Structure determination

To understand the molecular basis underpinning recognition of Clr-b by NKR-P1B, we expressed their corresponding CTLDs and determined the structure of the co-complex to 2.9 Å resolution (Table 1). The crystallographic asymmetric unit comprised eight protomers of NKR-P1B and sixteen protomers of Clr-b, which together formed eight highly similar NKR-P1B:Clr-b complexes (root mean square deviation (r.m.s.d) ~ 0.5 Å overall Cα atoms) (Supplementary Fig. 1). Within the crystal lattice, the molecules were tightly packed with no significant unaccounted electron density. Indeed, the structure refined very well, to R fac and R free values of 20.6 and 22.5 respectively, and continuous electron density was visible for the entire Clr-b chain (residues 74–194) and the vast majority of NKR-P1B (residues 91–215) (Supplementary Fig. 2) with the exception of a single loop (residues 177–179) that was distal to the Clr-b binding site. Clear electron density was also observed for a single N-acetlyglucosamine (GlcNAc) moiety attached to Asn169 of NKR-P1B (Fig. 1). This sugar chain did not impact any of the interactions discussed below. Surprisingly, each NKR-P1B:Clr-b complex was comprised of a single NKR-P1B monomer bound to a Clr-b homodimer, although a higher-order assembly was also apparent (discussed below).

Table 1 X-ray data collection statistics Full size table

Fig. 1 The CTLD fold of NKR-P1B and Clr-b. Comparison of the structures of NKR-P1B and Clr-b with other related receptors and ligands including mouse NKR-P1A (PDB ID: 3M9Z), Clr-g (PDB ID: 3RS1), CLEC9A (PDB ID: 3VPP) and LLT1 (PDB ID 4QKG). α-helices are shown in red and disulfide bonds are represented as green sticks. The single GlcNAc moiety attached to Asn169 of NKR-P1B is shown as black sticks Full size image

Structures of NKR-P1B and Clr-b

Both NKR-P1B and Clr-b adopted the classic CTLD fold comprising two central antiparallel β-sheets flanked by two α-helices (α1 and α2) (Fig. 1). Each of the β-sheets was comprised of four β-strands, which we denote β0, β1, β5, β1′ and β2′, β2, β3, β4 based on a strand assignment described previously25. The two intramolecular disulfide bonds that are highly conserved throughout CTLDs are present in NKR-P1B (Cys122–Cys210 and Cys189-202), whereas Clr-b only contains the former (Cys108–Cys190). Both NKR-P1B and Clr-b also possessed an additional disulfide bond (Cys94–Cys105 in NKR-P1B and Cys81–Cys91 in Clr-b) that tethered the N-terminus to the β1-strand. A structural similarity search using the Dali server revealed that NKR-P1B was most similar to members of the human CLEC family, including CLEC9A26 and CLEC5A27 (Z-score ≈ 20.2, r.m.s.d ≈ 1.6 Å over 119 aligned Cα residues), but also displayed considerable structural homology to other CTLD-containing proteins including human LOX-128, dectin-129, and mouse NKR-P1A30 (Z-score = 18.2, r.m.s.d = 1.7 Å over 121 aligned Cα residues) (Fig. 1). In contrast, Clr-b was most similar to the closely related family member Clr-g31, and also displayed considerable structural homology to the human NKR-P1A ligand LLT-132 (Z-score ≈ 22.8, r.m.s.d ≈ 1.0 Å over 117 aligned Cα residues). Notably, NKR-P1B and Clr-b were highly structurally similar to each other (r.m.s.d 1.6 Å over 87 aligned Cα atoms), with the main distinction being a reorientation of the α2-helix of NKR-P1B by ~10° (Supplementary Fig. 3A). Altogether, the structures of NKR-P1B and Clr-b highlight the conserved nature of this receptor-ligand pair, which are encoded within the same region of the NKC and likely arose by gene duplication from a common ancestral precursor.

NKR-P1B and Clr-b self-association

Both NKR-P1B and some Clr molecules have been reported to form disulfide-linked dimers on the cell surface15. Indeed, within the crystal lattice, Clr-b formed homodimers such that the N-termini were positioned in a manner that would enable the lateral association of two monomers in cis (within the plane of the same membrane) (Fig. 2a). The architecture of the Clr-b homodimer is reminiscent of that observed for a number of related proteins including Clr-g31, LLT-132, and KACL33, and thus could be described as a classic homodimer for CTLD-containing proteins. A similar arrangement has also been observed for some Ly49 receptors (e.g. Ly49A), where it is referred to as the closed conformation6.

Fig. 2 Clr-b and NKR-P1B self-association. a Overview of the Clr-b homodimer. Detailed interactions within the head, body, and tail regions are shown in shades of pink, green and blue respectively. Hydrogen bonds are represented as black dotted lines. b Overview of the non-classic NKR-P1B homodimer. Salt-bridges are shown as red dotted lines. c, d Sedimentation velocity analytical ultracentrifugation analysis of Clr-b (c) and NKR-P1B (d) self-association. The sedimentation coefficient distributions [c(s)] for a dilution series of each protein are shown as indicated. A magnified version of the NKR-P1B dimeric species is provided within the dashed box Full size image

Upon self-association, the two Clr-b monomers bury a total solvent accessible surface area (BSA) of ~1900 Å2, which is large compared to that of other classic CTLD homodimers such as KACL (1300 Å2) or Clr-g (1740 Å2)31,33. The interaction interface is comprised of three main segments that are analogous to head, body and tail regions. The central body stems from the β0 strands of each monomer, which associate via two main-chain hydrogen bonds between Ile85 and Gly86 to form an extended antiparallel β-sheet (Fig. 2a and Supplementary Table 1). These interactions are solidified by the head that is derived from the C-termini of the α2-helices. Here, Arg129 adopts a planar conformation that stacks against Tyr130 (Fig. 2a). The head and body interactions, which are relatively rich in H-bonds and symmetrical in nature, are further complemented by extensive hydrophobic interactions within the membrane proximal tails of Clr-b (Asn74-Trp84) that are asymmetric but account for 44% of the total BSA (Fig. 2a).

Unlike Clr-b, NKR-P1B was not observed to form a classic homodimer, most likely due to the orientation of the α2-helix, which protruded in a fashion that would sterically clash with a neighboring NKR-P1B protomer (Supplementary Fig. 3A). Indeed, an analysis of other related receptors and ligands for which structural data are available revealed that the extent to which the α2-helix protruded was directly correlated with the capacity of the molecule to form a classic CTLD homodimer (Supplementary Fig. 3B). Accordingly, this structural feature may be a key determinant governing oligomerization within CTLD-containing proteins.

Instead, two symmetry-related NKR-P1B molecules packed together to form a distinct non-classic homodimer (Fig. 2b). The NKR-P1B homodimer interface was modest in size (BSA ~700 Å2), with the interaction surface confined to the β2–β2′ loop, which primarily formed contacts with residues from the α1-helix (Supplementary Table 2). Here, Asp162 formed a salt-bridge interaction with Arg119, whereas Thr161, which was located at the tip of the β2–β2′ loop, was tightly packed at the juncture of the β2/β3 strands and the α1-helix (Fig. 2b). Within the NKR-P1B homodimer, the N-termini extend outwards within the same plane, but are not directly juxtaposed. Thus, it is possible that these remodel somewhat within the context of the native cysteine-containing stalk. Notably, within the NKp65:KACL crystal structure, the NKp65 receptor also formed a related homodimer via a similar interface (Supplementary Fig. 3C)33.

In order to assess the oligomeric status of NKR-P1B and Clr-b in solution, we performed analytical ultracentrifugation experiments at a range of protein concentrations. At low concentrations (~3–12 μM) Clr-b was evident as a single species at ~ 1.9S whose sedimentation profile correlated well with that of a monomer (frictional ratio (f/f 0 ) = 1.15)) (Fig. 2c). However, at higher concentrations (25–200 μM), an additional species, representing a dimeric form of Clr-b was visible at ~2.7S (f/f 0 = 1.28). In contrast, NKR-P1B was primarily monomeric (~ 2.1S) with only a small proportion of a dimeric species (~3.6S) being visible at the highest concentrations tested (100–200 μM) (Fig. 2d). Thus, Clr-b forms a relatively stable homodimer, while the potential self-assembly of NKR-P1B is likely to be extremely weak, at least in the absence of the membrane environment.

NKR-P1B:Clr-b stoichiometry and higher-order assembly

NKR-P1B engaged Clr-b via a head-to-head docking mode such that the N-terminus of each molecule protruded from opposite ends of the complex (Fig. 3a). This arrangement is consistent with a trans interaction, where NKR-P1B and Clr-b would be present on opposing cellular membranes. Surprisingly, a single NKR-P1B protomer engaged one half of the Clr-b homodimer, making contacts exclusively with only one Clr-b monomer (Fig. 3a and Supplementary Table 3). This unusual trimeric 1 NKR-P1B: 2 Clr-b stoichiometry was conserved in all eight of the complexes present within the crystallographic asymmetric unit (Supplementary Fig. 1A), and differs from the 2:2 stoichiometry observed for the related human NKp65:KACL complex, despite similarities in overall docking mode (Fig. 3b). The inability of Clr-b to be fully saturated by NKR-P1B was not due to differences in the conformation of the molecules, since both free and bound Clr-b were closely structurally homologous (r.m.s.d 0.4 Å over 112 aligned Cα atoms), with the exception of some side chain rearrangements that likely represent an induced-fit style mode of interaction (discussed below). Although an additional NKR-P1B molecule could potentially be accommodated within the NKR-P1B:Clr-b complex (Fig. 3c), the packing of molecules within the broader crystal lattice precluded this arrangement.

Fig. 3 Overview of the NKR-P1B:Clr-b complex. a Simplified representation showing how a single NKR-P1B monomer engages one half of the Clr-b homodimer in a 1:2 receptor-ligand stoichiometry. b Structure of the related NKp65 receptor bound to KACL (PDB ID: 4IOP) where two NKp65 monomers bind to the KACL homodimer. c Model of a fully saturated 2 NKR-P1B: 2 Clr-b complex. d Representation of the hexameric NKR-P1B:Clr-b assembly visible within the crystal structure, and how it could be accommodated at the NK cell-target cell interface. Membrane proximal stalks not visible within the crystal structure are depicted as dashed lines, with their length annotated. The position of inter-molecular cysteines in the NKR-P1B stalk are shown as green lines. e, f Close up views of the interactions at site I (e) and site II (f) within the NKR-P1B:Clr-b interface. Hydrogen bonds (black) and salt-bridges (red) are shown as dotted lines Full size image

Within the crystal, two trimeric NKR-P1B:Clr-b complexes were bridged via an NKR-P1B dimer to produce a hexameric arrangement (Fig. 3d) Here, each of the Clr-b N-termini lay planar to each other, indicating that these molecules could potentially be arranged in cis within a single target cell membrane. Notably, modeling of higher-order assemblies formed via full saturation of NKR-P1B:Clr-b binding sites results in a distinctly non-planar arrangement (Supplementary Fig. 4). Thus the largest arrangement that could be accommodated within a cellular membrane is the hexameric assembly observed within the crystal lattice.

The NKR-P1B:Clr-b interface

NKR-P1B and Clr-b interacted via an imperfect interface that was modest in size, burying a total solvent accessible surface area of 1450 Å2, and characterized by poor shape complementarity ((S c ) = 0.55, where 1 indicates a geometrically perfect fit)34. Despite this, the interface was densely packed with a large number of highly specific, polar interactions that included 18 H-bonds and 8 salt-bridges (Supplementary Table 3). The main interaction site (site I) comprised the β4–β5 loop of NKR-P1B, where a string of residues (Ser203-Arg207) lay on top of one of the Clr-b β-sheets in a perpendicular fashion, making extensive contacts with a variety of structural elements, including the β3 and β4 strands, and the β2′–β3 loop (Fig. 3e). Here, Asp205 of NKR-P1B played a central role. In addition to the 3 H-bonds that anchored its backbone, the side chain made multiple salt-bridge interactions with Arg164 and H-bonds to Asn173, Asn175, and Ser178 of Clr-b. Arg207 of NKR-P1B appeared equally important, forming a number of salt-bridge interactions with Clr-b residue Asp135. In addition, the hydroxyl groups of Ser203 and Ser204 of NKR-P1B both formed H-bonds with the Clr-b backbone whilst Asn206 bent downwards in order to facilitate an interaction with Arg186. The second, more peripheral interaction site (site II) was centered on the Clr-b β4–β5 loop which contacts the β2′–β3 loop as well as the β3 and β4 strands of NKR-P1B (Fig. 3f). In particular, four Clr-b residues, Arg181, Tyr183, Ser184, and Arg186 projected upwards, making hydrophilic interactions with NKR-P1B. Here, Arg181 and Tyr183 of Clr-b formed a multitude of H-bonds with the NKR-P1B main chain (including Thr184, Ser199, and Gly201) as well as the side chain of Ser188, while Ser184 and Arg186 flanked Asp200 from the NKR-P1B β4 strand. Notably, the interactions within site II were characteristic of an induced-fit style binding mode. In particular, Arg181 and Tyr183 of Clr-b moved toward NKR-P1B, which remodeled its β2′-β3 loop (compared to the m12-bound form) in order to accommodate these bulky residues (Supplementary Fig. 5).

Structural comparisons

The overall structure of the NKR-P1B:Clr-b complex is similar to that of the related NKp65:KACL receptor-ligand pair (r.m.s.d 1.9 Å over 218 aligned Cα atoms) (Fig. 3a–c), suggesting that an evolutionarily conserved docking topology underpins ligand recognition by both inhibitory and stimulatory members of the NKR-P1 family. However, a closer inspection of the molecular interactions reveals some differences. Notably, the NKR-P1B:Clr-b interface is considerably less ideal than that of NKp65:KACL, exhibiting poorer shape complementarity (S c 0.55 versus 0.69) and burying 14% less surface area (Fig. 4a). The major points of difference are the loop preceding the β2 strand (residues 131-4 in NKp65) and the β3–β4 loop, both of which are tightly packed against KACL forming a multitude of H-bond and Van der Waals interactions (Fig. 4b, c). In comparison, the same regions in NKR-P1B are withdrawn from Clr-b, such that the only contacts are derived from Tyr149 (Fig. 4). Despite being structurally dissimilar to Clr-b, the viral m12 ligand also targets the same NKR-P1B surface (Fig. 4a), although it does so more robustly than Clr-b, via an extensive surface (BSA 2,180 Å2) that exhibits high shape complementarity (S c 0.69). Overall, these structural observations explain why binding of m12 (K D ~ 6 μM) and KACL (K D ~ 0.7 nM) are relatively tight, while recognition of Clr-b by NKR-P1B is extremely weak (see below).

Fig. 4 NKR-P1B:Clr-b interactions. a Surface representation showing the binding footprint made by Clr-b (cyan) and m12 (blue) on NKR-P1B (salmon). For comparison, the surface of NKp65 (green) is shown in the same orientation, with the residues that contact KACL colored pink. b, c Close up views showing contacts that are present in the NKp65:KACL structure (pink and green respectively) but absent in the NKR-P1B:Clr-b interface (salmon and cyan respectively). Residues that form contacts are shown as sticks. H-bonds are represented by black dashed lines Full size image

The NKR-P1B:Clr-b interaction is extremely weak

Next, we sought to define the affinity of the NKR-P1B:Clr-b interaction using surface plasmon resonance (SPR). However, at the concentration range used, neither mammalian (570 μM) nor E. coli (200 μM) expressed Clr-b bound appreciably to NKR-P1B that was immobilized onto streptavidin-coated chips via a C-terminal BirA tag (Supplementary Fig. 6A). The absence of an interaction was not due to geometric constraints of the experimental setup, because m12, which interacts with the same surface of NKR-P1B, bound robustly in this assay. We also observed no interaction using extended forms of Clr-b (250 μM) and NKR-P1B that included the native cysteine residues involved in disulfide-mediated homodimerization (Supplementary Fig. 6B), although the latter did not form a homo-dimer despite containing two cysteine residues within the membrane proximal stalk (Supplementary Fig. 6C). Similar results were obtained using a different experimental setup (akin to that in described in ref. 35), whereby NKR-P1B tetramers were passed over immobilized Clr-b (Supplementary Fig. 6B). We did however observe a small fraction (6% of total) of a species consistent with a 1 NKR-P1B: 2 Clr-b complex (S 20,w = 4.2, f/f 0 = 1.2) using AUC (Supplementary Fig. 6D), although we cannot exclude the possibility that a 2:2 complex could also form at higher protein concentrations. Taken together, our data indicate that the NKR-P1B:Clr-b interaction was potentially extremely weak in solution. Notably, this is not the first example of a bona fide immune receptor-ligand interaction whose affinity lies outside of the detection limit of standard biophysical approaches36,37.

NKR-P1B:Clr-b binding is highly sensitive to mutation

Despite little/no apparent interaction in solution, NKR-P1B tetramers robustly stained both BWZ cells transduced with Clr-b, as well as HEK293T cells transfected with a Clr-b-expressing vector (Fig. 5a), in accordance with previous findings15. Thus, we utilized this assay to interrogate the energetic basis of the NKR-P1B:Clr-b interaction. To this end, we generated tetrameric forms of a panel of 15 individual NKR-P1B mutants, focusing on key amino acid residues located at the NKR-P1B:Clr-b interface. As a negative control, we mutated Asn150, which was close to the interface but did not directly contact Clr-b. Here, an N150A mutation did not impact NKR-P1B staining compared to wild type (Fig. 5b). As a positive control, we introduced a large, bulky charged residue into the central region of the interface (S204R). This non-conservative mutation completely abolished binding to the Clr-b transfectants (Fig. 5b). Of the remaining NKR-P1B residues examined, the vast majority (T184A, S188A, S199A, E200A, S203A, S204A, D205A, N206A, R207A) of mutant tetramers failed to stain HEK293T transfectants when substituted to alanine. Notably, all of these residues were centrally located at the interface and formed multiple contacts with Clr-b (Figs. 3, 5b). In contrast, mutation of residues at the periphery of the interface either had no effect (N113A and V183A) or reduced staining to a lesser extent (Y149A).

Fig. 5 Energetic basis of the NKR-P1B:Clr-b interaction. a BWZ cells (top) or HEK293T cells (bottom) were transduced or transfected, respectively, with empty vector (dashed line) or Clr-b-expressing vector (shaded gray), and stained using anti-Clr-b antibody (left) or NKR-P1B tetramers (right) and analyzed by flow cytometry. b HEK293T cells were transfected with vector expressing Clr-b (pIRES2-EGFP), and 48 h later were stained with NKR-P1B mutant tetramers. The GFP expression measures transfection efficiency and PE measures binding by PE-tetramers. Gates were set up using untransfected cells (HEK293T) and cells transfected with empty pIRES2-EGFP (Vector) that were stained with NKR-P1B tetramer (WT) or Streptavidin-PE (SA-PE). Labels on the top left correspond to point mutation on the NKR-P1B molecule, whereas italicized numbers correspond to mean fluorescence intensity of NKR-P1B. c Cells were transfected with constructs expressing NKR-P1B mutants, and 48 h later were used as stimulators to BWZ.CD3ζ/Clr-b reporters. Co-cultures were setup using a 1:1 stimulators: reporters ratio, and the next day were assayed for production of β-galactosidase using colorimetric assay. d, e NKR-P1B dimer mutants were transfected into HEK293T cells and used in BWZ assays using 3-fold dilutions of stimulators against BWZ.CD3ζ/Clr-b reporters (left) or BWZ.CD3ζ/m12Smith reporters (right). Significant differences are shown between the WT and mutant allele for each graph. Data were analyzed using (c) one-way ANOVA [F(15,48) = 51.88, p < 0.0001], (d) two-way ANOVA (Z.Clr-b: [F(12,40) = 64.14, p < 0.0001]; Z.m12: [F(12,40) = 21.71, p < 0.0001], or (e) two-way ANOVA (Z.Clr-b: [F(16,64) = 389.3, p < 0.0001]; Z.m12: [F(16,80) = 1297, p < 0.0001] with Bonferroni post-hoc tests. Significance intervals are depicted as ***p < 0.001. (b) is representative of 2 independent experiments. All other data are representative of at least 3 independent biological replicates. Data are presented as mean ± SEM Full size image

Secondly, we assessed the impact of the same NKR-P1B mutants in a cellular context using BWZ reporter assays. To this end, we generated a chimeric receptor expressing the extracellular domain of Clr-b fused with an intracellular CD3ζ signaling domain, and transduced BWZ.36 cells expressing β-galactosidase under NFAT elements with this construct38. These cells were sorted for high Clr-b expression using the anti-Clr-b antibody, 4A6 (Fig. 5a). To test these interactions, we then used site-directed mutagenesis to clone the panel of NKR-P1B mutants described above into the pIRES2-EGFP mammalian expression vector, transfected these constructs into HEK293T, and used these transfectants as stimulators for BWZ.CD3ζ/Clr-b reporters. Using this approach, we observed a similar pattern as described above for the tetramer staining experiments. More specifically, N113A, N150A, and V183A had no effect on binding, whereas weak interactions with the T184A and S204A mutants were observed (~ 60% reduction compared to wild type) (Fig. 5c). On the other hand, the Y149A, S188A, S199A, E200A, S203A, D205A, N206A, R207A, and the positive control S204R, all abolished the interaction (Fig. 5c). Importantly, all the NKR-P1B mutants were abundantly expressed at the cell surface as judged by anti-NKR-P1B mAb staining, although the levels of N206A and R207A were comparatively lower (Supplementary Fig. 7). Thus, both tetramer staining and cellular reporter data fully support the X-ray crystal structure, and demonstrate that the NKR-P1B:Clr-b interaction is underpinned by polar interactions that are uniquely sensitive to alterations, in line with the weak intrinsic affinity of the interaction.

The NKR-P1B homodimer is critical for Clr-b recognition

Finally, we sought to determine the importance of the NKR-P1B homodimer for receptor function. To this end, we generated two mutants (T161W and D162A) in residues located at the NKR-P1B homodimer interface and assessed their capacity to stimulate BWZ.Clr-b reporters. These transfectants resulted in a total loss (D162A) or dramatic reduction (T161W) in BWZ.Clr-b reporter signaling, indicating that the NKR-P1B homodimer was an important factor in the response to host Clr-b. To further support this conclusion, we also mutated the cysteine residues (Cys75 and Cys 88) within the NKR-P1B stalk region that have been implicated in the formation of disulfide-linked NKR-P1B homodimers on the cell surface. Here, mutation of both cysteine residues within the NKR-P1B stalk resulted in a total ablation of Clr-b reporter cell signaling, whereas mutation of either cysteine alone had little effect (Fig. 5e), indicating that both of the cysteine residues within the NKR-P1B stalk are involved in inter-molecular disulfide bond formation. Notably, all of the NKR-P1B dimer mutants tested had little to no effect on signaling from BWZ.m12 reporters relative to wild type (Fig. 5d, e), despite their moderately decreased level of expression on the cell surface, as judged by mAb staining (Supplementary Fig. 7). Thus, the non-classic NKR-P1B homodimer is critical for signaling in response to the weak affinity host-encoded Clr-b ligand, but is somewhat dispensable for signaling in response to the viral m12 decoy, which exhibits relatively high affinity for a single NKR-P1B molecule (Fig. 6).