Struttura della cellula NK umana NKR

a Dimerization interface of human NKR-P1. Subunits of human NKR-P1 are shown as Cα-trace (blue and cyan), and the dimer contact residues are shown as sticks with carbon atoms colored in light blue (blue subunit) and orange (cyan subunit); for clarity, only the residues of the blue subunit are labeled. The first GlcNAc unit N-linked to Asn116 and the carbohydrate chain N-linked to Asn169, observable in the NKR-P1_glyco structure, are shown with carbon atoms colored yellow and green, respectively. b Top view of the dimerization interface. The NKR-P1 subunits surfaces are colored blue and cyan. The GlcNAc units bound to Asn116 are shown as sticks with carbon atoms in yellow. Contact residues between the GlcNAc bound to chain A, and the chain B, are shown in yellow, whereas contact residues between the GlcNAc bound to chain B, and the chain A, are shown in purple. Hydrogen bonds are shown as green-dashed lines with a detailed view on the right-hand side. c Mixed glycosylation states at the dimer interface in the NKR-P1_deglyco structure. The GlcNAc unit N-linked to Asn157 of chain A is modeled with an occupancy of 0.5, while the second GlcNAc unit present at Asn116 of chain B is not modeled. Contours of 2mFo-DFc (2.8σ, cyan) and mFo-DFc (1σ, green) electron density maps are shown. d Small hydrophobic core in the central part of the NKR-P1 dimerization interface (subunits colored as in (a)). The central residues are shown as spheres with carbon atoms in yellow. The carbon atoms of Ile168 residues (whose mutation decreases the ability of NKR-P1 to bind LLT1)C polymorphism in the human KLRB1 gene alters ligand binding and inhibitory potential of CD161 molecules. PLoS One 10, e0135682 (2015)." href="/articles/s41467-022-32577-6#ref-CR51" id="ref-link-section-d57927382e2666"51 are shown in orange./p>

Y. Li and coworkers reported that the orientation of NKp65 bound to its ligand precludes the putative α2-centered dimerization of NKp6537. Similarly, a hypothetical NKp65 α1-centered dimer is also implausible based on steric hindrance and the lack of stabilizing interactions. This observation contrasts with the α1-centered dimerization of NKR-P1 present in both its unbound and complexed crystal structure. Interestingly, the single-nucleotide polymorphism (SNP) c.503 T > C of the human KLRB1 gene, causing the substitution of isoleucine 168 for threonine in the NKR-P1 CTLD, was reported to have a 37% frequency of the Thr168 alleleC polymorphism in the human KLRB1 gene alters ligand binding and inhibitory potential of CD161 molecules. PLoS One 10, e0135682 (2015)." href="/articles/s41467-022-32577-6#ref-CR51" id="ref-link-section-d57927382e3335">51. The authors showed that the Thr168 isoform of NKR-P1 has a lower affinity to LLT1 and a weaker inhibitory effect on NK cells. They proposed that Ile168 forms a part of the interaction interface between NKR-P1 and LLT1, directly affecting LLT1 recognition by NKR-P1C polymorphism in the human KLRB1 gene alters ligand binding and inhibitory potential of CD161 molecules. PLoS One 10, e0135682 (2015)." href="/articles/s41467-022-32577-6#ref-CR51" id="ref-link-section-d57927382e3339"51. However, the structure of the NKR-P1 homodimer shows that Ile168 is found at the dimerization interface rather than at the membrane-distal interaction interface – more specifically, in a small hydrophobic pocket within the dimerization interface (Fig. 3d). Therefore, we propose that substituting the nonpolar isoleucine residue with polar threonine caused by c.503 T > C SNP indirectly affects the binding affinity because this substitution destabilizes the α1-centered NKR-P1 homodimer. Glycosylation often significantly impacts receptor homooligomerization, as recently evidenced for, e.g., NK cell activation receptor NKp3052. NKR-P1 homodimerization is also regulated by its glycans; specifically, the glycans present on Asn116 and Asn157 (Fig. 3b). Core glycan chains present at these residues contribute partially to the α1-centered dimerization interface, but at the same time, the glycans clash together. As a result, the stability of the α1-centered NKR-P1 homodimer is improved by abrogating N-glycosylation on Asn157, leaving only the Asn116 glycan at the dimer interface (Supplementary Fig. 3b). Interestingly, a c.470 A > G SNP causing N157S mutation is also listed in the human genome variation database. However, the clinical significance of N157S mutation was not yet investigated, although it could significantly affect NKR-P1 signalization via stabilizing its ligand-bound state. The oligomeric state of the receptor might thus modulate the overall NKR-P1:LLT1 binding affinity. The NKp65:KACL complex stands out for its high affinity (Kd ~ 0.67 nM)37 – ca. 3000× stronger than that of NKp80:AICL (Kd ~ 2.3 µM)9 and 70,000–130,000× than that of NKR-P1:LLT1 (Kd ~ 48 µM39; this study 90 µM). Due to the exceptionally high NKp65:KACL binding affinity, any putative ancestral α1-centered dimerization interface may have been lost in NKp65. In contrast, the NKR-P1 and NKp80 receptors may have evolved to compensate for their low affinity to their ligands by utilizing the α1-centered dimerization and enabling an increased avidity effect./p>

a Representation of four adjacent asymmetric units within the NKR-P1:LLT1 complex crystal, excluding the additional unrelated NKR-P1 dimer. The NKR-P1 (blue and cyan) and LLT1 (green and lemon) dimers alternate in primary (cyan and green) and secondary (blue and lemon) interactions, forming a chain-like structure. The schematic depiction of this arrangement is shown in the inset with the same color code. The black and white triangles represent N-termini positions, pointing behind and in front of the display plane, respectively. b Depiction of the hypothetical arrangement of the chain-like structure upon contact of an NK cell (bottom, blue) with a target cell (top, green) showing the crystal structure of two NKR-P1 dimers (cyan and blue) interacting with two LLT1 dimers (green and lemon) in the primary (cyan and green) and secondary (blue and lemon) modes. The first three N-terminal residues in the structures are highlighted in red. The flexible stalk regions connecting the N-termini and cell membranes are represented as speckled lines of the corresponding color-coding. The view on the right-hand side is clipped for clarity at the plane indicated on the left-hand side view. c Schematic depiction of NKR-P1 extracellular domain dynamics and possible ligand binding arrangements. NKR-P1 is expressed as a disulfide-linked homodimer; however, its CTLDs may undergo conformation change similar to monomer-dimer equilibrium. Such putative equilibrium would be shifted towards monomeric species for the wild-type protein and its I168T allelic variantC polymorphism in the human KLRB1 gene alters ligand binding and inhibitory potential of CD161 molecules. PLoS One 10, e0135682 (2015)." href="/articles/s41467-022-32577-6#ref-CR51" id="ref-link-section-d57927382e3460"51. At the same time, a dimeric arrangement corresponding to the non-covalent dimer observed in the herein described crystal structures would be promoted for the S159A variant (left-hand side). Such NKR-P1 dimer could then interact with the cognate LLT1 ligand (itself being expressed as a disulfide-linked homodimer as well and forming stable non-covalent dimers with its CTLDs) in the previously suggested standard model of NK cell receptor – CTL ligand interaction (middle) or alternate with the dimeric ligand in the proposed chain-like arrangement based on NKR-P1:LLT1 complex crystal structure (right-hand side)./p> 0.05 are indicated as not significant; statistically significant p values are indicated with asterisks (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)./p>

3.0.CO;2-6" data-track-action="article reference" href="https://doi.org/10.1002%2F%28SICI%291521-4141%28199805%2928%3A05%3C1611%3A%3AAID-IMMU1611%3E3.0.CO%3B2-6" aria-label="Article reference 13" data-doi="10.1002/(SICI)1521-4141(199805)28:053.0.CO;2-6"Article CAS PubMed Google Scholar /p>C polymorphism in the human KLRB1 gene alters ligand binding and inhibitory potential of CD161 molecules. PLoS One 10, e0135682 (2015)./p>