Structure of NKp65 bound to its keratinocyte ligand reveals basis for genetically linked recognition in natural killer gene complex

Abstract

The natural killer (NK) gene complex (NKC) encodes numerous C-type lectin-like receptors that govern the activity of NK cells. Although some of these receptors (Ly49s, NKG2D, CD94/NKG2A) recognize MHC or MHC-like molecules, others (Nkrp1, NKRP1A, NKp80, NKp65) instead bind C-type lectin-like ligands to which they are genetically linked in the NKC. To understand the basis for this recognition, we determined the structure of human NKp65, an activating receptor implicated in the immunosurveillance of skin, bound to its NKC-encoded ligand keratinocyte-associated C-type lectin (KACL). Whereas KACL forms a homodimer resembling other C-type lectin-like dimers, NKp65 is monomeric. The binding mode in the NKp65–KACL complex, in which a monomeric receptor engages a dimeric ligand, is completely distinct from those used by Ly49s, NKG2D, or CD94/NKG2A. The structure explains the exceptionally high affinity of the NKp65–KACL interaction compared with other cell–cell interaction pairs ( K D = 6.7 × 10 −10 M), which may compensate for the monomeric nature of NKp65 to achieve cell activation. This previously unreported structure of an NKC-encoded receptor–ligand complex, coupled with mutational analysis of the interface, establishes a docking template that is directly applicable to other genetically linked pairs in the NKC, including Nkrp1–Clr, NKRP1A–LLT1, and NKp80–AICL.

Crystal structure of extracellular domain of human lectin‐like transcript 1 (LLT1), the ligand for natural killer receptor‐P1A

“…Instead, this region exhibits a wing-like structure and forms additional short β-strands, β2 and β2 , as in LLT1. In the structure of the KACL-NKp65 complex, the corresponding region of NKp65 similarly folds into a wing-like structure [28]. Taken together, these results support the idea that the regions may change their conformations according to their binding partners.…”

“…To discuss the binding topology of LLT1 and NKRP1A, the residues involved in the KACL binding to Nkp65 were structurally compared with those on the putative receptor binding face of LLT1 (Fig. 3) [28]. The characteristically conserved receptor-binding residues are Thr93/Thr, Leu158/Ile, Tyr165/Phe, Ser173/Ser, Arg175/Arg, Tyr177/Phe, and Glu179/Asp (LLT1/KACL).…”

“…They also form the typical dimer structure seen in the CTLR structures. Recently, the structure of the KACL-NKp65 complex was reported [28]. This was the first structure of a ligand-receptor complex with the CTLD.…”

HemITAM: A single tyrosine motif that packs a punch

“…NKp65 tightly binds to the C-type lectin-like glycoprotein KACL, which is encoded adjacently to KLRF2 in the NKC and restricted to keratinocytes (28,81). The exceptionally high affinity of the NKp65-KACL interaction (28, 82) is due to high shape complementary and a few "hotspot" amino acid residues, providing a high number of hydrogen bonds (29,82). NKp65 triggering stimulates calcium flux, degranulation, and cytotoxicity of NK-92 cells in a Sykdependent manner (28,29).…”

The Activating C-type Lectin-like Receptor NKp65 Signals through a Hemi-immunoreceptor Tyrosine-based Activation Motif (hemITAM) and Spleen Tyrosine Kinase (Syk)

“…The crystal structure of the (sNKp65) 2 -(sKACL) 2 tetrameric complex shows a symmetrical, butterfly-shaped assembly with a high shape complementarity of the receptor-ligand interface, explaining the high affinity in the nanomolar range (16). This interface comprises several hot spot amino acid residues that crucially contribute to the tight interaction (16,17).…”