Engineering and Characterization of a Stabilized α1/α2 Module of the Class I Major Histocompatibility Complex Product Ld

Jones, Lindsay L.; Brophy, Susan; Bankovich, Alexander J.; Colf, Leremy A.; Hanick, Nicole A.; García, K. Christopher; Kranz, David M.

doi:10.1074/jbc.m604343200

Cited by 50 publications

(63 citation statements)

References 59 publications

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“…A major enabling component to our experiments is that we used yeast surface display combined with directed protein engineering to produce a functional platform or ''mini'' H2-L d molecule composed of only the ␣1 and ␣2 domains of the heavy chain and peptide. This mini-MHC, which binds 2C with wild-type affinity (17,18), has a molecular mass of 21 kDa and refolded from E. coli together with the QL9 peptide (18). The structure of this mini-MHC determined at 2.4 Å shows it to be essentially identical to that of the full-length MHC (rms deviation for carbon-␣ atoms is 0.81 Å) (17).…”

Section: Resultsmentioning

confidence: 98%

“…Although isotopic 15 N-labeling of the protein of interest is needed for NMR, the source of 15 N for minimal media is inexpensive. Engineering the platform MHC (18) and assigning its NMR spectrum was the first necessary step and a major hurdle.…”

Section: Discussionmentioning

confidence: 99%

“…Yeast display was used to isolate the m6 high-affinity mutant by varying the sequence of the CDR3␣ of the 2C TCR (20). We subsequently conducted chemical-shift mapping experiments on wild-type 2C TCR with an affinity of 2 M for L d (described above) and high-affinity 2C TCR variant m6 with an affinity of 8 nM (CDR3␣ sequence 98-SGFASAL-104 in wild-type was changed to 98-SHQGRYL-104 in m6) (18). The advantage of analyzing the 2C TCR m6 was an improvement in the robustness of the NMR chemical shift spectral quality because of the higher affinity of the TCR, which results in a longer residence time of the TCR-pMHC complex.…”

Section: Comparison Of Binding Of Two Tcr Mutants With Different Affimentioning

confidence: 99%

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Solution mapping of T cell receptor docking footprints on peptide-MHC

Varani¹,

Bankovich²,

Liu

et al. 2007

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

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T cell receptor (TCR) recognition of peptide-MHC (pMHC) is central to the cellular immune response. A large database of TCR-pMHC structures is needed to reveal general structural principles, such as whether the repertoire of TCR/MHC docking modes is dictated by a ''recognition code'' between conserved elements of the TCR and MHC genes. Although Ϸ17 cocrystal structures of unique TCRpMHC complexes have been determined, cocrystallization of soluble TCR and pMHC remains a major technical obstacle in the field. Here we demonstrate a strategy, based on NMR chemical shift mapping, that permits rapid and reliable analysis of the solution footprint made by a TCR when binding onto the pMHC surface. We mapped the 2C TCR binding interaction with its allogeneic ligand H-2L d -QL9 and identified a group of NMR-shifted residues that delineated a clear surface of the MHC that we defined as the TCR footprint. We subsequently found that the docking footprint described by NMR shifts was highly accurate compared with a recently determined high-resolution crystal structure of the same complex. The same NMR footprint analysis was done on a highaffinity mutant of the TCR. The current work serves as a foundation to explore the molecular dynamics of pMHC complexes and to rapidly determine the footprints of many L d -specific TCRs.chemical shift mapping ͉ dynamics ͉ NMR ͉ cellular immunity ͉ protein-protein interaction T cells modulate the nature and extent of an immune response based primarily on specific T cell receptor (TCR) recognition of peptide in the context of a MHC molecule (1, 2). The TCR is a genetically recombined receptor, analogous to an antibody, that is composed of ␣ and ␤ chains. The binding site of the TCR comprises six loops called complementarity-determining regions (CDRs), with each chain contributing three loops, called CDR 1, 2, and 3. The CDR1 and CDR2 loop sequences are encoded by the variable (V) genes, but because there is no somatic mutation in TCR-V genes, these are referred to as ''germ line-derived.'' In contrast, the CDR3 loops, which are largely involved in antigenic peptide contact and thus specificity, are derived from recombination of V(D)J segments and vary in an almost unlimited fashion. MHC gene products present antigenic peptides to the TCR through a composite peptide-MHC (pMHC) surface composed of a highly variable element (i.e., peptide) in a groove surrounded by more conserved residues on the MHC helices (2).The interaction between TCR and pMHC has been studied through cocrystallization of Ϸ17 unique complexes (1, 3). Some general principles have emerged from the current database of complexes (4). Briefly, the TCR has an orientation over the pMHC surface that generally places the germline-encoded CDR1 and CDR2 loops in contact with the conserved helical residues of the MHC, whereas the highly variable, somatically recombined CDR3 loops primarily interact with the peptide. This docking orientation, also called the ''footprint,'' shows a high degree of variability (Ϯ60°) in different pMHC complex...

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Section: Resultsmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Section: Comparison Of Binding Of Two Tcr Mutants With Different Affimentioning

confidence: 99%

See 1 more Smart Citation

Solution mapping of T cell receptor docking footprints on peptide-MHC

Varani¹,

Bankovich²,

Liu

et al. 2007

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

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“…We have engineered a stable ␣1/␣2 L d molecule that lacks both the ␣ 3 domain of the class I H chain and ␤ 2 -microglobulin (␤ 2 m) (12 ϩ T cell lines, we were in a position to directly assess the impact of stimulating T cells through TCRs that might bind to non-MHC ligands (i.e., those that cannot bind to either CD4 or CD8).…”

Section: Cd8mentioning

confidence: 99%

“…The m31-L d protein was expressed and purified as described (12). Protein was biotinylated via a C-terminal biotinylation signal peptide sequence using a BirA ligase (Avidity).…”

Section: Expression and Purification Of Soluble L Dmentioning

confidence: 99%

Cutting Edge: Inhibitory Effects of CD4 and CD8 on T Cell Activation Induced by High-Affinity Noncognate Ligands

Chervin

Stone

Bowerman

et al. 2009

The Journal of Immunology

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It has been proposed that MHC restriction during thymocyte selection is controlled by coreceptor (CD4 or CD8) sequestration of the signaling molecule Lck. We explored this model as a mechanism for preventing peripheral T cell activation due to non-MHC ligand cross-reactivities of TCRs. TCRs that have a range of affinities for a class I MHC ligand were transduced into a T cell hybridoma in the absence or presence of coreceptors. High and intermediate affinity TCRs (KD = 17 and 540 nM) did not require CD8 for T cell activity, but CD4 acted as a potent inhibitor of the intermediate affinity TCR. These and other findings support the view that even high-affinity TCR:ligand interactions can be influenced by coreceptor sequestration of Lck. Thus, CD4 and CD8 act as “coreceptor inhibitors” to maintain appropriate TCR-mediated MHC restriction in peripheral T cell activity.

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Yeast Surface Display in Protein Engineering and Analysis

Hackel

Wittrup

2008

Protein Engineering Handbook

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Engineering and Characterization of a Stabilized α1/α2 Module of the Class I Major Histocompatibility Complex Product Ld

Cited by 50 publications

References 59 publications

Solution mapping of T cell receptor docking footprints on peptide-MHC

Solution mapping of T cell receptor docking footprints on peptide-MHC

Cutting Edge: Inhibitory Effects of CD4 and CD8 on T Cell Activation Induced by High-Affinity Noncognate Ligands

Yeast Surface Display in Protein Engineering and Analysis

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