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TAPBPR employs a ligand-independent docking mechanism to chaperone MR1 molecules

Nature Chemical Biology volume 18, pages 859–868 (2022)Cite this article

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Abstract

Chaperones tapasin and transporter associated with antigen processing (TAP)-binding protein related (TAPBPR) associate with the major histocompatibility complex (MHC)-related protein 1 (MR1) to promote trafficking and cell surface expression. However, the binding mechanism and ligand dependency of MR1/chaperone interactions remain incompletely characterized. Here in vitro, biochemical and computational studies reveal that, unlike MHC-I, TAPBPR recognizes MR1 in a ligand-independent manner owing to the absence of major structural changes in the MR1 α2-1 helix between empty and ligand-loaded molecules. Structural characterization using paramagnetic nuclear magnetic resonance experiments combined with restrained molecular dynamics simulations reveals that TAPBPR engages conserved surfaces on MR1 to induce similar adaptations to those seen in MHC-I/TAPBPR co-crystal structures. Finally, nuclear magnetic resonance relaxation dispersion experiments using 19F-labeled diclofenac show that TAPBPR can affect the exchange kinetics of noncovalent metabolites with the MR1 groove, serving as a catalyst. Our results support a role of chaperones in stabilizing nascent MR1 molecules to enable loading of endogenous or exogenous cargo.

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Fig. 1: Ligand dependence of MR1 stability and conformational plasticity.
Fig. 2: Biochemical characterization of TAPBPR interactions with empty or loaded MHC-I versus MR1.
Fig. 3: TAPBPR recognizes conserved interaction surfaces on MHC-I and MR1.
Fig. 4: Indirect assessment of TAPBPR/MR1 interactions in cells.
Fig. 5: Comparison of the molecular features of TAPBPR-bound MR1 versus MHC-I.
Fig. 6: TAPBPR influences ligand residence times in the MR1 groove.

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Data availability

Plasmids have been deposited to Addgene with accession IDs 178603, 178645, 178650 and 178653. NMR methyl resonance assignments of the free and TAPBPR-bound states of hpMR1 have been deposited into the Biological Magnetic Resonance Data Bank (https://bmrb.io/) under accession numbers 51044, 51045 and 51046. The atomic coordinates for the ten lowest-energy structures of the Ac-6-FP–hpMR1–bβ2m–TAPBPR complex have been deposited in the PDB (https://www.rcsb.org/) under the PDB ID 7RNO. The following PDB IDs for previously solved structures were also used in this study: 4GUP, 4PJ5, 1DUZ, 4PJ7, 5U1R, 5U2V, 5U16, 5WER, 6W9V and 6ENY. Source data for Figs. 4 and 5 and Extended Data Fig. 4 are provided with this paper. Other data are available from the corresponding author upon reasonable request.

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Acknowledgements

This research was supported through grants by the National Institute of Allergy and Infectious Diseases (NIAID) (no. 5R01AI143997) and NIGMS (no. 5R35GM125034) to N.G.S., grant no R35GM142505 to G.M.B. and NIGMS grant no. 3R35GM125034-05S to N.G.S. that funded a cryoprobe-equipped 600 MHz NMR spectrometer at UPenn. We acknowledge K. Natarajan and D. Margulies (NIAID) for helpful discussions, E. Adams (University of Chicago) for providing DNA plasmids for hpMR1, hMR1, hMR1 C262S and bβ2m as well as the 5-N-RU precursor compound. We thank J.P. Brady (Novartis) for providing the broadband 19F CPMG pulse sequence. We further acknowledge J. Cassel (Wistar Institute), A. Majumdar (Johns Hopkins University) and H. Roder/Takuya Mizukami (Fox Chase Cancer Center) for assistance with instrumentation and scheduling.

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Authors and Affiliations

  1. Center for Computational and Genomic Medicine, Department of Pathology and Laboratory Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Andrew C. McShan, Georgia F. Papadaki, Yi Sun, Giora I. Morozov & Nikolaos G. Sgourakis

  2. Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Andrew C. McShan, Georgia F. Papadaki, Yi Sun, Adam I. Green, Giora I. Morozov, George M. Burslem & Nikolaos G. Sgourakis

  3. Department of Biochemistry and Cancer Center at Illinois, University of Illinois, Urbana, IL, USA

    Christine A. Devlin & Erik Procko

  4. Department of Cancer Biology and Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    George M. Burslem

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Contributions

A.C.M., E.P. and N.G.S. designed all experiments in this study. A.C.M. and Y.S. prepared recombinant MR1 proteins and performed SPR experiment for hpMR1–MHC-I and hMR1, respectively. A.C.M. performed MD simulations, mass spectrometry, native gel shift assays, DSF, NMR, Rosetta modeling and determined the MR1–TAPBPR complex structure. C.A.D. and E.P. performed flow cytometry, surface expression analysis and in vitro binding assays. G.I.M. and G.F.P. expressed and purified recombinant TAPBPR proteins. A.I.G. and G.M.B. carried out chemical synthesis and validation of 19F-DCF. E.P., G.M.B. and N.G.S. acquired funding. N.G.S. supervised the project. A.C.M., C.A.D., E.P. and N.G.S. wrote the manuscript, with input from all the authors.

Corresponding author

Correspondence to Nikolaos G. Sgourakis.

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Competing interests

E.P. is an employee of Cyrus Biotechnology. N.G.S. is a cofounder of Tantigen Bio, Inc., MultiplexThera, Inc. and a named inventor on patents concerning the preparation of ligand-receptive MHC-I and MHC-like molecules; all companies had no role in this study. The other authors declare no competing interests.

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Nature Chemical Biology thanks Brian Baker, Christian Freund and Hamish McWilliam for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Mass spectroscopy characterization of empty and ligand bound hpMR1 states.

LC–MS analysis of purified a, empty hpMR1, b, DCF/hpMR1, c, 5-OP-RU/hpMR1, and d, Ac-6-FP/hpMR1 complexes. In each panel, the top graph shows the total ion chromatogram (TIC) while the bottom panel shows the deconvoluted m/z data (Da) of the selected TIC region highlighted in dotted boxes. The expected and observed molecular weights of each species are noted.

Extended Data Fig. 2 Complete chemical shift assignments of hpMR1 methyl resonances.

a, Comparison of 2D 1H-13C methyl HMQC spectra of AILV (250 µM) versus ILVproS (210 µM) labeled hpMR1 in complex with natural isotopic abundance Ac-6-FP and bβ2m recorded at a 1H field of 600 MHz at 25 °C. b, 2D 1H-13C methyl constant time HMQC spectra with a valine-selective decoupling pulse for ILV* (150 µM) labeled hpMR1 in complex with natural isotopic abundance Ac-6-FP and bβ2m recorded at a 1H field of 600 MHz at 25 °C. Unambiguous distinction between the isoleucine (red, ~8 to 16 ppm), valine (green) and leucine (red) resonances is achieved. c, 2D 13C-13C strips showing observed intramolecular NOEs and their assignment taken from 3D CM-CMHM HMQC-NOESY-HMQC experiments using 300 msec mixing time recorded at a 1H field of 800 MHz at 25 °C. d, The methyl NOE network centered on I139 δ1 from the RosettaCM model of hpMR1/bβ2m (PDB template 4PJ5, TCR removed).

Extended Data Fig. 3 Chemical shift profiles of Ile 13Cδ1 labeled hpMR1 loaded with different ligands.

a, 2D 1H-13C methyl HMQC spectra of 10 µM Ile 13Cδ1-labeled hpMR1 with natural isotopic abundance bβ2m in the absence (empty, yellow) and presence of 2 mM ligand recorded at a 1H field of 600 MHz at 25 °C. All samples contain 0.5% DMSO-d6. b, Ile δ1 methyl groups (grey spheres) mapped onto the MR1 groove in complex with 6-FP (PDB ID 4GUP, red), 3FSA (PDB ID 5U6Q, black, TCR removed), or 2OH1NA (PDB ID 5U16, brown, TCR removed). c, Overlay of 2D 1H-13C methyl HMQC spectra of Ile δ1 labeled hpMR1 with natural isotopic abundance bβ2m. Samples were derived from in vitro refolding of the Ac-6-FP/Ile labeled hpMR1/bβ2m complex (blue) or from titration of empty Ile δ1 labeled hpMR1/bβ2m with 2 mM Ac-6-FP (red). Refolded and titrated spectra were recorded at 25 °C at a 1H field of 800 MHz and 600 MHz, respectively.

Extended Data Fig. 4 Ligand independence of the interaction between hpMR1 and TAPBPR by native gel shift assay.

a, Native gel shift analysis for empty hpMR1/bβ2m/TAPBPR and empty MHC-I/TAPBPR complex formation. Each lane is loaded with free TAPBPR, free UVirrad/MHC-I, free empty hpMR1/bβ2m, or 1:1 molar ratio mixture with TAPBPR. Asterisk (*) denote complex formation. b, Native gel shift analysis where each lane is loaded with free TAPBPR, free hpMR1/bβ2m without (empty) or with ligands, or 1:1 mixtures with TAPBPR. Ligand/hpMR1 complexes were prepared by in vitro refolding as described in the main text. c, Quantification of the % of hpMR1/bβ2m/TAPBPR complexes formed in the presence of different ligands, relative to the free MR1 band, based on native gel electrophoresis and band quantification using ImageJ. Data are mean ± SD for n = 3 technical replicates.

Source data

Extended Data Fig. 5 Constructs for interaction of properly conformed MHC-I with TAPBPR.

a, Representative SPR sensorgram for varying concentrations of UV-irradiated HLA-A*01:01/hβ2m flowed over a streptavidin chip coupled with TAPBPRWT-biotin. RU = response units. b, Clustal Omega v1.2.4 alignment of HLA-A*02:01 peptides used in this study, processed using ESPript v3. Residues in red box, white character represent strict identity. Residues in red character represent similar group amino acids. c, In silico prediction of peptide binding to HLA-A*02:01 using netMHCpan-v4.1. d, Normalized DSF traces of purified empty and peptide-loaded loaded HLA-A*02:01/hβ2m. Fitted thermal stabilities (Tm, °C) are noted. Data are mean ± SD for n = 3 technical replicates. e, Summary of DSF determined thermal stability (Tm, °C) of empty (UVirrad) or peptide-loaded HLA-A*02:01/hβ2m complexes. Data are mean ± SD for n = 3 technical replicates. f, Representative SPR sensorgrams for varying concentrations of non-UV irradiated M89-97/HLA-A*02:01/hβ2m, TH/HLA-A*02:01/hβ2m, or NY-ESO-1/HLA-A*02:01/hβ2m flowed over a streptavidin chip coupled with TAPBPRWT-biotin. The concentration of analyte for the top sensorgram is noted. Fits from surface bound analysis are shown with dotted red lines. Data are mean ± SD for n = 3 technical replicates.

Extended Data Fig. 6 Ligand independence of the interaction between hpMR1 and TAPBPR by SPR.

a, SPR sensorgrams of varying concentrations of empty or ligand loaded hpMR1/bβ2m flowed over a streptavidin chip coupled with TAPBPRWT-biotin. The analyte concentrations are noted on the first three sensorgrams and are the same for every experiment. b, SPR sensorgrams of varying concentrations of Ac-6-FP loaded hMR1/hβ2m wild-type or hMR1 C262S/hβ2m flowed over a streptavidin chip coupled with TAPBPR-biotin. The analyte concentrations are noted on the sensorgram. The RU vs hMR1 concentration is also shown with fitting shown in the dotted lines. Data are mean ± SD for n = 3 technical replicates. The C262S mutation is a Cys->Ser mutation for the free hMR1 Cys on the α3 domain.

Extended Data Fig. 7 Flow cytometry analysis of indirect effects of TAPBPR on surface trafficking of MR1.

a, Expi293F cells were gated by FSC-SSC for the main cell population (magenta gate, ~60% of all events). b, Expi293F cells transfected with myc-tagged MR1 and FLAG-tagged TAPBPR were stained with anti-myc-Alexa 647 for surface MR1. For quantification of changes in myc-MR1 surface levels, the mean fluorescence for control (vector only) cells (black) was subtracted from the mean fluorescence of each sample.

Extended Data Fig. 8 The TAPBPR G24-R36 loop does not interact with the MR1 groove.

a, Surface representation of the groove of Ac-6-FP loaded MR1 (PDB ID 4PJ5, TCR removed) showing the open A’ pocket and closed F’ pocket. Ac-6-FP is shown as magenta spheres, MR1 is shown as a green surface. b, Plot of response units (RU) from SPR sensorgrams of varying concentrations of empty or Ac-6-FP loaded hpMR1/bβ2m flowed over a streptavidin chip coupled with TAPBPRWT-biotin (black) or TAPBPRΔG24-R36-biotin (red). The dotted lines indicate fits. Data are the mean±SD for n = 3 technical replicates. c, 2D 1H-13C methyl HMQC spectra of 100 µM Ac-6-FP/ILVproS labeled hpMR1/bβ2m in complex with TAPBPRWT (blue) or TAPBPRΔG24-R36 (red) recorded at a 1H field of 600 MHz at 25 °C. d, Comparison of chemical shift perturbation (CSP, ΔδCH3, ppm) analysis for residues of hpMR1 upon complex formation with TAPBPRWT versus TAPBPRΔG24-R36. CSPs plotted onto a RosettaCM model of hpMR1/bβ2m (PDB template 4PJ5, TCR removed). Methyl bearing residues affected for hpMR1 upon binding to TAPBPRWT (top) or TAPBPRΔG24-R36 (bottom) are shown in warm pink and unaffected residues in grey. hpMR1 and Ac-6-FP are colored green and magenta, respectively. e, and f, Structural models of the role of the TAPBPR G24-R36 loop for chaperone interactions with (e) Ac-6-FP/hpMR1 and (f) TAX9/HLA-A*02:01.

Extended Data Fig. 9 Measurement of 1HM-Γ2 PRE rates in the 89 kDa MR1/TAPBPR complex.

a, Melting temperature (Tm, °C) of TAPBPRWT (black) and TAPBPRL30C-MTSL (grey) obtained from differential scanning fluorimetry experiments. Data are mean ± SD for n = 3 technical replicates. b, 2D 1H-13C methyl HMQC spectra of 100 µM Ac-6-FP/ILVproS labeled hpMR1/bβ2m in complex with TAPBPRL30C-MTSL in the oxidized (paramagnetic, blue) or reduced (diamagnetic, red) state recorded at a 1H field of 600 MHz at 25 °C. Dotted boxes highlight a subset of the methyl resonances affected upon reduction of TAPBPRL30C-MTSL by ascorbic acid. c, Position of representative ILVproS methyl groups analyzed in panel d shown on the RosettaCM model of hpMR1 (PDB template 4PJ5, TCR removed). d, TAPBPRL30C-MTSL was complexed with Ac-6-FP/ILVproS hpMR1/bβ2m and 1HM R2 values were measured for oxidized (paramagnetic, blue) and reduced (diamagnetic, red) samples by recording a series of 2D methyl 1H-13C HMQC spectra and analyzing the NMR peak intensity as a function of a varied transverse relaxation delay (0, 6, 24, 36, 50 and 68 msec). The decay profiles for selected 1H methyl groups of L10 δ2, V28 γ2, I96 δ1 and L246 δ2 of hpMR1 are shown. A summary of all measured 1HM-Γ2 PRE rates is shown in Supplementary Table 5.

Extended Data Fig. 10 Comparison of the molecular interfaces of the TAPBPR bound MR1 versus MHC-I.

a, Interactions at the (Left) H2-Dd or (Right) hpMR1 α1/α2 interface with TAPBPR (TAPBPR in grey, H2-Dd or hpMR1 in green). The H2-Dd/TAPBPR interaction is shown for PDB ID 5WER, while the hpMR1/TAPBPR interaction is shown for the lowest energy hybrid NMR/MD model. Interacting residues are shown as sticks. b, Interactions at the (Left) H2-Dd or (Right) hpMR1 α3/β2m interface with TAPBPR (TAPBPR in grey, H2-Dd or hpMR1 in green; β2m in cyan). The H2-Dd/TAPBPR interaction is shown for PDB ID 5WER, while the hpMR1/TAPBPR interaction is shown for the lowest energy hybrid NMR/MD model. Interacting residues are shown as sticks. c, Overlay of Ac-6-FP in the hpMR1 groove for the TAPBPR chaperoned state (green, PDB 7RNO) versus Ac-6-FP in the hMR1 groove for the unchaperoned state (PDB ID 4PJ5, TCR removed). d, Structural overlays of unchaperoned 6-FP/hMR1 (PDB ID 4GUP, orange) compared with hybrid NMR/MD refined structures of Ac-6-FP/hpMR1/TAPBPR built using PDB ID 4PJ5, TCR removed (green) or 6-FP/hpMR1/TAPBPR built using PDB ID 4GUP (blue). The Cα RMSD between the green and blue structures is 1.769 Å.

Supplementary information

Supplementary Information

Supplementary Figs. 1–16 and Tables 1–8.

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Source data

Source Data Fig. 4

Unprocessed blots and statistical source data.

Source Data Fig. 5

Unprocessed blots and statistical source data.

Source Data Extended Data Fig. 4

Unprocessed native gels.

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McShan, A.C., Devlin, C.A., Papadaki, G.F. et al. TAPBPR employs a ligand-independent docking mechanism to chaperone MR1 molecules. Nat Chem Biol 18, 859–868 (2022). https://doi.org/10.1038/s41589-022-01049-9

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