A signal capture and proofreading mechanism for the KDEL-receptor explains selectivity and dynamic range in ER retrieval
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Evaluation Summary:
Binding of cargo to sorting receptors in membrane trafficking is essential to cellular organization. This work is significant because it generates a detailed model of the key residues accounting for specificity and affinity of binding by the KDEL receptor. Interestingly, it is not the affinity per se that accounts for the specificity of cargo binding but rather charge-based exclusion of potentially competing signals.
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Abstract
ER proteins of widely differing abundance are retrieved from the Golgi by the KDEL-receptor. Abundant ER proteins tend to have KDEL rather than HDEL signals, whereas ADEL and DDEL are not used in most organisms. Here, we explore the mechanism of selective retrieval signal capture by the KDEL-receptor and how HDEL binds with 10-fold higher affinity than KDEL. Our results show the carboxyl-terminus of the retrieval signal moves along a ladder of arginine residues as it enters the binding pocket of the receptor. Gatekeeper residues D50 and E117 at the entrance of this pocket exclude ADEL and DDEL sequences. D50N/E117Q mutation of human KDEL-receptors changes the selectivity to ADEL and DDEL. However, further analysis of HDEL, KDEL, and RDEL-bound receptor structures shows that affinity differences are explained by interactions between the variable −4 H/K/R position of the signal and W120, rather than D50 or E117. Together, these findings explain KDEL-receptor selectivity, and how signal variants increase dynamic range to support efficient ER retrieval of low and high abundance proteins.
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Reviewer #3 (Public Review):
The authors have have convincingly demonstrated that modifying residues in the KDEL signal peptide and/or receptor can have a dramatic effect on retrograde targeting and their binding affinities. At a simplistic level, the structure of the electrostatic surface of the KDEL is very positively charged and then transitions into a negatively charged surface. The transition point from negative to positive charge is exactly at the level of the -4 position. As such, a bunch of arginine residues in the receptor progressively engage the signal until its locked in place by salt-bridges (E-117/D50) to the "K/H/R"-residue. In addition, pi-cation interactions between a tryptophan residue and the "K/H/R"- residue in the -4 position are important. To validate and quantify interactions, crystal structures have been solved …
Reviewer #3 (Public Review):
The authors have have convincingly demonstrated that modifying residues in the KDEL signal peptide and/or receptor can have a dramatic effect on retrograde targeting and their binding affinities. At a simplistic level, the structure of the electrostatic surface of the KDEL is very positively charged and then transitions into a negatively charged surface. The transition point from negative to positive charge is exactly at the level of the -4 position. As such, a bunch of arginine residues in the receptor progressively engage the signal until its locked in place by salt-bridges (E-117/D50) to the "K/H/R"-residue. In addition, pi-cation interactions between a tryptophan residue and the "K/H/R"- residue in the -4 position are important. To validate and quantify interactions, crystal structures have been solved with the HDEL and RDEL peptides and computational studies have analysed the pi-cation interactions. Moreover, differences are discussed between the tighter (HDEL) vs. weaker (KDEL) binding peptides in context with the differences in pH between the Golgi and ER.
Strengths:
The authors build upon their previous crystal structure of the KDEL receptor and the Newstead and Barr groups team up to provide a strong scientific approach combining structure-function analysis with trafficking and cell biology to yield important molecular insights into the recognition of ER-retention signals by the KDEL receptor. This paper is technically strong and well-written in most parts. They are able to build a connection between the variation of ER-retention signals "K/H-DEL" binding affinities with their pH dependancies and the natural abundance of ER proteins.
Weaknesses:
The authors have not made detailed pH dependent profiles for the "R"-DEL retention signal. This is an important comparative control, because unlike lysine and histidine, arginine has a very high pKa and will therefore always remain protonated. The authors refer to the "acidic" Golgi versus the neutral pH of the ER. However, it would be more correct to refer to the mildy acidic Golgi vs neutral pH of the ER and give the pH values of 7.4 for ER and pH 6.2 for the Golgi lumen. This sets up the scientific question to be more nuanced, as its only a pH difference of around 0.5 to 1.0 pH units. The authors have not included the computational estimates for the pKa values of the "K/R/H"-DEL residues nor the comparative pH dependence of KDEL and RDEL binding affinities, which is needed to properly asses the influence of differences in pH between Golgi and ER organelles and functional significance of the RDEL variant in particular.
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Reviewer #2 (Public Review):
This study combines several different techniques to study the binding of the signal sequences of ER-resident protein to the KDEL receptor, which is required for their retrieval from later stages of the secretory pathway. The ER-resident proteins have a C-terminal four amino acid sequence, typically KDEL, HDEL or RDEL, which are bound by the KDEL receptor in the Golgi, leading to their return to the ER for another round of activities. Failure to retrieve the ER-proteins leads their secretion and loss of these valuable chaperones and enzymes. Structural work has highlighted the mode of binding for several variants of the signal sequence, here and in previous work. Binding studies are used to observe differences in affinity between the various signal sequences, showing that the HDEL sequence has the highest …
Reviewer #2 (Public Review):
This study combines several different techniques to study the binding of the signal sequences of ER-resident protein to the KDEL receptor, which is required for their retrieval from later stages of the secretory pathway. The ER-resident proteins have a C-terminal four amino acid sequence, typically KDEL, HDEL or RDEL, which are bound by the KDEL receptor in the Golgi, leading to their return to the ER for another round of activities. Failure to retrieve the ER-proteins leads their secretion and loss of these valuable chaperones and enzymes. Structural work has highlighted the mode of binding for several variants of the signal sequence, here and in previous work. Binding studies are used to observe differences in affinity between the various signal sequences, showing that the HDEL sequence has the highest affinity, but proteins containing KDEL or RDEL are more abundant. A system is set up where mScarlet proteins are tagged by a range of different C-terminal xDEL sequences to monitor the distribution of these proteins in the cell, looking at their retrieval from the Golgi. Next, a series of mutations are made in the KDEL receptor, targeting residues that are potentially involved in binding of the signal sequence and their ability to retrieve the different tagged mScarlet proteins is studied. Finally, a molecular dynamics simulation is carried out to monitor the binding process of the peptide sequence, showing a relay of positively charged residues involved in the consecutive binding of the negatively charged residues of the signal peptide and the carboxy terminus.
The paper is an excellent example of the use of a large number of different techniques, spanning structural, biophysical, cell biological and computational methods, to provide new and detailed insights into the binding mechanism of signal peptides to the KDEL receptor. It is one of the most complete papers I have had the pleasure to review, as it looks at this problem from so many different angles.
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Reviewer #1 (Public Review):
ER retrieval mediated by the KDEL receptor occurs for cargo present from great to minimal abundance and for cargo with different variations of the "KDEL" carboxy terminal sorting signal. Using a detailed structure/function approach, this study provides insight into the mechanism of cargo recognition by the receptor. A significant advance is the new structural data derived from co-crystals of the receptor with TAEHDEL and TAERDEL peptides that is now compared with the previous KDEL co-crystal structure. From there, the investigators use mutation of both receptor and cargo sequences as well as molecular simulation of the binding interaction. Altogether the findings identify charged receptor residues playing a role in specificity based on the -4 position of the signal, a receptor tryptophan that accounts for …
Reviewer #1 (Public Review):
ER retrieval mediated by the KDEL receptor occurs for cargo present from great to minimal abundance and for cargo with different variations of the "KDEL" carboxy terminal sorting signal. Using a detailed structure/function approach, this study provides insight into the mechanism of cargo recognition by the receptor. A significant advance is the new structural data derived from co-crystals of the receptor with TAEHDEL and TAERDEL peptides that is now compared with the previous KDEL co-crystal structure. From there, the investigators use mutation of both receptor and cargo sequences as well as molecular simulation of the binding interaction. Altogether the findings identify charged receptor residues playing a role in specificity based on the -4 position of the signal, a receptor tryptophan that accounts for higher affinity binding to HDEL, and binding pocket arginines that may be sequentially engaged by the carboxy terminus for capture of the three carboxyl groups in the DEL portion of the signal. The work is meticulously carried out and the findings will likely be of significant interest to the field.
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Evaluation Summary:
Binding of cargo to sorting receptors in membrane trafficking is essential to cellular organization. This work is significant because it generates a detailed model of the key residues accounting for specificity and affinity of binding by the KDEL receptor. Interestingly, it is not the affinity per se that accounts for the specificity of cargo binding but rather charge-based exclusion of potentially competing signals.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #2 agreed to share their name with the authors.)
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