Structural and functional aspects of the Ly49 natural killer cell receptors

Natural killer cells are part of the first line of innate immune defence against virus-infected cells and cancer cells in the vertebrate immune system. They are called 'natural' killers because, unlike cytotoxic T cells, they do not require a previous challenge and preactivation to become active. The Ly49 NK receptors are type II transmembrane glycoproteins, structurally characterized as disulphide-linked homodimers. They share extensive homology with C-type lectins, and they are encoded by a multigene family that in mice maps on chromosome 6. A fine balance between inhibitory and activating signals regulates the function of NK cells. Inhibitory Ly49 molecules bind primarily MHC class I ligands, whereas the ligands for activating Ly49 molecules may include MHC class I, but also interestingly MHC class I-like molecules expressed by viruses, as is the case for Ly49H, which binds the m157 gene product of murine cytomegalovirus. In this study, we review the function and X-ray crystal structure of the Ly49 NK cell receptors hitherto determined (Ly49A, Ly49C and Ly49I), and the structural features of the Ly49/MHC class I interaction as revealed by the X-ray crystal structures of Ly49A/H-2Dd and the recently determined Ly49C/H-2Kb.     

Binding of the natural killer cell inhibitory receptor Ly49A to its major histocompatibility complex class I ligand. Crucial contacts include both H-2Dd AND beta 2-microglobulin.

Ly49A, an inhibitory C-type lectin-like mouse natural killer cell receptor, functions through interaction with the major histocompatibility complex class I molecule, H-2D(d). The x-ray crystal structure of the Ly49A.H-2D(d) complex revealed that homodimeric Ly49A interacts at two distinct sites of H-2D(d): Site 1, spanning one side of the alpha1 and alpha2 helices, and Site 2, involving the alpha1, alpha2, alpha3, and beta(2)m domains. Mutants of Ly49A, H-2D(d), and beta(2)-microglobulin at intermolecular contacts and the Ly49A dimer interface were examined for binding affinity and kinetics. Although mutations at Site 1 had little affect, several at Site 2 and at the dimer interface hampered the Ly49A.H-2D(d) interaction, with no effect on gross structure or T cell receptor interaction. The region surrounding the most critical residues (in H-2D(d), Asp(122); in Ly49A, Asp(229), Ser(236), Thr(238), Arg(239), and Asp(241); and in beta(2)-microglobulin, Gln(29) and Lys(58)) of the Ly49A.H-2D(d) interface at Site 2 includes a network of water molecules, suggesting a molecular basis for allelic specificity in natural killer cell recognition.     

Crystal structure of the Ly49I natural killer cell receptor reveals variability in dimerization mode within the Ly49 family

Natural killer (NK) cells play a crucial role in the detection and destruction of virally infected and tumor cells during innate immune responses. The cytolytic activity of NK cells is regulated through a balance of inhibitory and stimulatory signals delivered by NK receptors that recognize classical major histocompatabilty complex class I (MHC-I) molecules, or MHC-I homologs such as MICA, on target cells. The Ly49 family of NK receptors (Ly49A through W), which includes both inhibitory and activating receptors, are homodimeric type II transmembrane glycoproteins, with each subunit composed of a C-type lectin-like domain tethered to the membrane by a stalk region. We have determined the crystal structure, at 3.0 A resolution, of the murine inhibitory NK receptor Ly49I. The Ly49I monomer adopts a fold similar to that of other C-type lectin-like NK receptors, including Ly49A, NKG2D and CD69. However, the Ly49I monomers associate in a manner distinct from that of these other NK receptors, forming a more open dimer. As a result, the putative MHC-binding surfaces of the Ly49I dimer are spatially more distant than the corresponding surfaces of Ly49A or NKG2D. These structural differences probably reflect the fundamentally different ways in which Ly49 and NKG2D receptors recognize their respective ligands: whereas the single MICA binding site of NKG2D is formed by the precise juxtaposition of two monomers, each Ly49 monomer contains an independent binding site for MHC-I. Hence, the structural constraints on dimerization geometry may be relatively relaxed within the Ly49 family. Such variability may enable certain Ly49 receptors, like Ly49I, to bind MHC-I molecules bivalently, thereby stabilizing receptor-ligand interactions and enhancing signal transmission to the NK cell.     

Human-specific evolution of killer cell immunoglobulin-like receptor recognition of major histocompatibility complex class I molecules

In placental mammals, natural killer (NK) cells are a population of lymphocytes that make unique contributions to immune defence and reproduction, functions essential for survival of individuals, populations and species. Modulating these functions are conserved and variable NK-cell receptors that recognize epitopes of major histocompatibility complex (MHC) class I molecules. In humans, for example, recognition of human leucocyte antigen (HLA)-E by the CD94:NKG2A receptor is conserved, whereas recognition of HLA-A, B and C by the killer cell immunoglobulin-like receptors (KIRs) is diversified. Competing demands of the immune and reproductive systems, and of T-cell and NK-cell immunity-combined with the segregation on different chromosomes of variable NK-cell receptors and their MHC class I ligands-drive an unusually rapid evolution that has resulted in unprecedented levels of species specificity, as first appreciated from comparison of mice and humans. Counterparts to human KIR are present only in simian primates. Observed in these species is the coevolution of KIR and the four MHC class I epitopes to which human KIR recognition is restricted. Unique to hominids is the emergence of the MHC-C locus as a supplier of specialized and superior ligands for KIR. This evolutionary trend is most highly elaborated in the chimpanzee. Unique to the human KIR locus are two groups of KIR haplotypes that are present in all human populations and subject to balancing selection. Group A KIR haplotypes resemble chimpanzee KIR haplotypes and are enriched for genes encoding KIR that bind HLA class I, whereas group B KIR haplotypes are enriched for genes encoding receptors with diminished capacity to bind HLA class I. Correlating with their balance in human populations, B haplotypes favour reproductive success, whereas A haplotypes favour successful immune defence. Evolution of the B KIR haplotypes is thus unique to the human species.     

Variable dimerization of the Ly49A natural killer cell receptor results in differential engagement of its MHC class I ligand

Natural killer (NK) cells play a vital role in the detection and elimination of virally infected and tumor cells. The Ly49 family of NK receptors regulates NK cell function by sensing major histocompatibility complex (MHC) class I molecules on target cells. Previous crystal studies revealed that the Ly49A homodimer binds one MHC molecule in an asymmetric interaction, whereas the Ly49C homodimer binds two MHC in a symmetrical fashion. Moreover, the bound receptors adopt distinctly different homodimeric forms: a "closed state" for Ly49A and an "open state" for Ly49C. Steric clashes between MHC molecules would preclude the closed Ly49A dimer from engaging two MHC in the manner of the open Ly49C dimer. To determine whether individual Ly49 receptors can undergo a conformational switch enabling them to bind MHC in different ways, we carried out a solution NMR study of unbound Ly49A, aided by dipolar coupling technology. This study reveals that, in solution, unligated Ly49A adopts a symmetric, open-state, homodimer conformation similar to that seen previously for Ly49C. Hence, Ly49A can assume both closed and open states. To address whether the Ly49A dimer can bind two MHC molecules in solution, besides the binding of one MHC observed in the crystal, we carried out analytical ultracentrifugation experiments. Velocity sedimentation demonstrates that the Ly49A dimer can engage two MHC molecules in solution, in agreement with NMR results showing that unbound Ly49A exists predominantly in the open state.     

The activating Ly49W and inhibitory Ly49G NK cell receptors display similar affinities for identical MHC class I ligands

The Ly49 receptor family plays an important role in the regulation of murine natural killer (NK) cell effector function. They recognize cell surface-expressed class I MHC (MHC-I) and are functionally equivalent to the killer Ig-related receptors (KIRs) in human NK cells. Ly49s exist in activating and inhibitory forms with highly homologous extracellular domains, displaying greater variability in the stalk regions. Inhibitory Ly49s can recognize self-MHC-I and therefore mediate tolerance to self. The role of activating Ly49 receptors is less clear. Some activating Ly49 receptors have been shown to recognize MHC-I molecules. The binding affinity of activating Ly49 receptors with MHC-I is currently unknown, and we sought to examine the affinities of two highly related receptors, an activating and an inhibitory Ly49 receptor, for their shared MHC-I ligands. The ectodomain of inhibitory Ly49G of the BALB/c mouse strain is highly similar to the Ly49W activating receptor in the nonobese diabetic (NOD) mouse. Recombinant soluble Ly49G and W were expressed, refolded, and analyzed for binding affinity with MHC-I by surface plasmon resonance. We found that Ly49G and Ly49W bound with similar affinity to the same MHC-I molecules. These results are a first determination of an activating Ly49 receptor affinity for MHC-I and show that, unlike prior results obtained with activating and inhibitory KIR receptors, functional homologues to Ly49 receptors, activating and inhibitory Ly49, can recognize common MHC-I ligands, with similar affinities.     

Peptide specificity of RT1-A1(c), an inhibitory rat major histocompatibility complex class I natural killer cell ligand

The rat major histocompatibility complex class Ia allelomorph RT1-A1(c) is a potent ligand for the recently identified inhibitory rLy-49 receptor, STOK-2. With the ultimate objective of studying the interactions of these molecules using structural and functional methods, we undertook a detailed study of its peptide specificity. The study revealed that designing an "ideal peptide" by choosing the most abundant residues in the "binding motif" obtained by pool sequencing does not necessarily yield an optimal binding peptide. For RT1-A1(c), as many as four positions, P2, P4, P5, and P9, were detected as putative anchors. Since this molecule displays a preference for highly hydrophobic peptides, we tested binding of peptides derived from the known leader peptide sequences of other rat histocompatibility complex class I molecules. One such peptide, found to bind well, requiring 1.6 microm peptide to achieve 50% stabilization, was searched for in vivo. Natural RT1-A1(c) binding peptides were purified from rat splenocytes and characterized by mass spectrometry using a combined matrix-assisted laser desorption ionization/time-of-flight and quadrupole time-of-flight approach. Results showed that the signal sequence-derived peptide was not detectable in the purified peptide pool, which was composed of a complex spectrum of peptides. Seven of these self-peptides were successfully sequenced.     

Molecular docking of superantigens with class II major histocompatibility complex proteins

The molecular recognition of two superantigens with class II major histocompatibility complex molecules was simulated by using protein– protein docking. Superantigens studied were staphylococcal enterotoxin B (SEB) and toxic shock syndrome toxin-1 (TSST-1) in their crystallographic assemblies with HLA-DR1. Rigid-body docking was performed sampling configurational space of the interfacial surfaces by employing a strategy of partitioning the contact regions on HLA-DR1 into separate molecular recognition units. Scoring of docked conformations was based on an electrostatic continuum model evaluated with the finite-difference Poisson– Boltzmann method. Estimates of nonpolar contributions were derived from the buried molecular surface areas. We found for both superantigens that docking the HLA-DR1 surface complementary with the SEB and TSST-1 contact regions containing a homologous hydrophobic surface loop provided sufficient recognition for the reconstitution of native-like conformers exhibiting the highest-scoring free energies. For the SEB complex, the calculations were successful in reproducing the total association free energy. A comparison of the free-energy determinants of the conserved hydrophobic contact residue indicates functional similarity between the two proteins for this interface. Though both superantigens share a common global association mode, differences in binding topology distinguish the conformational specificities underlying recognition. © 1998 John Wiley & Sons, Ltd.     

Mapping of the BALB/c Ly49 cluster defines a minimal natural killer cell receptor gene repertoire

The BALB/c inbred mouse strain is one of the most commonly used for immunological studies and is an animal model for natural killer (NK) cell function during pathogen infection and tumorigenesis. To understand better NK cell function in this strain, the complete BALB/c Ly49 haplotype was deduced. The BALB/c haplotype spans approximately 300 kb with a gene order and content of Ly49q, e, x, i, g, l, c, and a. Functional BALB/c alleles of Ly49q and e were isolated and found to be conserved. The BALB/c cluster represents a minimal haplotype as it contains many fewer functional genes than the 129 or B6 mouse strains. The small number of BALB/c Ly49 genes is due mainly to an absent group of genes (relative to B6 and 129) between Ly49x and i, although other smaller deletions are present. These gene deletions provide a genetic basis for the lack of certain Ly49-associated NK cell functions in this mouse strain. Finally, the mapping of a third Ly49 haplotype reveals that the basic murine Ly49 repertoire is composed of three framework gene pairs (Ly49q and e, Ly49i and g, and Ly49c and a) that are interspersed with variable numbers of strain-specific Ly49.     

Molecular organization of the canine major histocompatibility complex

The major histocompatibility complex (MHC) is composed of a tightly linked cluster of genes; in dogs, this is referred to as the dog leukocyte antigen (DLA) region. The canine MHC is located on chromosome 12, and several genes within the DLA region have been identified that have significant sequence similarity to their human counterparts. However, in order to characterize other loci in the DLA region, DNA sequencing has begun using a canine bacterial artificial chromosome (BAC) library. Initially 135 BAC clones were isolated from a BAC library using a mixture of human and canine probes. These BAC clones were screened with locus-specific primers in polymerase chain reactions (PCRs). Fifty-six BAC clones were subjected to FingerPrinted Contig (FPC) analysis and several overlapping clones were identified. One BAC clone RP81-231-G24 has been sequenced. Preliminary sequence analysis of this 150 kb clone indicates that it contains the region where the class I and class III regions are joined and encompasses DLA-12a, DLA-53, DLA-12, DLA-64, TNF-alpha, and a canine gene that appears to resemble the HLA class III gene HSPA1A (HSP70-1).     

Generation of major histocompatibility complex class I antigens

Presentation of antigenic peptides by major histocompatibility complex (MHC) class I molecules on the surface of antigen-presenting cells is an effective extracellular representation of the intracellular antigen content. The intracellular proteasome-dependent proteolytic machinery is required for generating MHC class I-presented peptides. These peptides appear to be derived mainly from newly synthesized defective ribosomal products, ensuring a rapid cytotoxic T lymphocyte-mediated immune response against infectious pathogens. Here we discuss the generation of MHC class I antigens on the basis of the currently understood molecular, biochemical and cellular mechanisms.     

Two structurally related rat Ly49 receptors with opposing functions (Ly49 stimulatory receptor 5 and Ly49 inhibitory receptor 5) recognize nonclassical MHC class Ib-encoded target ligands

The Ly49 family of lectin-like receptors in rodents includes both stimulatory and inhibitory members. Although NK alloreactivity in mice is regulated primarily by inhibitory Ly49 receptors, in rats activating Ly49 receptors are equally important. Previous studies have suggested that activating rat Ly49 receptors are triggered by polymorphic ligands encoded within the nonclassical class Ib region of the rat MHC, RT1-CE/N/M, while inhibitory Ly49 receptors bind to widely expressed classical class Ia molecules encoded from the RT1-A region. To further investigate rat Ly49-mediated regulation of NK alloreactivity, we report in this study the identification and characterization of two novel paired Ly49 receptors that we have termed Ly49 inhibitory receptor 5 (Ly49i5) and Ly49 stimulatory receptor 5 (Ly49s5). Using a new mAb (mAb Fly5), we showed that Ly49i5 is an inhibitory receptor that recognizes ligands encoded within the class Ib region of the u and l haplotypes, while the structurally related Ly49s5 is an activating receptor that recognizes class Ib ligands of the u haplotype. Ly49s5 is functionally expressed in the high NK-alloresponder PVG strain, but not in the low alloresponder BN strain, in which it is a pseudogene. Ly49s5 is hence not responsible for the striking anti-u NK alloresponse previously described in BN rats (haplotype n), which results from repeated alloimmunizations with u haplotype cells. The present studies support the notion of a complex regulation of rat NK alloreactivity by activating and inhibitory Ly49 members, which may be highly homologous in the extracellular region and bind similar class Ib-encoded target ligands.     

Cloning of the bovine major histocompatibility complex class II genes

Class II genes of the bovine major histocompatibility complex (MHC) have been cloned from a genomic library. The library was constructed in the bacteriophage lambda vector EMBL3 and comprises approximately 10 times the equivalent of the haploid genome. Half the library was screened with the human DQA, DQB, DRA and DRB cDNA probes. Of the 100 positively hybridizing phage clones, 37 were eventually fully characterized and mapped by means of Southern blot analysis. The exons encoding the first, second and transmembrane domain of all different A and B genes were subcloned and mapped in more detail. These analyses showed that these 37 clones were derived from five different A and 10 different B genes. The hybridization studies indicate that we have cloned and mapped two DQA genes, one DRA gene, two other A genes, four DQB genes, three DRB genes and three other B genes. Since the library was made from a heterozygous animal, this would suggest that there are at least one DQA, one DRA one other undefined A, two DQB, two DRB and one or two other undefined B genes in the haploid genome of Holstein Friesian cattle.