Molecular architecture of the major histocompatibility complex class I-binding site of Ly49 natural killer cell receptors

Natural killer (NK) cells play a vital role in the detection and destruction of virally infected and tumor cells during innate immune responses. The highly polymorphic Ly49 family of NK receptors regulates NK cell function by sensing major histocompatibility complex class I (MHC-I) molecules on target cells. Despite the determination of two Ly49-MHC-I complex structures, the molecular features of Ly49 receptors that confer specificity for particular MHC-I alleles have not been identified. To understand the functional architecture of Ly49-binding sites, we determined the crystal structures of Ly49C and Ly49G and completed refinement of the Ly49C-H-2K(b) complex. This information, combined with mutational analysis of Ly49A, permitted a structure-based classification of Ly49s that we used to dissect the binding site into three distinct regions, each having different roles in MHC recognition. One region, located at the center of the binding site, has a similar structure across the Ly49 family and mediates conserved interactions with MHC-I that contribute most to binding. However, the preference of individual Ly49s for particular MHC-I molecules is governed by two regions that flank the central region and are structurally more variable. One of the flanking regions divides Ly49s into those that recognize both H-2D and H-2K versus only H-2D ligands, whereas the other discriminates among H-2D or H-2K alleles. The modular design of Ly49-binding sites provides a framework for predicting the MHC-binding specificity of Ly49s that have not been characterized experimentally.     

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.     

Influence of xenogeneic beta2-microglobulin on functional recognition of H-2Kb by the NK cell inhibitory receptor Ly49C

NK cells maintain self-tolerance through expression of inhibitory receptors that bind MHC class I (MHC-I) molecules. MHC-I can exist on the cell surface in several different forms, including "peptide-receptive" or PR-MHC-I that can bind exogenous peptide. PR-MHC-I molecules are short lived and, for H-2K(b), comprise approximately 10% of total MHC-I. In the present study, we confirm that signaling through the mouse NK inhibitory receptor Ly49C requires the presence of PR-K(b) and that this signaling is prevented when PR-K(b) is ablated by pulsing with a peptide that can bind to it with high affinity. Although crystallographic data indicate that Ly49C can engage H-2K(b) loaded with high-affinity peptide, our data suggest that this interaction does not generate an inhibitory signal. We also show that no signaling occurs when the PR-K(b) complex has mouse beta(2)-microglobulin (beta(2)m) replaced with human beta(2)m, although replacement with bovine beta(2)m has no effect. Furthermore, we show that beta(2)m exchange occurs preferentially in the PR-K(b) component of total H-2K(b). These conclusions were reached in studies modulating the sensitivity to lysis of both NK-resistant syngeneic lymphoblasts and NK-sensitive RMA-S tumor cells. We also show, using an in vivo model of lymphocyte recirculation, that engrafted lymphocytes are unable to survive NK attack when otherwise syngeneic lymphocytes express human beta(2)m. These findings suggest a qualitative extension of the "missing self" hypothesis to include NK inhibitory receptors that are restricted to the recognition of unstable forms of MHC-I, thus enabling NK cells to respond more quickly to events that decrease MHC-I synthesis.     

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.     

The NK cell MHC class I receptor Ly49A detects mutations on H-2Dd inside and outside of the peptide binding groove

The NK cell inhibitory receptor Ly49A recognizes the mouse MHC class I molecule H-2D(d) and participates in the recognition of missing self. Previous studies indicated that the determinant recognized by Ly49A exists in alpha1/alpha2 domain of H-2D(d). Here we have substituted polymorphic as well as conserved residues of H-2D(d) alpha1/alpha2 domain (when compared with H-2K(d), which does not interact with Ly49A). We then tested the ability of the H-2D(d) mutants to interact with Ly49A by soluble Ly49A tetramer binding and NK cell cytotoxicity inhibition assays. Individual introduction of mutations converting the H-2D(d) residue into the corresponding H-2K(d) residue (N30D, D77S, or A99F) in H-2D(d) partially abrogated the interaction between Ly49A and H-2D(d). Introduction of the three mutations into H-2D(d) completely abolished Ly49A recognition. Individual introduction of D29N or R35A mutation into the residues of H-2D(d) that are conserved among murine MHC class I severely impaired the interaction. The crystal structure of H-2D(d) reveals that D77 and A99 are located in the peptide binding groove and that N30, D29, and R35 are in the interface of the three structural domains of MHC class I: alpha1/alpha2, alpha3, and beta(2)-microglobulin. These data suggest that Ly49A can monitor mutations in MHC class I inside and outside of the peptide binding groove and imply that inhibitory MHC class I-specific receptors are sensitive to mutations in MHC class I as well as global loss of MHC class I. Our results also provide insight into the molecular basis of Ly49A to distinguish MHC class I polymorphism.     

Cell-free synthesis and membrane insertion of mouse H-2Dd histocompatibility antigen and beta 2-microglobulin.

Messenger RNA from SL2 lymphoma cells was translated in a cell-free system in the presence of microsomal membranes. Mouse H-2Dd histocompatibility antigen was correctly assembled in the microsomal membranes, and transmembrane insertion of the nascent chain was accompanied by glycosylation and cleavage of the signal sequence H-2Kd antigens, synthesized in vivo, comprised a transmembrane glycoprotein and an unglycosylated protein in the cytoplasm. The glycosylated forms of the H-2Dd and H-2Kd antigens were modified during intracellular transport from the endoplasmic reticulum to the cell surface. beta 2-Microglobulin was also synthesized in vitro, and transfer of this protein into microsomal vesicles was accompanied by cleavage of its signal sequence. In the endoplasmic reticulum, beta-microglobulin can bind to newly synthesized H-2d glycoproteins. The mRNAs coding for beta 2-microglobulin and H-2Dd antigen could be separated on aqueous sucrose gradients.     

Photophysical analysis of class I major histocompatibility complex protein assembly using a xanthene-derivatized beta2-microglobulin

Spectral changes and a sixfold increase in the emission intensity were observed in the fluorescence of a single xanthene probe (Texas red) attached to beta2m-microglobulin (beta2m) upon assembly of beta2m into a ternary complex with mouse H-2Kd heavy chain and influenza nuclear protein peptide. Dissociation of the labeled beta2m from the ternary complex restored the probe's fluorescence and absorption spectra and reduced the emission intensity. Thus changes in xanthene probe fluorescence upon association/dissociation of the labeled beta2m molecule with/from the ternary complex provide a simple and convenient method for studying the assembly/dissociation mechanism of the class I major histocompatibility complex (MHC-I) encoded molecule. The photophysical changes in the probe can be accounted for by the oligomerization of free labeled beta2m molecules. The fluorescence at 610 nm is due to beta2m dimers, where the probes are significantly separated spatially so that their emission and excitation properties are close to those of xanthene monomers. Fluorescence around 630 nm is due to beta2m oligomers where xanthene probes interact. Minima in the steady-state excitation (550 nm) and emission (630 nm) anisotropy spectra correlate with the maxima of the high-order oligomer excitation and emission spectra, showing that their fluorescence is more depolarized. These photophysical features are explained by splitting of the first singlet excited state of interacting xanthene probes that can be modeled by exciton theory.     

The MHC class I molecule H-2Dp inhibits murine NK cells via the inhibitory receptor Ly49A.

MHC class I molecules strongly influence the phenotype and function of mouse NK cells. NK cell-mediated lysis is prevented through the interaction of Ly49 receptors on the effector cell with appropriate MHC class I ligands on the target cell. In addition, host MHC class I molecules have been shown to modulate the in vivo expression of Ly49 receptors. We have previously reported that H-2Dd and H-2Dp MHC class I molecules are able to protect (at the target cell level) from NK cell-mediated lysis and alter the NK cell specificity (at the host level) in a similar manner, although the mechanism behind this was not clear. In this study, we demonstrate that the expression of both H-2Dd and H-2Dp class I molecules in target cells leads to inhibition of B6 (H-2b)-derived Ly49A+ NK cells. This inhibition could in both cases be reversed by anti-Ly49A Abs. Cellular conjugate assays showed that Ly49A-expressing cells indeed bind to cells expressing H-2Dp. The expression of Ly49A and Ly49G2 receptors on NK cells was down-regulated in H-2Dp-transgenic (B6DP) mice compared with nontransgenic B6 mice. However, B6DP mice expressed significantly higher levels of Ly49A compared with H-2Dd-transgenic (D8) mice. We propose that both H-2Dd and H-2Dp MHC class I molecules can act as ligands for Ly49A.     

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.     

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.     

Purification and ligand binding of a soluble class I major histocompatibility complex molecule consisting of the first three domains of H-2Kd fused to beta 2-microglobulin expressed in the baculovirus-insect cell system.

A recombinant baculovirus encoding a single-chain murine major histocompatibility complex class I molecule in which the first three domains of H-2Kd are fused to beta 2-microglobulin (beta 2-m) via a 15-amino acid linker has been isolated and used to infect lepidopteran cells. A soluble, 391-amino acid single-chain H-2Kd (SC-Kd) molecule of 48 kDa was synthesized and glycosylated in insect cells and could be purified in the absence of detergents by affinity chromatography using the anti-H-2Kd monoclonal antibody SF1.1.1.1. We tested the ability of SC-Kd to bind antigenic peptides using a direct binding assay based on photoaffinity labeling. The photoreactive derivative was prepared from the H-2Kd-restricted Plasmodium berghei circumsporozoite protein (P.b. CS) peptide 253-260 (YIPSAEKI), a probe that we had previously shown to be unable to bind to the H-2Kd heavy chain in infected cells in the absence of co-expressed beta 2-microglobulin. SC-Kd expressed in insect cells did not require additional mouse beta 2-m to bind the photoprobe, indicating that the covalently attached beta 2-m could substitute for the free molecule. Similarly, binding of the P.b. CS photoaffinity probe to the purified SC-Kd molecule was unaffected by the addition of exogenous beta 2-m. This is in contrast to H-2KdQ10, a soluble H-2Kd molecule in which beta 2-m is noncovalently bound to the soluble heavy chain, whose ability to bind the photoaffinity probe is greatly enhanced in the presence of an excess of exogenous beta 2-m. The binding of the probe to SC-Kd was allele-specific, since labeling was selectively inhibited only by antigenic peptides known to be presented by the H-2Kd molecule.     

A novel receptor on allograft (H-2d)-induced macrophage (H-2b) toward an allogeneic major histocompatibility complex class I molecule, H-2Dd, in mice

The generation of knockout mice demonstrated that noncytotoxic CD4(+), but not cytotoxic CD8(+), T cells were essential for the rejection of skin or organ allografts. Earlier we reported that allograftinduced macrophages (AIM) in mice lysed allografts with H-2 haplotype specificity, implying screening of grafts by AIM. Here, we isolated a cDNA clone encoding a novel receptor on AIM (H-2D(b)) for an allogeneic major histocompatibility complex (MHC) class I molecule, H-2D(d), by using H-2D(d) tetramer and a monoclonal antibody (mAb; R15) specific for AIM. The cDNA (1,181-bp) encoded a 342-amino acid polypeptide with a calculated molecular mass of 45 kDa and was found to be expressed on AIM, but not on resident macrophages or other cells, infiltrating into the rejection site. HEK293T cells transfected with this cDNA reacted with R15 mAb and H-2D(d), but not H-2L(d), H-2K(d), H-2D(b), H-2K(b), H-2D(k), or H-2K(k), molecules; and the H-2D(d) binding was suppressed by the addition of R15 or anti-H-2D(d) mAb. AIM yielded a specific saturation isotherm in the presence of increasing concentrations of H-2D(d), but not H-2D(b) or H-2D(k), molecules. The dissociation constant of AIM toward H-2D(d) tetramers was 1.9 x 10(-9) M ; and the binding was completely inhibited by the addition of R15 or anti-H-2D(d) mAb. These results reveal that a novel receptor for an allogeneic H-2D(d) molecule was induced on effector macrophages responsible for allograft (H-2(d)) rejection in H-2(b) mice.     

Crystallization of murine major histocompatibility complex class I H-2Kb with single peptides.

X-ray quality crystals of a soluble murine class I H-2Kb molecule complexed with three different peptide antigens were grown in several forms by streak seeding and macroseeding methods. Co-crystals with VSV-8 (RGYVYGQL), OVA-8 (SIINFEKL) and SEV-9 (FAPGNYPAL) peptides were grown either from NaH2PO4/HPO4 or from polyethylene glycol 4000 within the pH range 5.0 to 7.5, with the use of 4-methyl-2-pentane diol (MPD) as an additive. The VSV-8 crystals grew in space groups P1, with cell dimensions a = 63.1 A, b = 69.1 A, c = 72.0 A, alpha = 89.9 degrees, beta = 77.1 degrees, gamma = 123.3 degrees and P2(1)2(1)2, with a = 138.1 A, b = 88.6 A, c = 45.7 A, and diffract to 2.9 and 2.3 A, respectively. Crystals of the SEV-9 complex grew from similar crystallization conditions to those of the orthorhombic VSV-8 complex with similar cell parameters and diffract to at least 2.5 A resolution. Crystals of the OVA-8 complex were obtained from either phosphate (space group C2, a = 118.7 A, b = 61.6 A, c = 85.3 A, beta = 108.4 degrees) or polyethylene glycol (space group P1, a = 64.5 A, b = 71.0 A, c = 66.3 A, alpha = 89.7 degrees, beta = 95.7 degrees, gamma = 123.3 degrees) and diffract to 3 A resolution. The crystallization procedures used here significantly increased the rate and production of X-ray quality crystals.     

Pregnancy and interferon tau regulate major histocompatibility complex class I and beta2-microglobulin expression in the ovine uterus

Major histocompatibility complex (MHC) class I molecules, consisting of an alpha chain and beta2-microglobulin (beta2MG), play an important role in immune rejection responses by discriminating self and nonself and are increased by type I interferons during antiviral responses. Interferon tau (IFNtau), the pregnancy-recognition signal in ruminants, is a type I interferon produced by the ovine conceptus between Days 11 and 21 of gestation. In study 1, expression of MHC class I alpha chain and beta2MG mRNA and protein was detected primarily in endometrial luminal epithelium (LE) and glandular epithelium (GE) on Days 10 and 12 of the estrous cycle and pregnancy. On Days 14-20 of pregnancy, MHC class I and beta2MG expression increased only in endometrial stroma and GE and, concurrently, was absent in LE and superficial ductal GE (sGE). Although neither MHC class I nor beta2MG proteins were detected in Day 20 trophectoderm, beta2MG mRNA was detected in conceptus trophectoderm. In study 2, cyclic ewes were ovariectomized on Day 5, treated daily with progesterone to Day 16, received intrauterine infusions between Days 11 and 16 of either control serum proteins or recombinant ovine IFNtau, and were hysterectomized on Day 17. The IFNtau increased MHC class I and beta2MG expression only in endometrial stroma and GE. During pregnancy, MHC class I and beta2MG gene expression is inhibited in endometrial LE and sGE but, paradoxically, is stimulated by IFNtau in the stroma and GE. The silencing of MHC class I alpha chain and beta2MG genes in the endometrial LE and sGE during pregnancy recognition and establishment may be a critical mechanism preventing immune rejection of the conceptus allograft.     

A dominant negative mutant beta 2-microglobulin blocks the extracellular folding of a major histocompatibility complex class I heavy chain

The major histocompatibility complex class I (MHC1) molecule plays a crucial role in cytotoxic lymphocyte function. beta 2-Microglobulin (beta 2m) has been demonstrated to be both a structural component of the MHC1 complex and a chaperone-like molecule for MHC1 folding. beta 2m binding to an isolated alpha 3 domain of MHC1 heavy chain at micromolar concentrations has been shown to accurately model the biochemistry and thermodynamics of beta 2m-driven MHC1 folding. These results suggested a model in which the chaperone-like role of beta 2m is dependent on initial binding to the alpha 3 domain interface of MHC1 with beta 2m. Such a model predicts that a mutant beta 2m molecule with an intact MHC1 alpha 3 domain interaction but a defective MHC1 alpha 1 alpha 2 domain interaction would block beta2m-driven folding of MHC1. In this study we generated such a beta 2m mutant and demonstrated that it blocks MHC1 folding by normal beta 2m at the expected micromolar concentrations. Our data support an initial interaction of beta 2m with the MHC1 alpha 3 domain in MHC1 folding. In addition, the dominant negative mutant beta 2m can block T-cell functional responses to antigenic peptide and MHC1.     

Class I major histocompatibility complex anchor substitutions alter the conformation of T cell receptor contacts

An immunogenic peptide (GP2) derived from HER-2/neu binds to HLA-A2.1 very poorly. Some altered-peptide ligands (APL) of GP2 have increased binding affinity and generate improved cytotoxic T lymphocyte recognition of GP2-presenting tumor cells, but most do not. Increases in binding affinity of single-substitution APL are not additive in double-substitution APL. A common first assumption about peptide binding to class I major histocompatibility complex is that each residue binds independently. In addition, immunologists interested in immunotherapy frequently assume that anchor substitutions do not affect T cell receptor contact residues. However, the crystal structures of two GP2 APL show that the central residues change position depending on the identity of the anchor residue(s). Thus, it is clear that subtle changes in the identity of anchor residues may have significant effects on the positions of the T cell receptor contact residues.     

Strategic mutations in the class I major histocompatibility complex HLA-A2 independently affect both peptide binding and T cell receptor recognition

Mutational studies of T cell receptor (TCR) contact residues on the surface of the human class I major histocompatibility complex (MHC) molecule HLA-A2 have identified a "functional hot spot" that comprises Arg(65) and Lys(66) and is involved in recognition by most peptide-specific HLA-A2-restricted TCRs. Although there is a significant amount of functional data on the effects of mutations at these positions, there is comparatively little biochemical information that could illuminate their mode of action. Here, we have used a combination of fluorescence anisotropy, functional assays, and Biacore binding experiments to examine the effects of mutations at these positions on the peptide-MHC interaction and TCR recognition. The results indicate that mutations at both position 65 and position 66 influence peptide binding by HLA-A2 to various extents. In particular, mutations at position 66 result in significantly increased peptide dissociation rates. However, these effects are independent of their effects on TCR recognition, and the Arg(65)-Lys(66) region thus represents a true "hot spot" for TCR recognition. We also made the observation that in vitro T cell reactivity does not scale with the half-life of the peptide-MHC complex, as is often assumed. Finally, position 66 is implicated in the "dual recognition" of both peptide and TCR, emphasizing the multiple roles of the class I MHC peptide-binding domain.     

Influence of major histocompatibility complex H-2LD class I molecules on spleen colony-forming units

To test whether the major histocompatibility complex class I genes are involved in the regulation of hemopoiesis, the stem cell activities of BALB/c-H-2dm2 (Dm2) mice, which are defective in the expression of H-2L antigens, have been compared with those of the wild-type, BALB/c-Kh, in in vivo and in vitro stem cell assays. In spleen colony-forming unit assays, Dm2 as hosts consistently supported a smaller number of colonies than did BALB/c-Kh. However, both Dm2 and BALB/c-Kh supported a comparable number of colonies in in vitro granulocyte-macrophage colony-forming unit and erythroid colony-forming unit assays. These observations together suggest that the mutation in Dm2 has not affected the hemopoietic potential of the stem cells but may probably affect the hemopoietic microenvironment for the development of the stem cells.     

Kinetics and thermodynamics of beta 2-microglobulin binding to the alpha 3 domain of major histocompatibility complex class I heavy chain

The major histocompatibility complex (MHC) class I molecule plays a crucial role in cytotoxic lymphocyte function. Functional class I MHC exists as a heterotrimer consisting of the MHC class I heavy chain, an antigenic peptide fragment, and beta2-microglobulin (beta2m). beta2m has been previously shown to play an important role in the folding of the MHC heavy chain without continued beta2m association with the MHC complex. Therefore, beta2m is both a structural component of the MHC complex and a chaperone-like molecule for MHC folding. In this study we provide data supporting a model in which the chaperone-like role of beta2m is dependent on initial binding to only one of the two beta2m interfaces with class 1 heavy chain. beta2-Microglobulin binding to an isolated alpha3 domain of the class I MHC heavy chain accurately models the biochemistry and thermodynamics of beta2m-driven refolding. Our results explain a 1000-fold discrepancy between beta2m binding and refolding of MHC1. The biochemical study of the individual domains of complex molecules is an important strategy for understanding their dynamic structure and multiple functions.