Variable MHC class I engagement by Ly49 natural killer cell receptors demonstrated by the crystal structure of Ly49C bound to H-2Kb

Dam, Julie; Guan, R.; Natarajan, Kannan; Dimasi, Nazzareno; Chlewicki, Lukasz K.; Kranz, David M.; Schuck, Peter; Margulies, David H.; Mariuzza, Roy A.

doi:10.1038/ni1006

Cited by 128 publications

(200 citation statements)

References 51 publications

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“…To further characterize MAM dimerization, we performed SV AUC experiments on MAM under various buffer conditions in the presence or absence of Zn 2+ , using a Beckman XL-1 analytical ultracentrifuge. Due to technical advances and software improvements, AUC has become a powerful tool with which to investigate the oligomerization state of proteins and protein complexes (Dam et al, 2003;Guan et al, 2004;Lebowitz et al, 2002;Schuck et al, 2002). Through monitoring of the sedimentation of a macromolecule in the centrifugal field, its hydrodynamic and thermodynamic properties can be characterized in solution, with the macromolecule in its native state.…”

Section: Analysis Of Mam Dimerization By Sedimentation Velocitymentioning

confidence: 99%

Mutagenesis, biochemical, and biophysical characterization of Mycoplasma arthritidis-derived mitogen

Li¹,

Zhao²,

Guo³

et al. 2007

Molecular Immunology

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Mycoplasma arthritidis -derived mitogen (MAM) is a superantigen (SAg) that can activate large fractions of T cells bearing particular TCR Vβ elements. Here we report the mutagenesis, biochemical and biophysical studies on the dimerization of MAM in solution. Our studies showed that although MAM mainly exists as a monomer in solution, a small percentage of MAM molecules form homodimer at high protein concentration, regardless of the presence of Zn 2+ . A distinct peak corresponding to a MAM homodimer was detected in the presence of EDTA, using both chemical cross-linking and analytical ultracentrifugation methods. Further mutagenesis studies revealed that single mutation of residues at the interface of the crystallographic dimer of MAM does not significantly affect the dimerization of MAM in solution. Circular dichroism (CD) analysis indicated that addition of Zn 2+ does not induce conformational changes of MAM from its apo-state. Thermal denaturation experiments indicated that addition of Zn 2+ to MAM solution resulted in a decrease of melting point (T m ), whereas addition of EDTA did not affect the T m of MAM. These results imply that there is no defined Zn 2+ -binding site on MAM.

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Section: Analysis Of Mam Dimerization By Sedimentation Velocitymentioning

confidence: 99%

Mutagenesis, biochemical, and biophysical characterization of Mycoplasma arthritidis-derived mitogen

Li¹,

Zhao²,

Guo³

et al. 2007

Molecular Immunology

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“…Mutagenic primers were designed to mutate to alanine, potential solvent-exposed interaction residues directly below or near the B pocket (residues 6, 8, 10, 23, 27, and 98), below the F pocket (residues 115 and 122), and in the ␣3 domain of the MHC I H chain (residues 223, 232, 243, 262) as defined by Ly49A/H-2D d and Ly49C/H-2K b cocrystals (30,31). Double mutants in the B subsite (R6A F8A), F subsite (Q115A D122A), C subsite (E232A Y262A, D223A K243A, E232A K243A, Y262A K243A), and between subsites (R6A D122A, R6A K243A, D122A K243A) were generated through an additional round of mutagenesis on RT1-A1 c constructs already containing the primary mutation.…”

Section: Mutagenesis Of Mhc Imentioning

confidence: 99%

“…Through mutagenesis studies and examination of KIR/MHC I cocrystal structures, it has been demonstrated that KIR bind MHC I on the top surfaces of the ␣1 and ␣2 helices of MHC I, and this principally involves interaction of polymorphic KIR residues with HLA-C residues 77 and 80 (21,22). The molecular interaction between mouse Ly49 and MHC I has also been studied by mutagenesis (23)(24)(25)(26)(27)(28)(29) and examination of cocrystal structures of Ly49A/H-2D d and Ly49C/H-2K b (30,31), and occurs primarily at a large interface known as site 2 that encompasses MHC I residues below the peptide-binding groove, within the ␣3 domain, and on the ␤ 2 -microglobulin (␤ 2 m). What has remained poorly understood is the mechanism by which this site 2 interface, which is highly conserved both within and between rat and mouse MHC I alleles, can engender the observed allele specificity of Ly49 receptors.…”

mentioning

confidence: 99%

“…Because cocrystal structures of Ly49A with H-2D d (30) and Ly49C with H-2K b (31) predict that both of these Ly49 interact with different residues in the ␣3 domain of their MHC I ligands, we also mutated ␣3 domain residues to determine whether conformational differences in MHC I might also affect how Ly49 are oriented in relation to the ␣3 domain, generating altered dependency on residues in this domain for recognition.…”

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confidence: 99%

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Distinctive Interactions at Multiple Site 2 Subsites by Allele-Specific Rat and Mouse Ly49 Determine Functional Binding and Class I MHC Specificity

Lavender

Chau

Kane

2007

The Journal of Immunology

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Rodent Ly49 exhibit allele-specific MHC I recognition, yet the interaction site, site 2, encompassing the area below the MHC peptide-binding groove, the α3 domain, and associated β2 microglobulin, is highly conserved among rat and mouse MHC I alleles. We previously demonstrated that allele-specific Ly49 recognition can be affected by polymorphisms specifically in the peptide anchor-binding and supertype-defining B pocket of MHC I, possibly through differential conformations assumed by solvent-exposed interaction residues when articulating with this pocket. Through mutagenesis of RT1-A1c and H-2Dd, we map for the first time the interaction site(s) on rat MHC I mediating rat Ly49i2 recognition and the previously unexamined Ly49GBALB/c interaction with H-2Dd. We demonstrate that rat Ly49i2 and mouse Ly49G use both unique and common interactions at three MHC I H chain subsites to mediate functional binding and allele-specific recognition. We find that the F subsite, formed by solvent-exposed residues below the more conserved C-terminal anchor residue-binding F pocket, acts as an anchoring location for both Ly49i2 and Ly49G, whereas these receptors exhibit distinctive reliance on solvent-exposed residues articulating with the polymorphic anchor-binding and supertype-defining pocket(s) at subsite B, as well as on interaction residues at subsite C in the MHC I α3 domain. Our findings, combined with previous Ly49A/H-2Dd and Ly49C/H-2Kb cocrystal data, suggest how allele-specific MHC I conformations and Ly49 polymorphisms may affect Ly49 placement on MHC I ligands and residue usage at site 2, thereby mediating allele-specific recognition at the highly conserved MHC I interface.

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“…The specificity of Ly49 receptors for the polymorphic MHC-I molecules has been determined using various approaches including bone marrow graft rejection experiments, functional in vitro tests, adhesion assays, and MHC multimer binding (5,6,(11)(12)(13). The available data are based to a significant extent on the transient overexpression of Ly49 receptors and/or staining with soluble MHC-I multimer complexed with human ␤ 2 m. However, recent data indicate that the binding of MHC-I multimers to Ly49 receptors is influenced by species-specific residues in ␤ 2 m (8,10,14). In addition, the cell surface levels of transiently transfected Ly49 receptors often exceed those of NK cells.…”

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confidence: 99%

Interactions of Ly49 Family Receptors with MHC Class I Ligands in trans and cis

Scarpellino

Oeschger

Guillaume

et al. 2007

The Journal of Immunology

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The Ly49A NK cell receptor interacts with MHC class I (MHC-I) molecules on target cells and negatively regulates NK cell-mediated target cell lysis. We have recently shown that the MHC-I ligand-binding capacity of the Ly49A NK cell receptor is controlled by the NK cells’ own MHC-I. To see whether this property was unique to Ly49A, we have investigated the binding of soluble MHC-I multimers to the Ly49 family receptors expressed in MHC-I-deficient and -sufficient C57BL/6 mice. In this study, we confirm the binding of classical MHC-I to the inhibitory Ly49A, C and I receptors, and demonstrate that detectable MHC-I binding to MHC-I-deficient NK cells is exclusively mediated by these three receptors. We did not detect significant multimer binding to stably transfected or NK cell-expressed Ly49D, E, F, G, and H receptors. Yet, we identified the more distantly related Ly49B and Ly49Q, which are not expressed by NK cells, as two novel MHC-I receptors in mice. Furthermore, we show using MHC-I-sufficient mice that the NK cells’ own MHC-I significantly masks the Ly49A and Ly49C, but not the Ly49I receptor. Nevertheless, Ly49I was partly masked on transfected tumor cells, suggesting that the structure of Ly49I is compatible in principal with cis binding of MHC-I. Finally, masking of Ly49Q by cis MHC-I was minor, whereas masking of Ly49B was not detected. These data significantly extend the MHC-I specificity of Ly49 family receptors and show that the accessibility of most, but not all, MHC-I-binding Ly49 receptors is modulated by the expression of MHC-I in cis.

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Variable MHC class I engagement by Ly49 natural killer cell receptors demonstrated by the crystal structure of Ly49C bound to H-2Kb

Cited by 128 publications

References 51 publications

Mutagenesis, biochemical, and biophysical characterization of Mycoplasma arthritidis-derived mitogen

Mutagenesis, biochemical, and biophysical characterization of Mycoplasma arthritidis-derived mitogen

Distinctive Interactions at Multiple Site 2 Subsites by Allele-Specific Rat and Mouse Ly49 Determine Functional Binding and Class I MHC Specificity

Interactions of Ly49 Family Receptors with MHC Class I Ligands in trans and cis

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