Differential scanning fluorimetry based assessments of the thermal and kinetic stability of peptide–MHC complexes

Hellman, Lance M.; Yin, Liusong; Wang, Yuan; Blevins, Sydney J.; Riley, Timothy P.; Belden, Orrin S.; Spear, Timothy T.; Nishimura, Michael I.; Stern, Lawrence J.; Baker, Brian M.

doi:10.1016/j.jim.2016.02.016

Cited by 61 publications

(73 citation statements)

References 57 publications

(74 reference statements)

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“…Replacement of the p1 Lys with alanine did not weaken binding of the peptide to A2, as the A2 complexes with both the native NS3 epitope and the K1A variant had identical melting temperatures (T m values) of 64°C when measured by differential scanning fluorimetry (DSF) (Fig. 1D), reflective of high-affinity peptide binding (24).…”

Section: Resultsmentioning

confidence: 97%

How an alloreactive T-cell receptor achieves peptide and MHC specificity

Wang

Singh

Spear

et al. 2017

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

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T-cell receptor (TCR) allorecognition is often presumed to be relatively nonspecific, attributable to either a TCR focus on exposed major histocompatibility complex (MHC) polymorphisms or the degenerate recognition of allopeptides. However, paradoxically, alloreactivity can proceed with high peptide and MHC specificity. Although the underlying mechanisms remain unclear, the existence of highly specific alloreactive TCRs has led to their use as immunotherapeutics that can circumvent central tolerance and limit graft-versus-host disease. Here, we show how an alloreactive TCR achieves peptide and MHC specificity. The HCV1406 TCR was cloned from T cells that expanded when a hepatitis C virus (HCV)-infected HLA-A2 − individual received an HLA-A2 + liver allograft. HCV1406 was subsequently shown to recognize the HCV nonstructural protein 3 (NS3):1406-1415 epitope with high specificity when presented by HLA-A2. We show that NS3/HLA-A2 recognition by the HCV1406 TCR is critically dependent on features unique to both the allo-MHC and the NS3 epitope. We also find cooperativity between structural mimicry and a crucial peptide "hot spot" and demonstrate its role, along with the MHC, in directing the specificity of allorecognition. Our results help explain the paradox of specificity in alloreactive TCRs and have implications for their use in immunotherapy and related efforts to manipulate TCR recognition, as well as alloreactivity in general.T-cell receptor | peptide-MHC | structure | alloreactivity | specificity

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Section: Resultsmentioning

confidence: 97%

How an alloreactive T-cell receptor achieves peptide and MHC specificity

Wang

Singh

Spear

et al. 2017

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

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“…While both 12mers formed complexes with B08 that were sufficiently stable to allow characterization (Supporting Figure 3), biophysical analysis suggested that the complexes are quite distinct in terms of thermodynamic and kinetic stability ( Figure 5A and Supporting Figure 6, Supporting Tables 1,2). Using the Differential Scanning Fluorimetry Assay (DSF) (41), the B08/LL12 complex displayed a Tm value of 57.7±0.4 °C. In contrast, the B08/AI12 complex, was markedly less stable with Tm value of 47.8±2.3 °C.…”

Section: Characterization Of Thermodynamic and Kinetic Stability Of Bmentioning

confidence: 99%

A systematic re-examination of processing of MHCI-bound antigenic peptide precursors by endoplasmic reticulum aminopeptidase 1

Mavridis

Arya

Domnick

et al. 2020

Journal of Biological Chemistry

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Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims antigenic peptide precursors to generate mature antigenic peptides for presentation by major histocompatibility complex class I (MHCI) molecules and regulates adaptive immune responses. ERAP1 has been proposed to trim peptide precursors both in solution and in pre-formed MHCI-peptide complexes, but which mode is more relevant to its biological function remains controversial. Here, we compared ERAP1-mediated trimming of antigenic peptide precursors in solution or when bound to three MHCI alleles, HLA-B*58, HLA-B*08 and HLA-A*02. For all MHCIpeptide combinations, peptide binding onto MHCI protected against ERAP1-mediated trimming. In only a single MHCI-peptide combination, trimming of an HLA-B*08-bound 12mer progressed at a considerable rate, albeit still slower than in solution. Results from thermodynamic, kinetic and computational analyses suggested that this 12mer is highly labile and that apparent on-MHC trimming rates are always slower than that of MHCI-peptide dissociation. Both ERAP2 and leucine aminopeptidase, an enzyme unrelated to antigen processing, could trim this labile peptide from pre-formed MHCI complexes as efficiently as ERAP1. A pseudopeptide analogue with high affinity for both HLA-B*08 and the ERAP1 active site could not promote the formation of a ternary ERAP1-MHCI-peptide complex. Similarly, no interactions between ERAP1 and purified peptide loading complex (PLC) were detected in the absence or presence of a pseudopeptide trap. We conclude that MHCI binding protects peptides from ERAP1 degradation and that trimming in solution, along with the dynamic nature of peptide binding to MHCI, are sufficient to explain ERAP1 processing of antigenic peptide precursors.

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“…We utilized well-characterized epitopes: the p29 peptide (YPNVNIHNF) for H2-L d (41), the HIV gp120 P18-I10 peptide (RGPGRAFVTI) for H2-D d (42), the neuroblastoma related NRAS Q61K peptide (ILDTAGKEEY) for HLA-A*01:01 (43), and the HTLV-1 TAX peptide (LLFGYPVYV) for HLA-A*02:01 (44). Using a differential scanning fluorimetry assay (45), we further confirmed that all purified samples displayed thermal stabilities that were characteristic of properly conformed, peptide-bound molecular species with T m values ranging from 51 to 63 °C (Table S1). Using a SEC assay, we observed formation of pMHC-I/TAPBPR complexes for both mouse pMHC-I (P18-I10/H2-D d /hβ2m and NIH/H2-L d /hβ2m), but neither of the human pMHC-I (NRAS Q61K /HLA-A*01:01/hβ2m and TAX/HLA-A*02:01/hβ2m) ( Fig.…”

Section: Tapbpr Recognizes Folded Pmhc-i Using a Conserved Binding Momentioning

confidence: 99%

Molecular determinants of chaperone interactions on MHC-I for folding and antigen repertoire selection

McShan¹,

Devlin²,

Overall³

et al. 2019

Preprint

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Highlights Deep mutagenesis identifies a conformational disulfide-linked epitope as the main requirement for association of nascent MHC-I molecules with the TAPBPR chaperone  Analysis of μs-ms timescale conformational dynamics by methyl NMR reveals allelespecific profiles at the TAPBPR interaction surfaces of peptide-loaded MHC-I molecules  μs-ms dynamics dictate the specificity of TAPBPR interactions for different MHC-I alleles through the sampling of a minor, "excited state" conformation  Restriction of dynamics though an engineered disulfide bond abrogates interactions with TAPBPR, both in solution and on a cellular membrane Keywords Major Histocompatibility Complex • MHC-I • NMR spectroscopy • protein dynamics • molecular chaperone • TAPBPR • peptide editing • deep mutagenesis mapping • conformational landscape • peptide repertoire Graphical Abstract AbstractThe interplay between a highly polymorphic set of MHC-I alleles and molecular chaperones shapes the repertoire of peptide antigens displayed on the cell surface for T cell surveillance.Here, we demonstrate that the molecular chaperone TAPBPR associates with a broad range of partially folded MHC-I species inside the cell. Bimolecular fluorescence complementation and deep mutational scanning reveal that TAPBPR recognition is polarized towards one side of the peptide-binding groove, and depends on the formation of a conserved MHC-I disulfide epitope in the α 2 domain. Conversely, thermodynamic measurements of TAPBPR binding for a representative set of properly conformed, peptide-loaded molecules suggest a narrower MHC-I specificity range. Using solution NMR, we find that the extent of dynamics at "hotspot" surfaces confers TAPBPR recognition of a sparsely populated MHC-I state attained through a global conformational change. Consistently, restriction of MHC-I groove plasticity through the introduction of a disulfide bond between the α 1 /α 2 helices abrogates TAPBPR binding, both in solution and on a cellular membrane, while intracellular binding is tolerant of many destabilizing MHC-I substitutions. Our data support parallel TAPBPR functions of i) chaperoning unstable MHC-I molecules at early stages of their folding process, akin to a holdase with broad allelespecificity, and ii) editing the peptide cargo of properly conformed MHC-I molecules en route to the surface, which demonstrates a narrower specificity. Our results suggest that TAPBPR exploits localized structural adaptations, both near and distant to the peptide-binding groove, to selectively recognize discrete conformational states sampled by MHC-I alleles, towards editing Sithe repertoire of displayed antigens. Significance StatementThe human population contains thousands of MHC-I alleles, showing a range of dependencies on molecular chaperones for loading of their peptide cargo, which are then displayed on the cell surface for T cell surveillance. Using the chaperone TAPBPR as a model, we combine deep mutagenesis with functional and biophysical data, especially solution NMR, to provide a compl...

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Differential scanning fluorimetry based assessments of the thermal and kinetic stability of peptide–MHC complexes

Cited by 61 publications

References 57 publications

How an alloreactive T-cell receptor achieves peptide and MHC specificity

How an alloreactive T-cell receptor achieves peptide and MHC specificity

A systematic re-examination of processing of MHCI-bound antigenic peptide precursors by endoplasmic reticulum aminopeptidase 1

Molecular determinants of chaperone interactions on MHC-I for folding and antigen repertoire selection

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