A signal capture and proofreading mechanism for the KDEL-receptor explains selectivity and dynamic range in ER retrieval

8 figures, 1 video, 2 tables and 4 additional files

Figures

Figure 1 with 2 supplements
ER retrieval signal abundance and affinity are not correlated.

(a) Sequence logos for ER resident proteins with C-terminal KDEL retrieval signals and variants thereof calculated using frequency or protein abundance (Itzhak et al., 2017; Itzhak et al., 2016). (b) Combined cellular concentrations of ER resident proteins with canonical KDEL, RDEL, and HDEL retrieval sequences in HeLa cells and mouse brain. (c) Competition binding assays for [3H]-TAEHDEL and unlabelled TAEKDEL, TAERDEL, and TAEHDEL to the KDEL receptor. IC50 values for the competing peptides were used to calculate the apparent KDwith the Cheng-Prusoff equation (Cheng and Prusoff, 1973). (d) Endogenous KDEL receptor redistribution was measured in COS-7 cells in the absence (-ligand) or presence of K/R/H/A/DDEL (mScarlet-xDELsec). TGN46 was used as a Golgi marker. Scale bar is 10 µm. (e) The mean difference for K/R/H/A/DDEL comparisons against the shared no ligand control are shown as Cummings estimation plots. The individual data points for the fraction of KDEL receptor fluorescence in the Golgi are plotted on the upper axes with sample sizes and p values.

Figure 1—source data 1

Source data for the ligand-induced KDELR receptor retrieval assays in Figure 1e.

https://cdn.elifesciences.org/articles/68380/elife-68380-fig1-data1-v3.xlsx
Figure 1—figure supplement 1
Abundance of ER resident proteins and chaperones in human cells and mouse brain.

(a) The mean concentration of ER resident chaperones with the indicated ER retrieval sequence variant is plotted in the bar graph (Itzhak et al., 2017; Itzhak et al., 2016). (b) Combined cellular concentrations of ER resident proteins with canonical KDEL, RDEL, and HDEL retrieval sequences in HeLa cells and mouse brain. (c) The mean concentration of KDELR1, KDELR2, and KDELR3 in HeLa cells and mouse brain is plotted in the bar graph (Itzhak et al., 2017; Itzhak et al., 2016). (d) Cells and media collected from HeLa S3 cells expressing the xDEL variants (mScarlet-xDELsec) indicated in the figure were Western blotted for resident ER chaperones and KDELR. (e) A bar graph of xDEL secretion showing mean ± SEM (n = 3). (f). Endogenous ER chaperone secretion was measured by western blotting after challenge with different retrieval signals, and plotted as a bar graph showing mean ± SEM (n = 3).

Figure 1—figure supplement 1—source data 1

Source data for analysis of ER protein levels in Figure 1—figure supplement 1a and c.

https://cdn.elifesciences.org/articles/68380/elife-68380-fig1-figsupp1-data1-v3.xlsx
Figure 1—figure supplement 1—source data 2

Source data for the western blots in Figure 1—figure supplement 1d showing the regions taken for the figure.

Individual blot files are provided as a ZIP archive.

https://cdn.elifesciences.org/articles/68380/elife-68380-fig1-figsupp1-data2-v3.pdf
Figure 1—figure supplement 1—source data 3

Source data for analysis of ER protein levels in Figure 1—figure supplement 1e and f.

https://cdn.elifesciences.org/articles/68380/elife-68380-fig1-figsupp1-data3-v3.xlsx
Figure 1—figure supplement 2
Retrieval specificity of KDELR1 and KDELR2.

(a) Distribution of human KDELR1 and (b) KDELR2 was measured in COS-7 cells in the absence (-ligand) or presence of K/R/H/A/DDEL retrieval signals (K/R/H/A/DDELsec). TGN46 was used as a Golgi marker. Scale bar is 10 µm. The fraction of KDEL receptor localised to the Golgi was measured before (no ligand) and after challenge with different retrieval signals as indicated. Golgi signal for KDEL receptor and ligand intensity are shown on the scatter plots with sample sizes. Effect sizes are shown as the mean difference for K/R/H/A/DDEL comparisons against the shared -ligand control with sample sizes and p-values.

Figure 1—figure supplement 2—source data 1

Source data for the ligand-induced KDELR receptor retrieval assays in Figure 1—figure supplement 2.

https://cdn.elifesciences.org/articles/68380/elife-68380-fig1-figsupp2-data1-v3.xlsx
Figure 2 with 2 supplements
Structures of the KDEL receptor bound to HDEL and RDEL retrieval signals.

(a) Crystal structure of chicken KDELR2 viewed from the side with the transmembrane helices numbered and coloured from N-terminus (blue) to C-terminus (red). The predicted membrane-embedded region of the receptor is indicated by a grey shaded box, with labels at the luminal and cytoplasmic faces. The TAEHDEL peptide is shown in stick format, coloured grey. (b) Close up views of bound TAEHDEL (this study), (c) TAERDEL (this study), and (d) TAEKDEL (PDB:6I6H) peptides bound to the receptor are shown with contributing side chains labelled. Hydrogen bonds are indicated as dashed lines. The molecular orbitals of W120 and the −4 histidine on the peptide are shown as a dotted surface. (e) Superposition of the HDEL, RDEL, and KDEL peptides reveals near identical binding position within the receptor. Retrieval signal side chains are numbered counting down from the C-terminus.

Figure 2—figure supplement 1
Analysis of pH-dependent interaction of HDEL, KDEL, and RDEL signals with KDELR2.

Thermal stability of chicken KDEL2 was measured at pH 5.4, 5.9, 6.4, and 7.0 in the presence of TAEHDEL, TAEKDEL, and TAERDEL peptides. The difference in melting temperature to a no ligand control is plotted in the bar graph as mean ± SEM (n = 3).

Figure 2—figure supplement 2
Polder difference density electron density maps for HDEL and RDEL peptides.

(a) The structure of the KDELR bound to the TAEHDEL peptide is shown as in Figure 2a. The mFo-DFc difference electron density used for model building is displayed (green mesh), contoured at 3σ. (b) Equivalent maps calculated for the RDEL peptide.

Figure 3 with 1 supplement
Roles of KDEL receptor E117 and W120 in retrieval signal binding and function in cells.

(a) Normalised binding of [3H]-TAEKDEL and (b) [3H]-TAEHDEL signals to purified WT and the indicated E117 and W120 mutant variants of chicken KDELR2. Bar graphs show mean binding ± SEM (n = 3). Line graphs show titration binding assays. (c) The fraction of WT, E117, and W120 mutant KDEL receptor localised to the Golgi in COS-7 cells was measured before (no ligand) and after challenge with different retrieval signals (K/R/HDEL) as indicated. Effect sizes are shown as the mean difference for K/R/HDEL comparisons against the shared -ligand control with sample sizes and p-values. Also see Figure 3—figure supplements 1—source data 1 files. (d) The π-π interactions between W120 and the histidine were visualised using reduced density gradient analysis. The wild-type W120 exhibit stronger π-π interactions compared with W120F, while W120A shows no π-π interactions. (e) When W120 is changed to phenylalanine, the protonated histidine has a higher root mean squared fluctuation (RMSF) in the binding pocket, which is further increased for the W120A substitution. (f) Binding of [3H]-TAEHDEL to the KDEL receptor was measured at pH 5.4–7.0 and is plotted as a function of receptor concentration.

Figure 3—figure supplement 1
Effect of KDEL receptor E117 and W120 mutants on retrieval signal function in cells.

(a) Distribution of WT, E117, and W120 mutant KDEL receptors was measured in COS-7 cells in the absence (-ligand) or presence of (b) KDEL, (c) RDEL, or (d) HDEL retrieval signals (K/R/HDELsec). TGN46 was used as a Golgi marker. Scale bar is 10 µm. The fraction of WT, E117 and W120 mutant KDEL receptor localised to the Golgi was measured before (no ligand) and after challenge with different retrieval signals (K/R/HDEL) as indicated. Golgi signal for KDEL receptor and ligand intensity are shown on the scatter plots with sample sizes. Effect sizes are shown as the mean difference for K/R/HDEL comparisons against the shared -ligand control with sample sizes and p-values. The Cumming estimation plots for this data are used in main Figure 3c.

Figure 3—figure supplement 1—source data 1

Source data for the ligand-induced KDELR receptor retrieval assays in Figure 3 and Figure 3—figure supplement 1.

https://cdn.elifesciences.org/articles/68380/elife-68380-fig3-figsupp1-data1-v3.xlsx
Figure 4 with 1 supplement
KDEL receptor E117 mutants show reduced selectivity for retrieval signals.

(a) E117Q, E117N, or E117A mutant KDEL receptors were tested for K/A/DDEL-induced redistribution from Golgi to ER in COS-7 cells. KDEL receptor distribution was followed in the absence (-ligand) or presence of K/A/DDELsec. TGN46 was used as a Golgi marker. Scale bar is 10 µm. (b) The fraction of E117Q, E117N or E117A mutant KDEL receptor localised to the Golgi was measured before (no ligand) and after challenge with different retrieval signals (K/A/DDEL). Effect sizes are shown as the mean difference for K/A/DDEL comparisons against the shared -ligand control with sample sizes and p values. .

Figure 4—source data 1

Source data for the ligand-induced KDELR receptor retrieval assays in Figure 4.

https://cdn.elifesciences.org/articles/68380/elife-68380-fig4-data1-v3.xlsx
Figure 4—figure supplement 1
Effect of ligand levels on the response of KDEL receptor E117 mutants to KDEL, ADEL, and DDEL signals.

Distribution of WT, E117A, E117Q, and E117N mutant KDEL receptors was measured in COS-7 cells in the absence (-ligand) or presence of KDEL, ADEL, and DDEL retrieval signals. The fraction of WT, E117 mutant KDEL receptor localised to the Golgi was measured before (no ligand) and after challenge with different retrieval signals. Ligand intensity was also measured and plotted against the Golgi fraction of KDEL receptor.

Figure 4—figure supplement 1—source data 1

Source data for the ligand-induced KDELR receptor retrieval assays in Figure 4—figure supplement 1.

https://cdn.elifesciences.org/articles/68380/elife-68380-fig4-figsupp1-data1-v3.xlsx
Figure 5 with 1 supplement
Charge distribution across the luminal entrance to the KDEL receptor binding pocket.

(a) KDEL receptor sequence alignment showing two regions centred around amino acid D50 and W120 of the human proteins. Cognate retrieval signal variants are shown to the right of the alignment. (b) The structure of the KDEL receptor with bound TAEHDEL highlighting key residues involved in ligand binding and variant residues D50, N54, and E117. (c) The charged surface for the WT KDEL receptor and (d) N50, N50/K54 and N50/K54/Q117 mutants is shown.

Figure 5—figure supplement 1
Comparison of human and yeast ER retrieval signals.

(a) Schematic of the ER retrieval construct showing the human growth hormone signal sequence (hGHss), linker, mScarlet fluorescent protein, and 16 amino acid extension carrying a C-terminal (CT) retrieval signal from known human and yeast ER proteins. A sequence alignment shows the conservation of the retrieval signal. (b) WT KDEL receptor distribution was followed in the absence (-ligand) or presence of human BIP derived signals (K/R/H/A/DDELsec). (c) WT KDEL receptor distribution was followed in COS-7 cells in the absence (-ligand) or presence of yeast BIP derived signals K/R/HA/DDELsec. (d) The fraction of KDEL receptor localised to the Golgi was measured before (no ligand) and after challenge with the different retrieval signals tested in b. and c. Effect sizes are shown as the mean difference for retrieval signal comparisons against the shared -ligand control with sample sizes and p-values. (e) WT KDEL receptor distribution was followed in COS-7 cells in the absence (-ligand) or presence of ER retrieval signals from human FKBP family proteins. In all image panels, TGN46 was used as a Golgi marker and the scale bar is 10 µm.

Figure 5—figure supplement 1—source data 1

Source data for the ligand-induced KDELR receptor retrieval assays in Figure 5—figure supplement 1.

https://cdn.elifesciences.org/articles/68380/elife-68380-fig5-figsupp1-data1-v3.xlsx
Figure 6 with 2 supplements
Re-engineering the selectivity of the human KDEL receptor for ADEL and DDEL signals.

(a) WT and a series of ‘K. lactis’-like mutant KDEL receptors were tested for K/A/DDEL-induced redistribution from Golgi to ER in COS-7 cells. KDEL receptor distribution was followed in the absence (-ligand) or presence of K/A/DDELsec. TGN46 was used as a Golgi marker. Scale bar is 10 µm. (b) The fraction of WT and mutant KDEL receptor localised to the Golgi was measured before (no ligand) after challenge with different retrieval signals (K/A/DDEL). Effect sizes are shown as the mean difference for K/A/DDEL comparisons against the shared -ligand control with sample sizes and p values.

Figure 6—source data 1

Source data for the ligand-induced KDELR receptor retrieval assays in Figure 6.

https://cdn.elifesciences.org/articles/68380/elife-68380-fig6-data1-v3.xlsx
Figure 6—figure supplement 1
Extended analysis of human KDEL receptor selectivity.

(a) WT KDEL receptors and a series of S. pombe like mutants were tested for K/A/DDEL-induced redistribution from Golgi to ER in COS-7 cells. KDEL receptor distribution was followed in the absence (-ligand) or presence of K/A/DDELsec. TGN46 was used as a Golgi marker. Scale bar is 10 µm. (b) The fraction of WT and mutant KDEL receptor localised to the Golgi was measured before (no ligand) after challenge with different retrieval signals (K/A/DDEL). Effect sizes are shown as the mean difference for K/A/DDEL comparisons against the shared -ligand control with sample sizes and p values.

Figure 6—figure supplement 1—source data 1

Source data for the ligand-induced KDELR receptor retrieval assays in Figure 6—figure supplement 1.

https://cdn.elifesciences.org/articles/68380/elife-68380-fig6-figsupp1-data1-v3.xlsx
Figure 6—figure supplement 2
Retrieval specificity of ‘K. lactis’ and ‘S. pombe’ triple mutant KDEL receptors.

(a) Triple mutant ‘K. lactis’ and (b) ‘S. pombe’-like KDEL receptors were tested for K/A/DDEL-induced redistribution from Golgi to ER in COS-7 cells. KDEL receptor distribution was followed in the absence (-ligand) or presence of the indicated ligands. TGN46 was used as a Golgi marker. Scale bar is 10 µm. The fraction of WT and mutant KDEL receptor localised to the Golgi was measured before (no ligand) after challenge with different retrieval signals.

Figure 6—figure supplement 2—source data 1

Source data for the ligand-induced KDELR receptor retrieval assays in Figure 6—figure supplement 2.

https://cdn.elifesciences.org/articles/68380/elife-68380-fig6-figsupp2-data1-v3.xlsx
Figure 7 with 1 supplement
Mechanism for initial retrieval signal capture by the KDEL receptor.

(a) Images depicting the key stages (i.-iv.) of TAEKDEL binding to the wild-type (WT) KDEL receptor simulated using molecular dynamics. Initial engagement of the C-terminus to R169 (i) is followed by transfer to R5 (ii), shortly followed by interaction of E −2 with R169 (iii). Finally, R47 engages the C-terminus allowing D −3 to interact with R169 (iv). See also Video 1. (b) A carton model depicting the key stages of retrieval signal binding and final pH-dependent locked state. (c) Occupancy of the hydrogen bonds between the C-terminus of the KDEL retrieval signal and R5, R47, and R169 is plotted as a function of signal position within the binding pocket. (d) The occupancy of potential hydrogen bonds between the different positions of the KDEL retrieval signal and D50, S54, and E117 is plotted as a function of signal position within the binding pocket. (e) Competition binding assays for [3H]-TAEHDEL and unlabelled TAEKDEL and TAEHDEL with a free (COOH) or amidated (CONH) C-terminus to chicken KDELR2 showing IC50 values for the competing peptides. (f) Normalised binding of [3H]-TAEHDEL and [3H]-TAEKDEL signals to the purified WT H12A, R169A, or R169K mutant chicken KDELR2. A mock binding control with no receptor indicates the background signal. (g) Distribution of WT, H12A, R169A, and R169K KDEL receptors was measured in COS-7 cells in the absence (-ligand) or presence of K/R/HDELsec. The mean differences for K/R/HDEL comparisons against the shared no ligand control are shown with sample sizes and p values. See also Figure 7—figure supplement 1 with accompanying source data.

Figure 7—figure supplement 1
R169 plays a crucial role in signal recognition.

(a) Distribution of WT, H12A, R169A, and R169K KDEL receptors was measured in COS-7 cells in the absence (-ligand) or presence of K/R/HDEL (mScarlet-xDELsec). TGN46 was used as a Golgi marker. Scale bar is 10 µm. (b) The raw data for the fraction of KDEL receptor fluorescence in the Golgi is plotted on the upper axes with sample sizes, with effect sizes shown in the lower graphs.

Figure 7—figure supplement 1—source data 1

Source data for the ligand-induced KDELR receptor retrieval assays in Figure 7 and Figure 7—figure supplement 1.

https://cdn.elifesciences.org/articles/68380/elife-68380-fig7-figsupp1-data1-v3.xlsx
A combined proofreading and relay handover model for signal capture by the KDEL receptor.

Newly synthesised secretory and ER luminal proteins are translocated into the ER and on to the Golgi. Those proteins with C-terminal retrieval signals are captured by the KDELR receptor and returned to the ER. Other proteins with different C-terminal sequences move on to be secreted. The retrieval signal can be broken down into two sections: the variable −4 passkey position and the −1 to −3 positions with free carboxyl-terminus. Signals are initially captured through their free carboxyl-terminus by the receptor R169. This is then handed over to R5 and finally R47 in a relay mechanism. Sequences are proofread for the residue at the −4 position by gatekeeper residues D50 and E117. Unwanted signal variants are rejected. Only signals that completely enter the binding pocket and engage R47 can undergo pH dependent capture and return to the ER.

Videos

Video 1
Stepwise engagement of the KDEL signal with the KDEL receptor.

Molecular dynamics simulation of TAEKDEL binding to the KDEL receptor simulated using molecular dynamics.

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional information
Strain, strain background (Escherichia coli)XL1-Blue Competent CellsAgilent Technologies200249Used to prepare plasmid DNA
Strain, strain background (Saccharomyces cerevisiae)Bj5460ATCC208285Used for KDELR protein expression
Cell line (African green monkey)COS-7 kidney fibroblast-like cell lineATCCCRL-1651ER retrieval assays
Cell line (Homo-sapiens)HeLa S3 cervical adenocarcinomaATCCCCL-2.2Protein secretion assays
AntibodyTGN46 sheep polyclonalBio-rad (AbD Serotec)AHP500GIF (1:1000)
AntibodyGRP78 BiP rabbit polyclonalAbcamab21685WB (1:1000)
AntibodyPDI rabbit polyclonalProteinTech#11245–1WB (1:1000)
AntibodyERp72 rabbit monoclonalCell Signalling Technology#5033SWB (1:1000)
AntibodyERp44 rabbit monoclonalCell Signalling Technology# 3798SWB (1:1000)
AntibodyKDEL receptor mouse monoclonalEnzo Life SciencesADI-VAA-PT048IF (1:1000)
WB (1:1000)
AntibodyRFP mouse monoclonalChromotek6G6WB (1:1000)
Detects mScarlet on Western blot
AntibodyDonkey anti-Mouse IgG Alexa Fluor 488InvitrogenA-21202IF (1:2000)
Secondary
AntibodyDonkey anti-Sheep IgG Alexa Fluor 647InvitrogenA-21448IF (1:2000)
Secondary
AntibodyPeroxidase-AffiniPure Donkey Anti-Rabbit IgGJackson Immuno Research711-035-152-JIRWB (1:2000)
Secondary
AntibodyPeroxidase-AffiniPure Donkey Anti-Mouse IgGJackson Immuno Research711-035-152-JIRWB (1:2000)
Secondary
AntibodyPeroxidase-AffiniPure Donkey Anti-Sheep IgGJackson Immuno Research713-035-147-JIRWB (1:2000)
Secondary
Recombinant DNA reagentpcDNA3.1 hGHss-mScarlet-H. sapiens BiP639-654Bräuer et al., 2019KDELSECPMID:30846601
Recombinant DNA reagentpcDNA3.1 hGHss-mScarlet-H. sapiens BiP639-654 K651RThis paperRDELSECMaterial and methods. Available from Barr lab
Recombinant DNA reagentpcDNA3.1 hGHss-mScarlet-H. sapiens BiP639-654 K651HThis paperHDELSECMaterial and methods. Available from Barr lab
Recombinant DNA reagentpcDNA3.1 hGHss-mScarlet-H. sapiens BiP639-654K651A (ADELSEC)This paperADELSECMaterial and methods. Available from Barr lab
Recombinant DNA reagentpcDNA3.1 hGHss-mScarlet-H. sapiens BiP639-654K651D (DDELSEC)This paperDDELSECMaterial and methods. Available from Barr lab
Recombinant DNA reagentpcDNA3.1 hGHss-mScarlet-S. cerevisiae BiP667-682This paperYeast BiPMaterial and methods. Available from Barr lab
Recombinant DNA reagentpcDNA3.1 hGHss-mScarlet-S. pombe BiP648-663This paperS. pombe BiPMaterial and methods. Available from Barr lab
Recombinant DNA reagentpcDNA3.1 hGHss-mScarlet-S. pombe BiP648-663 A660KThis paperS. pombe BiP A > KMaterial and methods. Available from Barr lab
Recombinant DNA reagentpcDNA3.1 hGHss-mScarlet-K. lactis BiP664-679This paperK. lactis BIPMaterial and methods. Available from Barr lab
Recombinant DNA reagentpcDNA3.1 hGHss-mScarlet-K. lactis BiP664-679 D676KThis paperK.lactis BiP D > KMaterial and methods. Available from Barr lab
Recombinant DNA reagentpcDNA3.1 hGHss-mScarlet-H. sapiens FKBP7207-222This paperFKBP7Material and methods. Available from Barr lab
Recombinant DNA reagentpcDNA3.1 hGHss-mScarlet-H. sapiens FKBP9555-570This paperFKBP9Material and methods. Available from Barr lab
Recombinant DNA reagentpcDNA3.1 hGHss-mScarlet-H. sapiens FKBP10567-582This paperFKBP10Material and methods. Available from Barr lab
Recombinant DNA reagentpcDNA3.1 hGHss-mScarlet-H. sapiens FKBP14196-211This paperFKBP14Material and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1-GFPBräuer et al., 2019KDELR1PMID:30846601
Recombinant DNA reagentpEF5/FRT human KDELR2-GFPThis paperKDELR2Material and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 H12A-GFPBräuer et al., 2019H12AExpression in mammalian cells for functional assays; PMID:30846601
Recombinant DNA reagentpEF5/FRT human KDELR1 D50N-GFPThis paperD50NMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 N54K-GFPThis paperN54KMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 N54R-GFPThis paperN54RMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 E117Q-GFPThis paperE117QMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 E117D-GFPThis paperE117DMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 E117A-GFPThis paperE117AMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 E117N-GFPThis paperE117NMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 W120F-GFPThis paperW120FMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 W120A-GFPThis paperW120AMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 R169K-GFPThis paperR169KMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 R169A-GFPThis paperR169AMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 E117A/W120A-GFPThis paperE117A/W120AMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 D50N/N54K-GFPThis paperD50N/N54KMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 D50N/N54R-GFPThis paperD50N/N54RMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 D50N/E117Q-GFPThis paperD50N/E117QMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 D50N/E117N-GFPThis paperD50N/E117NMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 N54K/E117Q-GFPThis paperN54K/E117QMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 D50N/N54K/E117Q-GFPThis paperD50N/N54K/E117QMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 N54R/E117N-GFPThis paperN54R/E117NMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 D50N/N54R/E117N-GFPThis paperD50N/N54R/E117NMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 D50N/N54K/E117Q/W120A-GFPThis paperD50N/N54K/E117Q/W120AMaterial and methods. Available from Barr lab
Recombinant DNA reagentpEF5/FRT human KDELR1 D50N/N54R/E117N/W120A-GFPThis paperD50N/N54R/E117N/W120AMaterial and methods. Available from Barr lab
Recombinant DNA reagentpDDGFP-Leu2d-GgKDELR2Addgene123618Protein expression in yeast for biochemical assays and structures
Recombinant DNA reagentpDDGFP-Leu2d-GgKDELR2_H12AThis paperKDELR2_H12AMaterial and methods. Available from Newstead lab
Recombinant DNA reagentpDDGFP-Leu2d-GgKDELR2_E117AThis paperKDELR2_E117AMaterial and methods. Available from Newstead lab
Recombinant DNA reagentpDDGFP-Leu2d-GgKDELR2_E117DThis paperKDELR2_E117DMaterial and methods. Available from Newstead lab
Recombinant DNA reagentpDDGFP-Leu2d-GgKDELR2_E117QThis paperKDELR2_E117QMaterial and methods. Available from Newstead lab
Recombinant DNA reagentpDDGFP-Leu2d-GgKDELR2_E127AThis paperKDELR2_E127AMaterial and methods. Available from Newstead lab
Recombinant DNA reagentpDDGFP-Leu2d-GgKDELR2_E127QThis paperKDELR2_E127QMaterial and methods. Available from Newstead lab
Recombinant DNA reagentpDDGFP-Leu2d-GgKDELR2_W120AThis paperKDELR2_W120AMaterial and methods. Available from Newstead lab
Recombinant DNA reagentpDDGFP-Leu2d-GgKDELR2_W120FThis paperKDELR2_W120FMaterial and methods. Available from Newstead lab
Recombinant DNA reagentpDDGFP-Leu2d-GgKDELR2_R169AThis paperKDELR2_R169AMaterial and methods. Available from Newstead lab
Recombinant DNA reagentpDDGFP-Leu2d-GgKDELR2_R169KThis paperKDELR2_R169KMaterial and methods. Available from Newstead lab
Peptide, recombinant proteinTEV ProteaseMerckT4455-10KU
Peptide, recombinant protein3H-TAEHDELCambridge Research Biochemicalscustom synthesis185 MBq 106 Ci/mmol
Peptide, recombinant protein3H-TAEKDELCambridge Research Biochemicalscustom synthesis185 MBq 128 Ci/mmol
Peptide, recombinant proteinTAEHDELCambridge peptidescustom synthesis
Peptide, recombinant proteinTAEKDELCambridge peptidescustom synthesis
Peptide, recombinant proteinTAERDELCambridge peptidescustom synthesis
Peptide, recombinant proteinTAEDDELCambridge peptidescustom synthesis
Peptide, recombinant proteinTAEKDEL-CONHCambridge peptidescustom synthesisC-amidated peptide variant.
Peptide, recombinant proteinTAEHDEL-CONHCambridge peptidescustom synthesisC-amidated peptide variant.
Chemical compound, drugSodium phosphate monobasic (NaH2PO4)SigmaS8282
Chemical compound, drugSodium phosphate dibasic (Na2HPO4)Sigma71640
Chemical compound, drugSodium periodate (NaIO4)Sigma311448
Chemical compound, drug16% (w/v) FormaldehydeThermo Fisher Scientific28908
Chemical compound, drugSaponinSigmaS7900
Chemical compound, drugL-Lysine monohydrochlorideSigma62929
Chemical compound, drugMowiol 4–88Millipore475904
Chemical compound, drugTrichloroacetic acidSigmaT6399
Chemical compound, drugDodecyl maltoside (DDM)GlyconD97002-C
Chemical compound, drugCholesteryl hemisuccinate (CHS)SigmaC6512
Chemical compound, drugMonooleinSigmaM7765
Software, algorithmMetamorph 7.5Molecular Dynamics Inchttp://www.moleculardevices.comMicroscope image data acquisition
Software, algorithmFiji 2.0.0-rc-49/1.52iNIH Imagehttp://fiji.sc/Microscope image data analysis
Software, algorithmGraphPad Prism 7GraphPad Softwarehttp://www.graphpad.comGraph plotting
Software, algorithmRR project for statistical computinghttps://www.r-project.orgStatistical analysis and graph plotting
Software, algorithmAdobe Illustrator CCAdobe Systems Inchttp://www.adobe.comFigure preparation
Software, algorithmAdobe Photoshop CCAdobe Systems Inchttp://www.adobe.comFigure preparation
Software, algorithmCOOTEmsley et al., 2010https://www2.mrc-lmb.cam.ac.uk/personal/pemsley/cootMacromolecular structure model building; PMID:20383002
Software, algorithmPyMOLSchrodingerhttps://pymol.org/2Molecular visualisation
Software, algorithmBusterGlobal Phasinghttps://www.globalphasing.comStructure refinement
Software, algorithmGROMACSAbraham et al., 2015https://www.gromacs.orgMolecular dynamics
Software, algorithmGMX_lipid17.ff: GROMACSWu and Biggin, 2020http://doi.org/10.5281/zenodo.3610470Port of the Amber LIPID17 force field
Software, algorithmMDAnalysis 1.0SciPy2016https://conference.scipy.org/proceedings/scipy2016/oliver_beckstein.htmlAnalysis of molecular dynamics simulations
Software, algorithmModeller 9.21Webb and Sali, 2016https://salilab.org/modeller/PMID:27322406
OtherUltima Gold Scintillation FluidPerkin Elmer6013326
OtherHisPurTMThermo Fisher Scientific25214
OtherHisTrap HPCytiva17-5248-01
OtherSuperdex 200 10/300 GLCytiva28-9909-44
OtherUltra-15 Centrifugal Filter Unit, 50K NMWCAmiconUFC905024
OtherYeast Drop Out media -UraFormediumDCS0169
OtherYeast Drop Out media -LeuMerckY1376-20G
OtherTunair FlasksSigmaZ710822-4EA
OtherDulbecco's modified Eagle's mediumThermo Fisher Scientific31966–047
OtherFoetal Bovine SerumSigmaF9665
OtherTrypLE Express EnzymeThermo Fisher Scientific12605036
OtherOpti-MEMThermo Fisher Scientific11058021
OtherEZ-PCR Mycoplasma Test KitGeneflowK1-0210
OtherTransIT-LT1Mirus Bio LLCMIR 2306
OtherECL western blotting reagentCytivaRPN2106
Author response table 1
Calculated pKa values for retrieval signal -4 side chains.
WTW120FW120A
HDEL8.9+/-0.57.6+/-0.36.5+/-0.1
KDEL11.1+/-0.49.7+/-0.29.0+/-0.1

Additional files

Supplementary file 1

Crystallographic data collection statistics.

Values in parentheses are for the highest resolution shell.

https://cdn.elifesciences.org/articles/68380/elife-68380-supp1-v3.docx
Supplementary file 2

Free energy differences for HDEL protonation states and KDEL.

The free energy difference is expressed in kcal/mol and the sum is the ensemble free energy difference between KDEL and HDEL. HID signifies that the histidine is protonated at δ nitrogen and HIE means that the histidine is protonated at ε nitrogen, while HIP means that both positions are protonated. The occupancy of protonation state is computed at pH seven and expressed as a percentage (%).

https://cdn.elifesciences.org/articles/68380/elife-68380-supp2-v3.docx
Supplementary file 3

Cation-π and π-π contributions between receptor W120 and the −4 histidine in the retrieval signal.

The cation-π interaction is the energy resulting from induction calculated at the level of sSAPT0/jun-cc-pVDZ. The π-π interaction is the sum of exchange and correlation energy. The sum is the sum of cation-π and π-π energy.

https://cdn.elifesciences.org/articles/68380/elife-68380-supp3-v3.docx
Transparent reporting form
https://cdn.elifesciences.org/articles/68380/elife-68380-transrepform-v3.docx

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  1. Andreas Gerondopoulos
  2. Philipp Bräuer
  3. Tomoaki Sobajima
  4. Zhiyi Wu
  5. Joanne L Parker
  6. Philip Biggin
  7. Francis A Barr
  8. Simon Newstead
(2021)
A signal capture and proofreading mechanism for the KDEL-receptor explains selectivity and dynamic range in ER retrieval
eLife 10:e68380.
https://doi.org/10.7554/eLife.68380