant domain (CRTAMIgC-stalk) (c) of CRTAM to extracellular region of Necl2 (Necl2EC). The kinetic parameters are shown in the upper part of each sensorgram and was determined by SPR analysis described in the materials and methods. The fit curves are shown with dotted lines. The concentration gradient of the proteins that were injected on the CM5 chip is denoted in the right part of the sensorgram, from highest to lowest concentration according to the response curve. The 0.0 μM concentration represents only to HBS-P+ buffer that was injected on the CM5 chip.

Fig. 3

4 Discussion

In this study, we described that the recombinant CRTAMEC and CRTAMIgC-stalk, after refolding, were purified by gel chromatography within their expected dimeric molecular weight. We observe the dimer of CRTAMEC or CRTAMIgC-Stalk. There is a free sulfhydryl group of Cys177, a conserved residue in CRTAM of different species, but not in other nectin-like molecules (Supplementary Figs. 5 and 6). Therefore, a disulphide bond could be formed between two CRTAM molecules, as previously found on the surface of activated CD8 T cells [17]. However, in activated human CD8+ cells, only a tiny fraction of surface-expressed CRTAM is detected as a dimer [11]. On a T cell hybridoma transfected with CRTAM gen, dimers were detected if the cells were treated with the cross-linker Bis (sulfosuccinimidyl) surfactant [13]. These results suggest that weak interactions are involved in dimer formation rather than a disulphide bond. Therefore, the 3D model of the IgC domain suggests that a hydrophobic region with aromatic residues such as phenylalanine, proline, and tyrosine located at the interface participates in the stabilization of the cis interaction of the CRTAM molecules, similar to the crystal structure of the IgV domain of CRTAM reported, where hydrophobic residues located on the antiparallel beta sheet participate in the formation of the homodimer [25]. This kind of interaction is possible because the Ig-like domains of CRTAM line up along their longitudinal axis as other Ig-like molecules. However, we hypothesize about the interaction for the formation of CRTAM dimers. The answer to this point will be solved by analyzing the whole molecule by X-ray diffraction or other techniques that determine the protein structure. On the other hand, CRTAMIgV-stalk dimers and monomers were recovered, implying the contribution of the IgV domain to the cis-interaction, through hydrophobic residues as reported [15,25]. The aggregation of immunoglobulin proteins expressed in a bacterial system and refolded post expression is common due to impaired disulphide bonds, through intramolecular scrambling and/or intermolecular crosslinking [26], but these aggregates were excluded by molecular size in gel filtration chromatography.

We found that the affinity of the CRTAMEC-Necl2EC interaction was 2.16 nM. This high affinity drops to 0.97 μM when the IgC of CRTAM is not present and the complex dissociates rapidly (Fig. 3B). These results agree with the affinities of the heterophilic interaction between Nectin members and Nectin-like families: they are in the nanomolar range while the homophilic ones are in micromolar values [5,7,8]. The result also indicates the participation of the IgC domains during the interaction with the constant domains of Necl2, but further experiments are needed to define if the interaction occurs with C1 or C2.

Other techniques to obtain the 3D structure of the complex Necl2-CRTAM could help to better understand how the constant domains of both molecules interact during cell adhesion. For example, nuclear magnetic resonance (NMR) would help elucidate the homodimer structure of the IgC domain in its free form in solution, as reported with the membrane-proximal constant domain of mouse Nectin-1 [25,26]. Other techniques, such as X-ray crystallography, cryogenic electron microscopy (Cryo-EM), small-angle X-ray or neutron scattering (SAX/SANS), could be utilized to elucidate whether CRTAM-Necl2 forms chain oligomerization in solution, as has been demonstrated in the structures of Nectin-like 5 (also called PVR; Poliovirus receptor), Nectin-1, and Nectin-3 [7,[27], [28], [29], [30]].

Takeshi Ito et al. [31] reported the interaction of CRTAM-Necl2 with a value of 3.3 nM (Kon = 3.1 × 104 M−1s−1, Koff = 1.1x10−4s−1), which is quite close to our results (Kd = 2.16 nM, Kon = 6.04x104 M−1S−1, Koff = 1.30x10−4s−1). They expressed CRTAM and Necl2 as chimeric molecules of Fc in mammalian cells, which could cause a background by dimerization of Fc, while our kinetics involved non-chimerized recombinant proteins [31]. However, the similarity of these results confirms that the interaction between CRTAM and Necl2 is very stable and dissociates very slowly. Takeshi Ito et al. speculate on the possible participation of N-glycosylation in the interaction of those molecules, but for our analysis we used recombinant proteins produced in E. coli. Thus, it is not the glycosylation, but the IgC domain that is essential to establish a high-affinity interaction of those molecules. It should also be mentioned that several binding affinities of nectin and nectin-like molecules have been calculated by SPR, using recombinant proteins expressed in the bacterial system, for which glycosylations, although important, are not essential for high affinity adhesion, as observed in this work and also as reported for the variable domain of CRTAM by Zhang (2013) [7,15,[32], [33], [34], [35]].

The results of this work suggest that before cell adhesion, CRTAM and Necl2 are expressed as homodimers on the cell surface of a T-cell and an antigen-presenting cell, respectively (Fig. 4a). Subsequently, when both cells interact, the homodimer of CRTAM is switched to a heterodimer with Necl2 where the constant domain of CRTAM probably binds by proximity to the first constant domain of Necl2 (Fig. 4b). Thus, these surface interactions occur first, followed by the recruitment of other intracellular proteins, such as scaffold proteins involved in polarity, such as Scrib [14]. This would explain how the high-affinity CRTAM-Necl2 interaction helps prolong cellular activation together with other costimulation molecules [8,13,14].Fig. 4 Hypothetical proposed model of CRTAM dimer formation and binding to Necl2. a) CRTAM and Necl2 dimers expressed on the surface of T cell and APC cells, respectively. b) CRTAM binds strongly to Necl2 to form a heterodimer through extracellular domains, where the IgC domain of CRTAM could bind with the de constant domain of Necl2. Intracellular motifs for protein recruitment are not necessary for this surface interaction.

Fig. 4

Funding

The present work was supported by the Cinvestav to Vianney Ortiz-Navarrete. Barragan-Gálvez Juan Carlos is a postdoctoral researcher and was supported by 10.13039/501100003141 Consejo Nacional de Humanidades, Ciencias y Tecnologías (CONAHCYT) scholarship CVU 489981 .

Data availability

The data supporting this study's findings are available from the corresponding author V. O–N.

CRediT authorship contribution statement

Juan Carlos Barragan-Galvez: Writing – original draft, Validation, Methodology, Investigation, Formal analysis. Araceli Hernandez-Flores: Methodology, Investigation, Formal analysis. Orestes Lopez-Ortega: Validation, Methodology. Adriana A. Rodriguez-Alvarez: Methodology, Investigation, Formal analysis. Jose Luis Maravillas-Montero: Writing – review & editing, Writing – original draft, Investigation, Formal analysis. Vianney Ortiz-Navarrete: Writing – review & editing, Writing – original draft, Validation, Supervision, Formal analysis, Conceptualization.

Declaration of competing interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.

Appendix A Supplementary data

The following is the Supplementary data to this article:Multimedia component 1

Multimedia component 1

Acknowledgements

Benjamin Ortiz-Lopez for his support for cell culture of transfected cells.

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.bbrep.2024.101813.
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