Misfolding Ectodomain Mutations of the Lutropin Receptor Increase Efficacy of Hormone Stimulation.

We demonstrate 2 novel mutations of the LHCGR, each homozygous, in a 46,XY patient with severe Leydig cell hypoplasia. One is a mutation in the signal peptide (p.Gln18_Leu19ins9; referred to here as SP) that results in an alteration of the coding sequence of the N terminus of the mature mutant receptor. The other mutation (p.G71R) is also within the ectodomain. Similar to many other inactivating mutations, the cell surface expression of recombinant human LHR(SP,G71R) is greatly reduced due to intracellular retention. However, we made the unusual discovery that the intrinsic efficacy for agonist-stimulated cAMP in the reduced numbers of receptors on the cell surface was greatly increased relative to the same low number of cell surface wild-type receptor. Remarkably, this appears to be a general attribute of misfolding mutations in the ectodomains, but not serpentine domains, of the gonadotropin receptors. These findings suggest that there must be a common, shared mechanism by which disparate mutations in the ectodomain that cause misfolding and therefore reduced cell surface expression concomitantly confer increased agonist efficacy to those receptor mutants on the cell surface. Our data further suggest that, due to their increased agonist efficacy, extremely small changes in cell surface expression of misfolded ectodomain mutants cause larger than expected alterations in the cellular response to agonist. Therefore, for inactivating LHCGR mutations causing ectodomain misfolding, the numbers of cell surface mutant receptors on fetal Leydig cells of 46,XY individuals exert a more exquisite effect on the relative severity of the clinical phenotypes than already appreciated.

Crystal structure of the human natural killer cell inhibitory receptor KIR2DL1-HLA-Cw4 complex.

Inhibitory natural killer (NK) cell receptors down-regulate the cytotoxicity of NK cells upon recognition of specific class I major histocompatibility complex (MHC) molecules on target cells. We report here the crystal structure of the inhibitory human killer cell immunoglobulin-like receptor 2DL1 (KIR2DL1) bound to its class I MHC ligand, HLA-Cw4. The KIR2DL1-HLA-Cw4 interface exhibits charge and shape complementarity. Specificity is mediated by a pocket in KIR2DL1 that hosts the Lys80 residue of HLA-Cw4. Many residues conserved in HLA-C and in KIR2DL receptors make different interactions in KIR2DL1-HLA-Cw4 and in a previously reported KIR2DL2-HLA-Cw3 complex. A dimeric aggregate of KIR-HLA-C complexes was observed in one KIR2DL1-HLA-Cw4 crystal. Most of the amino acids that differ between human and chimpanzee KIRs with HLA-C specificities form solvent-accessible clusters outside the KIR-HLA interface, which suggests undiscovered interactions by KIRs.

A disulfide-linked natural killer cell receptor dimer has higher affinity for HLA-C than wild-type monomer.

Inhibitory receptors on the surface of natural killer (NK) cells recognize specific MHC class I molecules on target cells and prevent the target cell lysis by NK cells. The killer cell immunoglobulin-related receptors (KIR), KIR2D, found in human, specifically interact with polymorphic HLA-C molecules. The crystal structure of the inhibitory receptor, KIR2DL1, revealed a relationship to the hematopoietic receptor family, suggesting that the signaling mechanism of KIR2D molecules may resemble that of the hematopoietic receptors, and involve KIR2D dimerization. We have engineered a disulfide-linked dimer of KIR2DL1 by introducing a free cysteine at the C-terminal stem region of the receptor. The disulfide-linked KIR2DL1 dimer binds to HLA-Cw4 at a molar ratio of one dimer to one HLA-Cw4 molecule. Furthermore, the covalently-linked KIR2DL1 dimer binds more tightly to HLA-Cw4 than the wild-type monomer, suggesting the occurrence of a second binding event that increases the overall affinity of KIR dimer for HLA-C.

Cobalt-mediated dimerization of the human natural killer cell inhibitory receptor.

Upon engagement of specific class I major histocompatibility complex (MHC) molecules on target cells, inhibitory receptors on natural killer (NK) cells deliver a negative signal that prevents the target cell lysis by NK cells. In humans, killer cell immunoglobulin-related receptors (KIR) with two immunoglobulin-like domains (KIR2D) modulate the lysis of target cells bearing specific HLA-C alleles (Moretta, A., Vitale, M., Bottino, C., Orengo, A. M., Morelli, L., Augugliaro, R., Barbaresi, M., Ciccone, E., and Moretta, L. (1993) J. Exp. Med. 178, 597-604). The transduction of inhibitory signals by KIR2D molecules is impaired by the zinc chelator, 1,10-phenanthroline, and mutation of a putative zinc-binding site (Rajagopalan, S., and Long, E. O. (1998) J. Immunol. 161, 1299-1305), but the mechanism by which zinc may affect the function of KIR remains unknown. In this study, the inhibitory NK receptor KIR2DL1 was discovered to dimerize in the presence of Co(2+) as observed on native gel electrophoresis and by gel filtration column chromatography. Furthermore, Co(2+)-mediated KIR2DL1 dimer binds to HLA-Cw4 with higher affinity than the wild type KIR2DL1 monomer. Replacement of the amino-terminal His residue by Ala abolishes the ability of KIR2DL1 to bind Co(2+), indicating that Co(2+)-mediated KIR2DL1 dimerization involves pairing of the D1 domain. Although not observed on native gels, the inhibitory receptor KIR2DL1 can be chemically cross-linked into dimers in the presence of Zn(2+) and its related divalent metal ions, suggesting that Co(2+)-mediated dimerization of KIR2DL1 may mimic a weaker interaction between KIR2DL1 and zinc in vivo.

Structure of human histocompatibility leukocyte antigen (HLA)-Cw4, a ligand for the KIR2D natural killer cell inhibitory receptor.

The crystal structure of the human class I major histocompatibility complex molecule, human histocompatibility leukocyte antigen (HLA)-Cw4, the ligand for a natural killer (NK) cell inhibitory receptor, has been determined, complexed with a nonameric consensus peptide (QYDDAVYKL). Relative to HLA-A2, the peptide binding groove is widened around the COOH terminus of the alpha 1 helix, which contains residues that determine the specificity of HLA-Cw4 for the inhibitory NK receptor, KIR2D. The structure reveals an unusual pattern of internal hydrogen bonding among peptide residues. The peptide is anchored in four specificity pockets in the cleft and secured by extensive hydrogen bonds between the peptide main chain and the cleft. The surface of HLA-Cw4 has electrostatic complementarity to the surface of the NK cell inhibitory receptor KIR2D.

A mutant human beta2-microglobulin can be used to generate diverse multimeric class I peptide complexes as specific probes for T cell receptors.

Antigen-specific receptors (TCR) on CD8 T lymphocytes form relatively short-lived complexes with their natural ligands: peptides in association with major histocompatibility complex (MHC) class I molecules, which consist of a polymorphic heavy chain and a conserved light chain, beta2-microglobulin (beta2-M). To produce soluble MHC-peptide complexes in a form that would bind more stably and could be used to identify, count, and isolate CD8 T cells having the appropriate TCR, we prepared multimeric MHC-peptide complexes. Our work builds on the assembly of recombinant MHC class I peptide complexes using a mutant human beta2-M chain (Tyr 67 > Cys) which can form stable heterodimers with diverse MHC heavy chains. With biotin added to the SH group, the assembled MHC-peptide monomers formed multimers with avidin linked to a fluorochrome. The specific reactivity of the multimeric reagents with human and mouse cytotoxic T cells (CTL) is described. The present approach permits the production of class I multimers, without the necessity of genetic engineering each heavy chain, a significant advantage in view of the enormous polymorphism of MHC heavy chains. Because human beta2-M forms stable heterodimers with diverse class I heavy chains from various species (human and non human primates, mouse, etc.), this procedure is a general method for producing multimers of MHC-peptide complexes as T cell receptor-specific probes.

Structure of the inhibitory receptor for human natural killer cells resembles haematopoietic receptors.

Abnormal cells deficient in class I major histocompatibility complex (MHC) expression are lysed by a class of lymphocytes called natural killer (NK) cells. This lysis provides a defence against pathogens and tumour cells that downregulate MHC expression to avoid an MHC-restricted, T-cell immune response. Normal cells escape lysis because their MHC molecules are recognized by NK-cell inhibitory receptors, which inhibit lysis. Several such inhibitory receptor families have been described in humans and mice. In the human killer-cell inhibitory receptor family, individual p58 members are specific for a subset of class I human leukocyte antigen (HLA)-C molecules. The human p58 natural killer-cell inhibitory receptor clone 42 recognizes HLA-Cw4, -Cw2 and -Cw6, but not HLA-Cw3, -Cw2, -Cw7 or -Cw8, which are recognized by p58 killer-cell inhibitor receptor clone 43. We have determined the X-ray structure of the p58 NK-cell inhibitory receptor clone 42 at 1.7-A resolution. The structure has tandem immunoglobulin-like domains positioned at an acute, 60-degree angle. Loops on the outside of the elbow between the domains form a binding site projected away from the NK-cell surface. The topology of the domains and their arrangement relative to each other reveal a relationship to the haematopoietic receptor family, with implications for the signalling mechanism in NK cells.

Structure of the complex between human T-cell receptor, viral peptide and HLA-A2.

Recognition by a T-cell antigen receptor (TCR) of peptide complexed with a major histocompatibility complex (MHC) molecule occurs through variable loops in the TCR structure which bury almost all the available peptide and a much larger area of the MHC molecule. The TCR fits diagonally across the MHC peptide-binding site in a surface feature common to all class I and class II MHC molecules, providing evidence that the nature of binding is general. A broadly applicable binding mode has implications for the mechanism of repertoire selection and the magnitude of alloreactions.

Direct binding of a soluble natural killer cell inhibitory receptor to a soluble human leukocyte antigen-Cw4 class I major histocompatibility complex molecule.

Natural killer (NK) cells expressing specific p58 NK receptors are inhibited from lysing target cells that express human leukocyte antigen (HLA)-C class I major histocompatibility complex molecules. To investigate the interaction between p58 NK receptors and HLA-Cw4, the extracellular domain of the p58 NK receptor specific for HLA-Cw4 was overexpressed in Escherichia coli and refolded from purified inclusion bodies. The refolded NK receptor is a monomer in solution. It interacts specifically with HLA-Cw4, blocking the binding of a p58-Ig fusion protein to HLA-Cw4-expressing cells, but does not block the binding of a p58-Ig fusion protein specific for HLA-Cw3 to HLA-Cw3-expressing cells. The bacterially expressed extracellular domain of HLA-Cw4 heavy chain and beta2-microglobulin were refolded in the presence of a HLA-Cw4-specific peptide. Direct binding between the soluble p58 NK receptor and the soluble HLA-Cw4-peptide complex was observed by native gel electrophoresis. Titration binding assays show that soluble monomeric receptor forms a 1:1 complex with HLA-Cw4, independent of the presence of Zn2+. The formation of complexes between soluble, recombinant molecules indicates that HLA-Cw4 is sufficient for specific ligation by the NK receptor and that neither glycoprotein requires carbohydrate for the interaction.

DNA binding by an amino acid residue in the C-terminal half of the Rel homology region.

BACKGROUND: Members of the Rel family of transcription factors are important in regulating the inflammatory, acute phase, and immune responses in mammals. The structural basis for sequence-specific binding by Rel proteins is poorly understood, however. In the studies reported here, a new photoaffinity labeling procedure has been used to probe DNA contacts established by a Rel protein, the p50 homodimer of NF-kappa B. RESULTS: Using a novel post-synthetic modification method, 8-azido-2'-deoxyadenosine (N3dA), a photoactive analog of 2'-deoxyadenosine, was introduced at a specific site within a consensus DNA binding site for the NF-kappa B p50 homodimer. Upon irradiation with ultraviolet light, the N3dA-substituted DNA was efficiently photocrosslinked to p50. The crosslinked amino acid of p50 was identified as K244, which lies in the carboxy-terminal half of the Rel homology region (RHR). Mutation of K244 exerts strong effects on DNA binding, confirming the importance of this residue for p50-DNA interactions. CONCLUSIONS: We have used N3dA-containing DNA to identify a residue of NF-kappa B p50 that contacts DNA illustrating the value of this approach in studies of protein-DNA interactions. K244 appears not to contact a DNA base, but rather a phosphate moiety that lies nearby. The segment of protein sequence in which K244 resides has been implicated in dimerization. The results presented here suggest that the DNA-binding domain extends farther toward the carboxy-terminus than previously thought.