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Stimulation or tolerance of natural killer (NK) cells is achieved through a cross-talk of signals derived from cell surface activating and inhibitory receptors. Killer cell immunoglobulin-like receptors (KIR) are a family of highly polymorphic activating and inhibitory receptors that serve as key regulators of human NK cell function. Distinct structural domains in different KIR family members determine function by providing docking sites for ligands or signalling proteins. Here, we review a growing body of literature that has identified important structural elements on KIR that contribute to function through studies of engineered mutants, natural polymorphic sequence variants, crystal structure data and the conservation of protein sequences throughout primate evolution. Extensive natural polymorphism is associated with both human KIR and their ligands, MHC class I (HLA-A, -B and -C) molecules, and numerous studies have demonstrated associations between inheritance of certain combinations of KIR and HLA genes and susceptibility to several diseases, including viral infections, autoimmune disorders and cancers. In addition, certain KIR/HLA combinations can influence pregnancy and the outcome of haematopoietic stem cell transplantation. In view of the significant regulatory influences of KIR on immune function and human health, it is essential to fully understand the impacts of these polymorphic sequence variations on ligand recognition, expression and function of the receptor. Natural killer (NK) cells are critical effectors of the early innate immune response toward transformed and virus-infected cells.1 Whereas T and B cells are triggered upon detecting foreign invaders through the expression of antigen-specific receptors, the activation status of NK cells is regulated by a balance of intracellular signals received from an array of germ-line-encoded activating and inhibitory receptors.2 When an NK cell encounters an abnormal cell (e.g. tumour or virus-infected) and activating signals predominate, the NK cells can rapidly induce apoptosis of the target cell through directed secretion of cytolytic granules containing perforin and granzymes or engagement of death domain-containing receptors.3 Activated NK cells can also secrete cytokines, such as interferon-γ, tumour necrosis factor-α and granulocyte–macrophage colony-stimulating factor (GM-CSF), which activate both innate and adaptive immune cells.4 The human killer cell immunoglobulin-like receptors (KIR; also known as CD158) are a family of transmembrane glycoproteins expressed on NK cells and a subset of T cells.5 The KIR are key regulators of the development, tolerance and activation of NK cells.1 The major ligands for KIR are MHC class I (HLA-A, -B or -C) molecules, which are expressed on the surface of nearly every normal nucleated cell in the body, are encoded by the most polymorphic genes in humans, and define immune ‘self’. Tolerance of NK cells toward normal cells is achieved through their expression of MHC-I-binding inhibitory receptors, which include KIR, NKG2A/CD94 and CD85j (ILT2, LIR1). The NK cells preferentially attack abnormal cells that have down-regulated surface MHC-I molecules, termed ‘missing self recognition’.6 The loss of MHC-I expression is a rare event that occurs in certain tumour and virus-infected cells to avoid recognition by cytolytic T cells, which use T-cell receptor (TCR) recognition of antigenic peptides bound to MHC-I molecules to identify abnormal target cells. In this way NK cells intervene to eliminate MHC-I-deficient target cells. The KIR-mediated regulation of NK cells can significantly impact their responsiveness during viral infection, cancer, haematopoietic stem cell transplantation and pregnancy.7–11 On the other hand, the expression of self MHC-I-reactive KIR is also critical for the maturation of functionally responsive NK cells, through a process referred to as ‘education’, ‘licensing’, or ‘arming’.12–16 The KIR family is encoded by 14 highly polymorphic genes (2DL1 to 2DL5, 3DL1 to 3DL3, 2DS1 to 2DS5, and 3DS1], and distinct family members can transduce either activating or inhibitory signals (Table 1). Nomenclature of KIR is based upon the number of C2-type immunoglobulin-like domains in the extracellular region (2D for two domains, 3D for three domains) and by the length of the cytoplasmic domain (L for long-tailed receptors and S for short ones) (Fig. 1).21 All inhibitory KIR have long cytoplasmic domains possessing immunoreceptor tyrosine-based inhibitory motifs (ITIMs; I/VxYxxL/V), which recruit protein tyrosine phosphatases that are critical for mediating inhibitory function.2 In contrast, KIR with short cytoplasmic domains associate with a transmembrane signalling adaptor protein, DAP12 (also called KARAP). Consistent with antigen receptor signalling, DAP12-dependent activation occurs through the recruitment of Syk/ZAP-70 tyrosine kinases by immunoreceptor tyrosine-based activation motifs ITAM; Yxx(L/I/V)x6–8Yxx(L/I/V).2 The only exception to this short/long-tailed rule is KIR2DL4, which is a unique long-tailed activating KIR. Compared with other KIR family members, 2DL4 is only expressed on CD56high NK cells, functions as a more potent activator of cytokine production rather than cytotoxicity, and associates with ITAM-containing FcεRI-γ adaptor instead of DAP12 (Fig. 1).22–24 The major structural differences between different killer cell immunoglobulin-like receptor (KIR) family members. The KIR can be divided into three major subgroups: the inhibitory receptors, the activating receptors, and a unique activating receptor known as KIR2DL4. Nomenclature of KIR is based upon the number of extracellular immunoglobulin-like domains (2D or 3D) and the length of the cytoplasmic domain (L for long and S for short). Receptors with long cytoplasmic domains contain one or two immunoreceptor tyrosine-based inhibitory motif sequences (orange) that provide inhibitory function. The short cytoplasmic KIR and the long-tailed KIR2DL4 are activating receptors, which contain a basic amino acid (+) within the transmembrane domain that interacts with an acidic amino acid (−) within the transmembrane domains of signalling adaptor proteins DAP12 (blue) or FcεRI-γ (green), respectively. These adaptors provide intracellular immunoreceptor tyrosine-based activator motif sequences (purple) that allow for activating function. Individual KIR recognize distinct subsets of MHC-I allotypes (Table 1) with inhibitory KIR always having higher avidity than activating KIR for MHC-I.25 Activating KIR appear to respond best when they encounter allogeneic MHC-I, as was demonstrated by Chewning et al.,26 which could potentially be a mechanism by which NK cells promote anti-tumour responses after haematopoietic transplantation. The ligands for 2DL5, 3DL3 and 2DS5 are currently unknown. Intriguingly, CpG-oligodeoxynucleotides were recently shown to bind to 3DL2 for subsequent internalization to TLR9-containing endosomes, demonstrating that non-MHC-I molecules may also serve as KIR ligands.27 The KIR genes evolved in mammals from a single ancestral gene (KIR3D) that duplicated to form 3DL and 3DX (3DL0) lineages.28–30KIR3DL subsequently evolved rapidly in response to extensive MHC-I diversity and pathogen challenges to establish four separate lineages of KIR genes in modern humans.28–30 The most conserved KIR orthologue in primates is 2DL4, which is present in most human haplotypes and found in Old World, but not New World, monkeys.28,29 In a remarkable example of convergent evolution, a functionally similar, but structurally distinct, family of type II transmembrane C-type lectin receptors, called Ly49 (also known as KLRA), are expanded in mice (approximately 15 genes) and rats (approximately 26 genes).31 Alternatively, only one to three KIR genes are found in these rodent species, and murine KIR genes are not even expressed in NK cells.32 Conversely, the KIR locus has rapidly expanded in primates, and humans carry a single remnant Ly49 gene, which does not encode a functional protein.33 In contrast, intermediate species, such as pigs and seals, have evolved with minimal expansion of either KIR or Ly49 gene loci.34,35 The sequences of human KIR within the extracellular, transmembrane and cytoplasmic domains are remarkably conserved, yet the KIR genes have evolved to produce a highly polymorphic family of receptors. Genetic evidence indicates that the genes expanded through duplication and recombination, which was probably accelerated by their close proximity of head-to-tail orientation within the 19q13.4 chromosomal locus in humans.36 The KIR gene products attain a high level of diversity based upon four levels of variation:9 (i) the product of each KIR gene specifically recognizes a distinct subset of the available MHC-I allotypes (Table 1),5,17,37 (ii) different combinations of the 14 KIR genes are inherited as distinct haplotypes by individuals within the human population, and different haplotypes can vary in proportion of activating and inhibitory KIR,38,39 (iii) many distinct alleles of individual KIR genes have arisen through point mutations encoding minor sequence variations of one to several amino acids, which can affect receptor surface expression level, recognition by anti-KIR antibodies, and affinity/avidity for MHC-I,37,40–42 and (iv) diversity of the NK cell repertoire in peripheral blood is generated through the stochastic expression of different combinations of the available KIR gene products on the surface of individual NK cells.43–45 Here we review our current understanding of the structure/function relationships within human KIR. Our understanding of how the molecular structure of KIR influences function is derived from studies of specifically engineered mutants, natural polymorphic variants, crystal structures, and the conservation of functionally important sequences in KIR throughout mammalian evolution, particularly among primates. The structure/function relationships in KIR will be organized by focusing separately on (i) the extracellular/ligand-binding domain, (ii) the transmembrane domain and (iii) the cytoplasmic domain, which each share responsibility for a distinct aspect of KIR function. The KIR extracellular domains are chiefly responsible for ligand recognition and comprise two or three closely related immunoglobulin-like domain structural units (designated D1 and D2 in most KIR2D receptors; D0, D1 and D2 in KIR3D receptors; and D0 and D2 in 2DL4 and 2DL5). Domains D1 and D2 form a V-shaped orientation, as revealed from crystal structures of 2DL1, 2DL2 and 2DL3,46–48 and the angle formed between D1 and D2 varies significantly in different KIR2DL structures (from 66° for 2DL1 to 81° for 2DL2). Moesta et al. showed that the higher avidity of 2DL2, compared with 2DL3, for group 1 HLA-C ligands (which include Cw1, Cw3, Cw7 and Cw8) is the result of variations at two residues that appear to interact with each other near the hinge between D1 and D2 to alter the angle between the two domains.17 KIR binding to MHC-I is dependent upon hydrogen bonding and charge complementarity, whereas TCR/MHC interactions rely more upon hydrogen bonding and van der Waal’s forces.49 The D1 and D2 domains contact the exposed surface of the HLA molecule at a site that straddles the C-terminal end of the bound antigenic peptide.49 As a result of this binding overlap, certain bound peptides can decrease the affinity of inhibitory KIR for MHC-I, and binding is especially influenced by amino acids at positions 7 or 8 of the peptides.50,51 Interestingly, Stewart et al. provided evidence that Epstein–Barr virus infection of cells promotes 2DS1 binding to HLA-C, and although the affinity was lower than that of inhibitory 2DL1 (which recognizes the same MHC-I allotypes), the interaction of both receptors was significantly impacted by alterations at position 7 or 8 in the peptide bound to HLA-C.25 Crystal structures have revealed at least 16 residues in the D1 and D2 domains of KIR2DL that provide primary contacts with HLA-C46,49 (Table 2). These residues in 2DL2 form four salt bridges and five hydrogen bonds with conserved residues in the α1 and α2 domains of group 1 HLA-C, the cognate ligands for 2DL2.46,49 Although 16 residues are conserved between 2DL2 and 2DL3, which share ligand these individual residues between other KIR. In this the differences in these 16 residues define the individual toward certain conserved residues on distinct subsets of the highly polymorphic MHC-I amino acid position of HLA-C is a critical KIR contact which is either to define the group 1 HLA-C allotypes by or in the group HLA-C allotypes by Although the structure of KIR3D receptors has not yet by et that the D1 D2 domains of 3DL1 binding to MHC-I, the D0 domain ligand by et that D0 may also in contact with the three immunoglobulin-like domains, D0 is the most polymorphic in residues in the D0 domain and in are of of their through evolution, as as residues which are between human 3DL1 alleles The D0 residues can also influence 3DL1 function by cell surface such as natural at position and position which surface expression of proteins encoded by and The 3DL1 gene is the most polymorphic of the KIR with at least alleles and studies of extracellular 3DL1 sequence provide significant into understanding structure/function as in The high polymorphism of 3DL1 is not is the most polymorphic group of MHC-I in distinct alleles within the same the 3DL1 and genes three lineages that within their extracellular and residues that 3DL1 from are within or near the 16 residues that contact with alleles of 3DL1 produce a protein of the cell surface and as whereas other 3DL1 gene products are expressed at high or levels on the cell Extensive point of 3DL1 by et al. identified numerous extracellular that surface expression and binding for et al. that positions in the 3DL1 extracellular domain for amino acid in and human nearly of these positions within the D1 and D2 domains either or positions to the 16 amino acids primary contact with MHC-I (Table polymorphic alleles and point of 3DL1 that within these ligand interaction residues significantly influence KIR ligand avidity and inhibitory function 2). In to inhibitory KIR, the transmembrane domains of activating KIR contain a basic critical for with DAP12 or a in the transmembrane domains of and receptors a docking site for and 2DL4 an that associates with indicates that of the activating receptors with of DAP12 upon in the As both DAP12 and FcεRI-γ KIR with cytoplasmic that activation signalling (Fig. 1). As shown in the basic transmembrane residues and their positions between the of activating KIR and the protein Consistent with other immune receptors and an near the end of the 2DL4 transmembrane domain is to interact with a acid in In contrast, the in and other receptors and is for interaction with a acid in of the in 2DL4 or the in associations with FcεRI-γ and of the acid in DAP12 with As basic residues in receptor transmembrane domains with acidic residues in transmembrane it is that receptors and 2DL4 can the cell surface in the of DAP12 or Interestingly, the of FcεRI-γ with 2DL4 to be of expression levels of FcεRI-γ in NK cells, and of FcεRI-γ cell surface expression of the basic residues in KIR and the acidic in proteins each other and promote and surface of human killer cell immunoglobulin-like receptor (KIR) transmembrane transmembrane domain sequences are shown for each human KIR, based upon the most amino acid encoded in alleles for each amino acids within the activating KIR are in amino acids within the transmembrane domains are and in The a in the sequence that was for Although minor polymorphic within the transmembrane domains of KIR they are between amino The significant alterations that eliminate the unique and residues found in the activating KIR and positions are at the were derived from the The amino acids found within the transmembrane domains of human inhibitory KIR are conserved (Fig. 2). In contrast, a is and conserved within the transmembrane domains of the activating KIR and could potentially contribute to protein interactions within the The transmembrane and are to amino acids in and this unique natural significantly DAP12 and of activating Interestingly, residues are also found within the transmembrane domains of activating KIR in receptors and position 16 in as as 3DL3 the structure of a transmembrane domain, a et al. demonstrated that of the transmembrane in with the same group found that of transmembrane residues other than the in with either or not alter with whereas of the to significantly In the is and for of receptors with although other amino acids may contribute to the of the transmembrane studies have identified transmembrane polymorphic variations that significantly influence KIR function. most 3DL1 alleles encode an at the position in the transmembrane domain (Fig. alleles encode including which has inhibitory function. et al. found that of this to inhibitory function of toward ligands, that even a minor between two residues can significantly impact inhibitory a is found at position in the transmembrane domains of and 2DS5, other KIR contain at that position (Fig. 2). et al. found that surface expression of was significantly when the is to cell surface expression levels of 2DS5 are the at position and surface expression of 2DS5 is instead in alleles by variations in extracellular variations in transmembrane residues can influence function and but their impacts vary between KIR. the most sequence elements in inhibitory KIR are the engagement with MHC-I ligands, KIR are by family kinases to docking sites for domain-containing protein tyrosine and of and is to activation toward target cells by critical tyrosine that activation and have distinct for recruitment to KIR, of the tyrosine of 3DL1 in a inhibitory function that is through recruitment of of by this KIR was by significant in the receptor could recruit of the these these that of both is to recruit and both and in to KIR inhibitory function. As activating KIR cytoplasmic domains, inhibitory KIR have long cytoplasmic KIR2DL4 is a exception of an activating KIR containing a long cytoplasmic As shown in the length of the cytoplasmic domains of long-tailed KIR is although most contain two which are to inhibitory function. the of activating KIR the cytoplasmic which inhibitory function. is also important to that a found in of the alleles of 2DL4 a cytoplasmic domain that cell surface expression and of human killer cell immunoglobulin-like receptor (KIR) cytoplasmic cytoplasmic domain sequences are shown for each human KIR, based upon the most amino acid encoded in alleles for each were derived from the sites and in protein tyrosine-based inhibitory motif and immunoreceptor tyrosine-based motif sequences are and in The cytoplasmic domains of four long domain KIR 2DL5, and contain only one The C-terminal tyrosine in both and 3DL2 is instead in a sequence of an immunoreceptor tyrosine-based motif which is a sequence known to recruit and the adaptor can recruit both and but not although inhibitory function of was preferentially by protein, but not The inhibitory of the the cytoplasmic domain was also shown to be potent than that of These data that the to rely more on recruitment than to inhibitory function. In with these by et al. demonstrated that the cytoplasmic domain of 3DL2 than that of 2DL4 and 3DL3 contain only the (Fig. receptor containing the extracellular and transmembrane domains of 3DL1 and the cytoplasmic domain of 2DL4 demonstrated a potent inhibitory through recruitment of Interestingly, 2DL4 is an activating receptor that does not appear to be influenced by the the functional of recruitment to 2DL4 least of the activation signalling through 2DL4 is of with FcεRI-γ and to be through the cytoplasmic unique signalling may activation of by 2DL4 within early As 3DL3 only the it is to function as an inhibitory receptor through recruitment of although this has not Although it was that tyrosine of the two cytoplasmic was the only functionally event on KIR, several cytoplasmic and sites have also In level was significantly higher than tyrosine in an NK cell and was significantly in primary NK cells by protein with The major sites in 3DL1 are two of the and as as distinct and sites between the two and (Fig. in and in that these individual sites could be by 1 protein and an to be a based upon of the to a include protein kinases and although evidence of by of these kinases is In studies showed that could be by several subsequent these sites will be referred to by the for which they serve as and three of these four and sites extensive conservation within KIR sequences from humans and other mammals When the human KIR the site is present in KIR, the site is found in human KIR with long cytoplasmic domains, including 2DL4, but is in KIR, of the In contrast, the site is conserved in KIR, On the other hand, the site is present among KIR. the and sites are also conserved in most primate KIR, whereas their varies in KIR from other mammals (Fig. Interestingly, the site in KIR from and is with which is known to a functional for such a at that of mammalian killer cell immunoglobulin-like receptor (KIR) cytoplasmic cytoplasmic domain sequences are shown for each KIR, based upon the most amino acid encoded in alleles for each sites and in protein tyrosine-based inhibitory immunoreceptor tyrosine-based and related sequences are and in The a into the sequence for in the site that could as residues are and sequence human and the many sequence identified a of positions in human KIR, only a single polymorphism has within the and polymorphism is a of the site to in which eliminate the it is that the and sites on KIR cytoplasmic domains are conserved, especially within primates, that they have evolved to important in KIR function. evidence in NK cell indicates that at least and can KIR surface expression levels by internalization and of 3DL1 at either the and sites or the site to KIR surface expression by On the other hand, of the site to acid not surface expression and or toward target cells, which was to the higher receptor expression On the other hand, at the site on 3DL1 in a inhibitory surface expression levels when compared with These data that has the to influence KIR inhibitory function. of the site on 3DL1 to surface receptor whereas at the same site the site was at a than either 3DL1 or an these data that of KIR at a highly conserved site between the two in to the receptor on the cell The functional impact of the to polymorphism in the site of has not but is to be to the Interestingly, an demonstrated that engagement can KIR expression on T that activation may induce KIR to this et al. found that surface levels of a receptor containing the KIR cytoplasmic domain were upon of through a mechanism surface was at least by of the site In that surface of inhibitory KIR are essential to tolerance of NK cells and the subset of T cells, and a growing body of evidence indicates that the cytoplasmic site a in surface These in inhibitory KIR are with and known to the surface internalization and intracellular of other studies are to the functional impacts of KIR at the and as as other cytoplasmic and which could also potentially be of these could influence KIR expression NK cell In the KIR are a highly polymorphic family of human receptors, and many sequence variations can significantly impact their ligand affinity and function. The KIR are key regulators of immune especially on NK cells, and the level of KIR surface expression can significantly influence the balance of signals NK cell maturation and growing literature indicates that distinct combinations of KIR and HLA class I genes can contribute to and the outcome of haematopoietic stem cell Although we have a basic understanding of how major KIR structural elements contribute to our present of the array of polymorphic influences is to our are the influences of the polymorphism among HLA class I ligands and the ligands for several KIR. These are currently our to fully establish many of KIR/HLA class I interactions on human we a more understanding of the influences of polymorphic variations in KIR sequences on surface signalling and HLA In to the influences of we are only to understand how protein and can impact KIR expression and function. understanding of these may provide to surface expression of KIR and NK cell function. is that the interactions between polymorphic KIR and HLA ligands are more to our immune responses to infections, and transplantation than is currently and for critical of this The for our to of was by and and by of from the of The was also in by an from the of The of this is the responsibility of the and does not the of the The have
Campbell et al. (Fri,) studied this question.