cells Article Molecular Dynamics Simulations Reveal Canonical Conformations in Different pMHC/TCR Interactions Josephine Alba 1,* , Lorenzo Di Rienzo 2,3, Edoardo Milanetti 2,3, Oreste Acuto 4 and Marco D’Abramo 1,* 1 Department of Chemistry, University of Rome Sapienza, P.le A.Moro 5-00185 Rome, Italy 2 Department of Physics, University of Rome Sapienza, 5-00185 Rome Italy; [email protected] (L.D.R.); [email protected] (E.M.) 3 Center for Life Nano Science@Sapienza, Istituto Italiano di Tecnologia, 000161 Rome, Italy 4 Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK; [email protected] * Correspondence: [email protected] (J.A.); [email protected] (M.D.); Tel.: +39-0649-693263 (J.A.) Received: 25 February 2020; Accepted: 8 April 2020; Published: 10 April 2020 Abstract: The major defense system against microbial pathogens in vertebrates is the adaptive immune response and represents an effective mechanism in cancer surveillance. T cells represent an essential component of this complex system. They can recognize myriads of antigens as short peptides (p) originated from the intracellular degradation of foreign proteins presented by major histocompatibility complex (MHC) proteins. The clonotypic T-cell antigen receptor (TCR) is specialized in recognizing pMHC and triggering T cells immune response. It is still unclear how TCR engagement to pMHC is translated into the intracellular signal that initiates T-cell immune response. Some work has suggested the possibility that pMHC binding induces in the TCR conformational changes transmitted to its companion CD3 subunits that govern signaling. The conformational changes would promote phosphorylation of the CD3 complex ζ chain that initiates signal propagation intracellularly. Here, we used all-atom molecular dynamics simulations (MDs) of 500 ns to analyze the conformational behavior of three TCRs (1G4, ILA1 and ILA1α1β1) interacting with the same MHC class I (HLA-A*02:01) bound to different peptides, and modelled in the presence of a lipid bilayer. Our data suggest a correlation between the conformations explored by the β-chain constant regions and the T-cell response experimentally determined. In particular, independently of the TCR type involved in the interaction, the TCR activation seems to be linked to a specific zone of the conformational space explored by the β-chain constant region. Moreover, TCR ligation restricts the conformational space the MHC class I groove. Keywords: molecular dynamics; biophysics; protein-membrane; T cell antigen receptor 1. Introduction Cytotoxic T cells recognize and kill virus-infected cells and cancer cells upon T-cell antigen receptor (TCR) interaction with major histocompatibility complex (MHC) class I proteins presenting viral and tumor antigens, respectively [1–3]. MHCs class I are composed of one α-chains, which form the binding site for a nine/ten residue-long peptide, and of a non-covalent bound β2 microglobulin (Figure1). To recognize pMHC, T cells use a clonally distributed αβ dimer with Ig-like variable domains, Vα and Vβ. Together, Vα and Vβ form the pMHC binding site composed of six loops homologous to antibody complementarity determining regions (CDRs) 1, 2 and 3 [4,5]. CDR1 and CDR2 have limited variability, while CDR3s are hypervariable. VαVβ orientates diagonally relative to Cells 2020, 9, 942; doi:10.3390/cells9040942 www.mdpi.com/journal/cells Cells 2020, 9, 942 2 of 15 the long axis of the peptide-binding groove [4,5] in such a way that the CDR3s make contacts mostly with the peptide whereas the CDR1s and CDR2s contact mainly the MHC. Vα and Vβ connect to Ig-like Cellsconstant 2020, 9, 942 domains (Cα and Cβ) that are linked to transmembrane regions (TMRs) via a connecting stalk2 of 15 (CP). pMHC binding is signaled intracellularly by four non-covalently associated subunits (γ, δ, " and intoζ), three called dimers CD3, that (γε, are δε, organized ζζ) [6]. δε into and three γε dimers ectodomains (γε, δε, ζζinterface)[6]. δε witandh γεCαectodomains and Cβ ectodomains, interface respectively,with Cα and while Cβ ectodomains,ζζ has a very respectively, short ectodomain. while ζζ Allhas CD3 a very subunits short ectodomain.possess short All and CD3 unstructured subunits intracellularpossess short tails and that unstructured are phosphorylated intracellular soon tails after that pMHC are phosphorylated binding at the soon immunoreceptor after pMHC binding tyrosine at - basedthe activation immunoreceptor motifs (ITAMs). tyrosine-based Early activationbiochemical motifs work (ITAMs). [7] suggested Early TCR biochemical-CD3 allosteric work [ 7regulation] suggested and fluorescenceTCR-CD3- allostericbased studies regulation has suggested and fluorescence-based a movement studiesof a Cα has loop suggested upon pMHC a movement binding. of a Moreover, Cα loop recentupon NMR pMHC investigations binding. Moreover, of soluble recent αβ dimer NMR ectodomain investigations alone of has soluble suggestedαβ dimer that ectodomainTCR-CD3 signaling alone is governedhas suggested by allosteric that TCR-CD3 regulation signaling upon ispMHC governed binding by allosteric [8,9]. regulation upon pMHC binding [8,9]. FigureFigure 1. 1.ModelModel of of one one of thethe pMHC-TCR pMHC-TCR simulated simulated complexes. complexes. The different The different regions regions are labelled: are Vlabelled:α/Vβ Vα/Vβand C αand/Cβ referCα/Cβ to the refer TCR Variableto the TCR regions Variable (alpha/beta) regions and TCR (alpha/beta) Constant regions and (alphaTCR /beta),Constant respectively. regions (alpha/beta), respectively. Combining experimental and theoretical studies, we recently found that the bound peptide can affCombiningect the conformation experimental of the MHCand theoretical I binding groove, studies, suggesting we recently a diff founderent presentation that the bound of the peptide antigens, can affectwhich the seems conformation to be related of tothe di ffMHCerent CTLsI binding responses groove, [10]. suggesting However, in a that different study wepresentation did not provide of the antigens,a modelling which of seems the pMHC to be interacting related to withdifferent the TCR. CTLs To responses better characterize [10]. However, such a complexin that study molecular we did notnetwork, provide herea mode welling present of the the pMHC first exhaustive interacting computational with the TCR. study To better of five characterize pMHC-TCR such complexes, a complex modelled in a heterogeneous lipid bilayer (see Video S1 in SI, and Figure1). molecular network, here we present the first exhaustive computational study of five pMHC-TCR complexes,2. Materials model andled Methods in a heterogeneous lipid bilayer (see Video S1 in SI, and Figure 1). 2. Materials2.1. Complexes and Modelling Methods Using the crystallographic structures (PDB ID: 4MNQ; PDB ID: 2BNR) the transmembrane 2.1. Complexes Modelling alpha helices were built by means of the Modeller Software version 9.19 (Accelerys, San Diego, CA, USA)Using [11 ],the following crystallographic the amino structures acid sequences (PDB provided ID: 4MNQ; by thePDB Uniprot ID: 2BNR) database the transmembrane [12] (Uniprot entries alpha helicesP01848 were and built P01850 by for means the TCR of αtheand Modellerβ chains respectively;Software version Q9MY51 9.19 for (Accelerys, the α chain San of the Diego, HLA-A* CA, USA02:01).)[11], The following heterogeneous the amino lipid acid bilayer sequences was built provided by means by ofthe the Uniprot CHARMM-GUI database [ Membrane12] (Uniprot Builder entries P01848web softwareand P01850 (Harvard, for the Cambridge, TCR α and MA,β chains USA) respectively; [13], with a composition Q9MY51 for of: the 1-palmitoyl-2-oleoyl-sn- α chain of the HLA-A* 02:0glycero-3-phosphocholine1). The heterogeneous lipid (POPC) bilayer at 90%, was phosphatidylinositolbuilt by means of the (4,5)-bisphosphate CHARMM-GUI Membrane (PIP2) at 7% Builder and web1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine software (Harvard, Cambridge, MA, USA) [13 (POPS)], with a at composition 3%. Such a lipid of: composition1-palmitoyl- is2- basedoleoyl on-sn- glycerothe work-3-phosphocholine of Chavent et al. [(POPC)14] and Zechat 90%, et al. phosphatidylinositol [15] to approximate the (4,5) lipid-bisphosphate composition of(PIP2) a mammalian at 7% and cell membrane [16]. These studies reported an essential role of the PIP2 in receptor activation and 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS) at 3%. Such a lipid composition is based TCR triggering. Note that recent findings reported that cholesterol can inhibit T cell activation and on the work of Chavent et al. [14] and Zech et al. [15] to approximate the lipid composition of a mammalian cell membrane [16]. These studies reported an essential role of the PIP2 in receptor activation and TCR triggering. Note that recent findings reported that cholesterol can inhibit T cell activation and thus, this lipid was not included in our membrane model [17,18]. The modelled complexes were then manually inserted in the membrane using the VMD Software version 1.9.3 [19] (University of Ilinois, Champaign, IL. USA). Cells 2020, 9, 942 3 of 15 thus, this lipid was not included in our membrane model [17,18]. The modelled complexes were then manually inserted in the membrane using the VMD Software version 1.9.3 [19] (University of Ilinois, Champaign, IL, USA). 2.2. MD Simulations The systems were solvated with the TIP3P water model [20] and neutralized with Na+ and Cl- ions at physiological concentration (0.15 M). The exceeding solvent was manually removed, excluding the water molecules within a range of 2 Å from lipids. An energy minimization step was performed using the steepest descent algorithm without position restraints. After the minimization, a series of equilibration steps were performed: (1) a NPT equilibration of 40 ps was run to allow the packing of the lipids around the protein, using an integration time step of 0.2 fs; then the NPT equilibration was extended until 2 ns. (2) an NVT equilibration of 40 ps was performed and then extended until 4 ns, increasing the time step at 1 fs.