The class II MHC homolog HLA-DM catalyzes exchange of peptides bound to class II MHC proteins, and is an important component of the Ag presentation machinery. The mechanism of HLA-DM-mediated ...catalysis is largely obscure. HLA-DM catalyzes exchange of peptides of varying sequence, suggesting that a peptide sequence-independent component of the MHC-peptide interaction could be involved in the catalytic process. Twelve conserved hydrogen bonds between the peptide backbone and the MHC are a prominent sequence-independent feature of the MHC-peptide interaction. To evaluate the relative importance of these hydrogen bonds toward HLA-DM action, we prepared peptide variants that lacked the ability to form one or more of the hydrogen bonds as a result of backbone amide N-methylation or truncation, and tested their ability to be exchanged by HLA-DM. We found that disruption of hydrogen bonds involving HLA-DR1 residues alpha51-53, a short extended segment at the N terminus of the alpha subunit helical region, led to heightened HLA-DM catalytic efficacy. We propose that those bonds are disrupted in the MHC conformation recognized by HLA-DM to allow structural transitions in that area during DM-assisted peptide release. These results suggest that peptides or compounds that bind MHC but cannot form these interactions would be preferentially edited out by HLA-DM.
Many persistent viruses have evolved the ability to subvert MHC class I antigen presentation. Indeed, human cytomegalovirus (HCMV) encodes at least four proteins that down-regulate cellsurface ...expression of class I. The HCMV unique short (US)2 glycoprotein binds newly synthesized class I molecules within the endoplasmic reticulum (ER) and subsequently targets them for proteasomal degradation. We report the crystal structure of US2 bound to the HLA-A2/Tax peptide complex. US2 associates with HLA-A2 at the junction of the peptide-binding region and the α3 domain, a novel binding surface on class I that allows US2 to bind independently of peptide sequence. Mutation of class I heavy chains confirms the importance of this binding site in vivo. Available data on class I-ER chaperone interactions indicate that chaperones would not impede US2 binding. Unexpectedly, the US2 ER-luminal domain forms an Ig-like fold. A US2 structure-based sequence alignment reveals that seven HCMV proteins, at least three of which function in immune evasion, share the same fold as US2. The structure allows design of further experiments to determine how US2 targets class I molecules for degradation.
The structure of a bacterial superantigen, Staphylococcus aureus enterotoxin B, bound to a human class II histocompatibility complex molecule (HLA-DR1) has been determined by X-ray crystallography. ...The superantigen binds as an intact protein outside the conventional peptide antigen-binding site of the class II major histocompatibility complex (MHC) molecule. No large conformational changes occur upon complex formation in either the DR1 or the enterotoxin B molecules. The structure of the complex helps explain how different class II molecules and superantigens associate and suggests a model for ternary complex formation with the T-cell antigen receptor (TCR), in which unconventional TCR-MHC contacts are possible.
The recent determination of the structure of a class II MHC molecule complexed to a specific peptide reveals both similarities and differences with peptide binding by class I MHC.
Class II major histocompatibility complex (MHC) proteins are essential for normal immune system function but also drive many autoimmune responses. They bind peptide antigens in endosomes and present ...them on the cell surface for recognition by CD4+ T cells. A small molecule could potentially block an autoimmune response by disrupting MHC-peptide interactions, but this has proven difficult because peptides bind tightly and dissociate slowly from MHC proteins. Using a high-throughput screening assay we discovered a class of noble metal complexes that strip peptides from human class II MHC proteins by an allosteric mechanism. Biochemical experiments indicate the metal-bound MHC protein adopts a 'peptide-empty' conformation that resembles the transition state of peptide loading. Furthermore, these metal inhibitors block the ability of antigen-presenting cells to activate T cells. This previously unknown allosteric mechanism may help resolve how gold(I) drugs affect the progress of rheumatoid arthritis and may provide a basis for developing a new class of anti-autoimmune drugs.
Before exit from the endoplasmic reticulum (ER), MHC class I molecules transiently associate with the transporter associated with antigen processing (TAP1/TAP2) in an interaction that is bridged by ...tapasin. TAP1 and TAP2 belong to the ATP-binding cassette (ABC) transporter family, and are necessary and sufficient for peptide translocation across the ER membrane during loading of MHC class I molecules. Most ABC transporters comprise a transmembrane region with six membrane-spanning helices. TAP1 and TAP2, however, contain additional N-terminal sequences whose functions may be linked to interactions with tapasin and MHC class I molecules. Upon expression and purification of human TAP1/TAP2 complexes from insect cells, proteolytic fragments were identified that result from cleavage at residues 131 and 88 of TAP1 and TAP2, respectively. N-Terminally truncated TAP variants lacking these segments retained the ability to bind peptide and nucleotide substrates at a level comparable to that of wild-type TAP. The truncated constructs were also capable of peptide translocation in vitro, although with reduced efficiency. In an insect cell-based assay that reconstituted the class I loading pathway, the truncated TAP variants promoted HLA-B*2705 processing to similar levels as wild-type TAP. However, correlating with the observed reduction in tapasin binding, the tapasin-mediated increase in processing of HLA-B*2705 and HLA-B*4402 was lower for the truncated TAP constructs relative to the wild type. Together, these studies indicate that N-terminal domains of TAP1 and TAP2 are important for tapasin binding and for optimal peptide loading onto MHC class I molecules.
The two subunits of the human class I histocompatibility antigen (HLA)-A2 have been expressed at high levels (20-30 mg/liter) as insoluble aggregates in bacterial cells. The aggregates were dissolved ...in 8 M urea and then refolded to form an HLA-A2-peptide complex by removal of urea in the presence of an antigenic peptide. Two peptides from the matrix protein and nucleoprotein of influenza virus are known to bind to HLA-A2, and both support the refolding of the recombinant HLA-A2 molecule. An additional peptide, a nonamer from the gp120 envelope protein of human immunodeficiency virus type 1, also supported refolding. Yields of purified recombinant HLA-A2 are 10-15%. In the absence of an HLA-A2-restricted peptide, a stable HLA-A2 complex was not formed. Monoclonal antibodies known to bind to native HLA-A2 also bound to the recombinant HLA-A2-peptide complex. Three purified HLA-A2-peptide complexes refolded from bacterially produced protein aggregates crystallize under the identical conditions as HLA-A2 purified from human lymphoblastoid cells. Crystals of the recombinant HLA-A2 molecule in complex with the influenza matrix nonamer peptide, Mp(58-66), diffract to >1.5-Å resolution.
Most poxviruses, including variola, the causative agent of smallpox, express a secreted protein of 35 kDa, vCCI, which binds CC-chemokines with high affinity. This viral protein competes with the ...host cellular CC-chemokine receptors (CCRs), reducing inflammation and interfering with the host immune response. Such proteins or derivatives may have therapeutic uses as anti-inflammatory agents. We have determined the crystal structure to 1.85- angstrom resolution of vCCI from cowpox virus, the prototype of this poxvirus virulence factor. The molecule is a β -sandwich of topology not previously described. A patch of conserved residues on the exposed face of a β -sheet that is strongly negatively charged might have a role in binding of CC-chemokines, which are positively charged.
Cell surface complexes of class I MHC molecules and bound peptide antigens serve as specific recognition elements controlling the cytotoxic immune response. The 2.1 A structure of the human class I ...MHC molecule HLA-B27 provides a detailed composite image of a co-crystallized collection of HLA-B27-bound peptides, indicating that they share a common main-chain structure and length. It also permits direct visualization of the conservation of arginine as an "anchor" side chain at the second peptide position, which is bound in a potentially HLA-B27-specific pocket and may therefore have a role in the association of HLA-B27 with several diseases. Tight peptide binding to class I MHC molecules appears to result from the extensive contacts found at the ends of the cleft between peptide main-chain atoms and conserved MHC side chains, which also involve the peptide in stabilizing the three-dimensional fold of HLA-B27. The concentration of binding interactions at the peptide termini permits extensive sequence (and probably some length) variability in the center of the peptide, where it is exposed for T cell recognition.
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.
PDF
