Engineering protein for X‐ray crystallography: The murine major histocompatibility complex class II molecule I‐A<sup>d</sup>

Scott, Christopher A.; García, K. Christopher; Stura, E.A.; Peterson, Per A.; Wilson, Ian A.; Teyton, Luc

doi:10.1002/pro.5560070222

Cited by 20 publications

(24 citation statements)

References 39 publications

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“…Binding to peptides was determined by ELISA using immobilized MEM antibodies, biotinylated peptide (bio-GGVTELGRPDAEYWNSQKDL for MEM-264, Ϫ265, and Ϫ267, and bio-GGSPLTVEWRA for MEM-266) followed by streptavidin detection. teins with careful control of culture conditions, although some alleles are unstable in the absence of peptide (42)(43)(44)(45), and sometimes the MHC proteins co-purify with mixtures of adventitiously bound endogenous insect or medium-derived peptides (41,45). Peptide-free preparations of HLA-DR1, HLA-DR4 (carrying the protein product of the HLA-DRB1*0401 gene), and HLA-DR52a (DRB3*0101 gene) were obtained using insect cell expression systems, and the corresponding peptide-loaded forms by in vitro loading with appropriate peptides (HA for DR4, and PLG, an integrin variant for DR52a (46)).…”

Section: Table I Antibody Characterizationmentioning

confidence: 99%

Monoclonal Antibodies Specific for the Empty Conformation of HLA-DR1 Reveal Aspects of the Conformational Change Associated with Peptide Binding

Carven

Chitta

Hilgert

et al. 2004

Journal of Biological Chemistry

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Class II major histocompatibility complex (MHC) proteins bind peptides and present them at the cell surface for interaction with CD4؉ T cells as part of the system by which the immune system surveys the body for signs of infection. Peptide binding is known to induce conformational changes in class II MHC proteins on the basis of a variety of hydrodynamic and spectroscopic approaches, but the changes have not been clearly localized within the overall class II MHC structure. To map the peptideinduced conformational change for HLA-DR1, a common human class II MHC variant, we generated a series of monoclonal antibodies recognizing the ␤ subunit that are specific for the empty conformation. Each antibody reacted with the empty but not the peptide-loaded form, for both soluble recombinant protein and native protein expressed at the cell surface. Antibody binding epitopes were characterized using overlapping peptides and alanine scanning substitutions and were localized to two distinct regions of the protein. The pattern of key residues within the epitopes suggested that the two epitope regions undergo substantial conformational alteration during peptide binding. These results illuminate aspects of the structure of the empty forms and the nature of the peptide-induced conformational change. Major histocompatibility complex (MHC)1 molecules are heterodimeric cell-surface proteins that play an important role in the initiation of antigen-specific immune responses. Class II MHC proteins bind peptides derived from extracellular, endosomal, and internalized cell-surface antigens, and present them at the cell surface for inspection by CD4 ϩ T cells (1). Three-dimensional structures have been determined for peptide complexes of several polymorphic variants of both human and murine class II MHC molecules (reviewed in Ref.2). Both the MHC ␣ and ␤ chains contribute to the peptide binding site, which is made up of a ␤ sheet floor topped by two roughly parallel ␣ helical regions. Each subunit contributes an immunoglobulin-like domain below the peptide binding site, as well as short transmembrane and cytoplasmic domains. Peptides bind in an extended conformation in the groove between the two helices, with ϳ10 residues able to interact with the MHC protein, and the peptide termini extending from the binding site. This conformation, similar to a polyproline type II helix, has a 2.7-residue repeat and appears to be dictated by a network of conserved hydrogen bonding interactions between the MHC and bound peptide (3). The conformation places 4 -6 of the peptide side chains into pockets within the overall groove. The residues lining these pockets vary between allelic variants, providing different peptide-sequence binding specificity. Overall the interaction buries ϳ70% of the peptide surface area in the central region of a bound peptide, leaving the remainder available for interaction with antigen receptors on T cells (4).Although the canonical structure visualized by x-ray crystallography is relatively stereotyped, a number of studies h...

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Section: Table I Antibody Characterizationmentioning

confidence: 99%

Monoclonal Antibodies Specific for the Empty Conformation of HLA-DR1 Reveal Aspects of the Conformational Change Associated with Peptide Binding

Carven

Chitta

Hilgert

et al. 2004

Journal of Biological Chemistry

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“…The wild-type cDNA for the I-A g7 ␤-chain was modified to generate two constructs to express empty and peptide-filled molecules, as previously reported for I-A d (26,27). For the expression of empty molecules, the cDNA was interrupted after the codon for the last tryptophan before the transmembrane segment and extended by a linker (SSADL), a thrombin site (LVPRGS), a basic zipper, and a hexa-histidine tail.…”

Section: Construction Expression and Purification Of I-a G7 Moleculesmentioning

confidence: 99%

“…All constructs were subcloned into the metallothionein promoter-based fly expression vector pRMHa3 (29). Modified ␤-chains were transfected along with the soluble I-A d acid zipper ␣-chain or soluble I-A d acid zipper biotin ␣-chain (26,27) to produce empty and peptide-filled I-A g7 molecules. I-A g7 /Ii complexes were expressed by transfecting cDNAs for the ␣-and ␤-chain without histidine coding sequences and the human Ii construct.…”

Section: Construction Expression and Purification Of I-a G7 Moleculesmentioning

confidence: 99%

The I-Ag7 MHC Class II Molecule Linked to Murine Diabetes Is a Promiscuous Peptide Binder

Stratmann

Apostolopoulos

Mallet-Designe

et al. 2000

The Journal of Immunology

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120

106

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Susceptibility to insulin-dependent diabetes mellitus is linked to MHC class II genes. The only MHC class II molecule expressed by nonobese diabetic (NOD) mice, I-Ag7, shares a common α-chain with I-Ad but has a peculiar β-chain. As with most β-chain alleles linked to diabetes susceptibility, I-Ag7 contains a nonaspartic residue at position β57. We have produced large amounts of empty I-Ag7 molecules using a fly expression system to characterize its biochemical properties and peptide binding by phage-displayed peptide libraries. The identification of a specific binding peptide derived from glutamic acid decarboxylase (GAD65) has allowed us to crystallize and obtain the three-dimensional structure of I-Ag7. Structural information was critical in evaluating the binding studies. I-Ag7, like I-Ad, appears to be very promiscuous in terms of peptide binding. Their binding motifs are degenerate and contain small and/or small hydrophobic residues at P4 and P6 of the peptide, a motif frequently found in most globular proteins. The degree of promiscuity is increased for I-Ag7 over I-Ad as a consequence of a larger P9 pocket that can specifically accommodate negatively charged residues, as well as possibly residues with bulky side chains. So, although I-Ad and I-Ag7 are structurally closely related, stable molecules and good peptide binders, they differ functionally in their ability to bind significantly different peptide repertoires that are heavily influenced by the presence or the absence of a negatively charged residue at position 57 of the β-chain. These characteristics link I-Ag7 with autoimmune diseases, such as insulin-dependent diabetes mellitus.

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“…These latter problems have been overcome by methods for producing soluble MHC molecules bearing single peptides (1)(2)(3)(4) and for producing fluorescent multimeric versions of these molecules (5)(6)(7)(8)(9)(10). The cooperative binding achieved with these multivalent ligands produces an avidity high enough that antigen-specific T cells can be detected by flow cytometry.…”

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confidence: 99%

An inverse relationship between T cell receptor affinity and antigen dose during CD4⁺T cell responsesin vivoandin vitro

Rees

Bender

Teague

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

244

225

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Multimeric peptide͞class II MHC staining reagents were synthesized and shown to bind with appropriate specificity to T cell hybridomas. A small, expanded population of T cells detected with one of these reagents in peptideimmunized C57BL͞10 mice persisted for several months. This population expanded further on secondary immunization. Equating the extent of binding of this reagent to T cell receptor affinity, we saw little correlation of immunizing peptide dose to T cell receptor affinity at the peak of the primary response. However, there was an inverse relation between peptide dose and the apparent receptor affinity of the T cells that were present several months after a primary response or after a secondary stimulation either in vivo or in vitro.Tracking the cellular dynamics of antigen-specific T cell responses in vivo has been difficult, not only because the responding precursors occur at low frequency, but also because the peptide͞MHC ligand for the T cell receptor (TCR) is cell bound and of low affinity. These latter problems have been overcome by methods for producing soluble MHC molecules bearing single peptides (1-4) and for producing fluorescent multimeric versions of these molecules (5-10). The cooperative binding achieved with these multivalent ligands produces an avidity high enough that antigen-specific T cells can be detected by flow cytometry. In examining the binding of these types of reagents to T cells with receptors of known affinity for monovalent peptide͞MHC, we observed a direct correlation between the extent of multimer binding and receptor affinity (10).The phenomenon of affinity maturation is well established in B cell responses. With multiple immunizations of T celldependent antigens, B cells bearing Ig receptors of steadily increasing affinity grow to dominate the B cell pool (11, 12). These cells appear to arise by selection of somatically mutated receptors as antigen becomes limiting (13)(14)(15). Recent studies have demonstrated that T cells having higher average affinity for peptide͞MHC are selected after multiple exposures to antigen (16,17). Whether this affinity maturation is primarily the result of the experience of repeated exposure to antigen or a shaping of the T cell repertoire during antigen waning (18-20) is unknown.We examined these questions for CD4 ϩ T cells responding to a peptide presented by the mouse class II MHC molecule, IA b . We tracked the frequency and relative affinities of peptide͞MHC-binding T cells during both primary and secondary immune responses. Our results show that with repeated or prolonged exposure to antigen, limiting doses of antigen select for T cells with higher-affinity receptors.

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Engineering protein for X‐ray crystallography: The murine major histocompatibility complex class II molecule I‐A^d

Cited by 20 publications

References 39 publications

Monoclonal Antibodies Specific for the Empty Conformation of HLA-DR1 Reveal Aspects of the Conformational Change Associated with Peptide Binding

Monoclonal Antibodies Specific for the Empty Conformation of HLA-DR1 Reveal Aspects of the Conformational Change Associated with Peptide Binding

The I-Ag7 MHC Class II Molecule Linked to Murine Diabetes Is a Promiscuous Peptide Binder

An inverse relationship between T cell receptor affinity and antigen dose during CD4⁺T cell responsesin vivoandin vitro

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Engineering protein for X‐ray crystallography: The murine major histocompatibility complex class II molecule I‐Ad

Cited by 20 publications

References 39 publications

Monoclonal Antibodies Specific for the Empty Conformation of HLA-DR1 Reveal Aspects of the Conformational Change Associated with Peptide Binding

Monoclonal Antibodies Specific for the Empty Conformation of HLA-DR1 Reveal Aspects of the Conformational Change Associated with Peptide Binding

The I-Ag7 MHC Class II Molecule Linked to Murine Diabetes Is a Promiscuous Peptide Binder

An inverse relationship between T cell receptor affinity and antigen dose during CD4+T cell responsesin vivoandin vitro

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Engineering protein for X‐ray crystallography: The murine major histocompatibility complex class II molecule I‐A^d

An inverse relationship between T cell receptor affinity and antigen dose during CD4⁺T cell responsesin vivoandin vitro