Cryo-EM structures of the caspase-activated protein XKR9 involved in apoptotic lipid scrambling
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Curated by eLife
Evaluation Summary:
This paper reports the atomic structure of XKR9, a membrane protein that is implicated in initiating the process to get rid of cells that are undergoing programmed cell death (apoptosis). The protein of interest was originally proposed to be a lipid channel, but the work presented here suggests that it is unlikely to function in this capacity alone. As a first step in this nascent field, the paper should be of interest to membrane structural biologists, and those working on lipid transport and apoptosis.
(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 #3 agreed to share their name with the authors.)
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Abstract
The exposure of the negatively charged lipid phosphatidylserine on the cell surface, catalyzed by lipid scramblases, is an important signal for the clearance of apoptotic cells by macrophages. The protein XKR9 is a member of a conserved family that has been associated with apoptotic lipid scrambling. Here, we describe structures of full-length and caspase-treated XKR9 from Rattus norvegicus in complex with a synthetic nanobody determined by cryo-electron microscopy. The 43 kDa monomeric membrane protein can be divided into two structurally related repeats, each containing four membrane-spanning segments and a helix that is partly inserted into the lipid bilayer. In the full-length protein, the C-terminus interacts with a hydrophobic pocket located at the intracellular side acting as an inhibitor of protein function. Cleavage by caspase-3 at a specific site releases 16 residues of the C-terminus, thus making the pocket accessible to the cytoplasm. Collectively, the work has revealed the unknown architecture of the XKR family and has provided initial insight into its activation by caspases.
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Author Response:
Reviewer #1:
Straub et al., present the first structures of membrane proteins from the XKR family of lipid scramblases. While structures of lipid scramblases from the TMEM16 family have been solved previously, it is the XKR family of proteins that have been identified as the scramblases involved in the dissipation of phosphatidyl-serine asymmetry in the plasma membrane to signal apoptosis. As such, the molecular details of these proteins has been highly sought after. Through the development of a synthetic nanobody that binds to XKR9 from Rattus norvegicus, the authors solved the full-length structure of this small 43 kDa protein by cryo-EM, with a resolution of 3.66 Å. This structure reveals a novel topology, adding to the growing repertoire of membrane protein folds. In addition, they were able to determine the …
Author Response:
Reviewer #1:
Straub et al., present the first structures of membrane proteins from the XKR family of lipid scramblases. While structures of lipid scramblases from the TMEM16 family have been solved previously, it is the XKR family of proteins that have been identified as the scramblases involved in the dissipation of phosphatidyl-serine asymmetry in the plasma membrane to signal apoptosis. As such, the molecular details of these proteins has been highly sought after. Through the development of a synthetic nanobody that binds to XKR9 from Rattus norvegicus, the authors solved the full-length structure of this small 43 kDa protein by cryo-EM, with a resolution of 3.66 Å. This structure reveals a novel topology, adding to the growing repertoire of membrane protein folds. In addition, they were able to determine the structure of the caspase-3 treated protein at a resolution of 4.3 Å, which cleaves a C-terminal peptide that has been proposed to be involved with scramblase activation. In addition, both structures possess densities that are suggestive of lipids, with densities embedded within the protein core, thus mapping out a putative lipid site or pathway. There has been very little structural information about XKR proteins so far, thus, this work is impactful to the field and pushes forward our ability to investigate a new class of lipid scramblases.
A limitation of this study is that the structures do not clearly inform on the mechanism quite yet. Unfortunately, transport function was not observable in the reconstituted liposomes, and so the connection between structure and function are limited. Certainly, it can be challenging to reconstitute function from purified proteins, but given that the previous studies of this protein are based on cellular activity, such as the rescue of PS scrambling of XKR8 knockouts by XKR9 and mutant constructs (Suzuki et al., JBC 2014), it is still not clear whether this protein provides the basic unit for transport or whether other components are required. With this, it is unclear whether the caspase cleaved protein informs on a mechanistically active structure. Thus, the paper needs to be clarified by focusing on the novel structure, potential lipid pathways and the difference in the caspase treated vs. full-length structures without speculating on the molecular mechanism.
We appreciate the comments of the reviewer and agree that we can at this point not demonstrate the function of XKR9 as scrambling unit beyond doubt. We thus toned down all hypotheses concerning molecular mechanisms.
Reviewer #2:
In this report by Straub and colleagues, they describe cryo-EM structures of a rat ortholog of XKR9 in full-length and caspase-9 activated states. The structure is a technical achievement due to the small size of XKR9 and provides a first view into this family of proteins, of which three members, XKR4, XKR8, and XKR9, participate in lipid scrambling. The structures are determined in complex with a synthetic monobody, resulting in an interpretable density map. To begin to understand the role of caspase cleavage in activation, a structure is determined following caspase activation. Notably, no changes could be detected in the cleaved form and it thus remains unclear how caspase activates XKR9 or how activated XKR9 mediates lipid scrambling. Overall, these results will be of broad interest and will likely serve as a foundation for future studies into this interesting family of proteins.
We appreciate the comments of the reviewer but would like to correct that the binder is a synthetic nanobody and not a monobody.
Reviewer #3:
This is a characteristically high quality report from the Dutzler lab of the atomic structure (via cryo EM, using synthetic single chain antibody for size enhancement) of a class of membrane proteins that was identified as being important for the exposure of the signaling lipid phosphatidylserine at the surface of apoptotic cells. These Xk-related proteins were proposed as caspase-activated lipid scramblases. The Dutzler paper reveals the structure of the Xkr9 homolog but the data do not allow conclusions about scramblase activity. No activity was detected and the protein in its two very similar conformations before and after caspase treatment offers no obvious clue as to its function. Nevertheless this is an important first step in this nascent field.
We thank the reviewer for these supportive comments.
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Evaluation Summary:
This paper reports the atomic structure of XKR9, a membrane protein that is implicated in initiating the process to get rid of cells that are undergoing programmed cell death (apoptosis). The protein of interest was originally proposed to be a lipid channel, but the work presented here suggests that it is unlikely to function in this capacity alone. As a first step in this nascent field, the paper should be of interest to membrane structural biologists, and those working on lipid transport and apoptosis.
(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 #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Straub et al., present the first structures of membrane proteins from the XKR family of lipid scramblases. While structures of lipid scramblases from the TMEM16 family have been solved previously, it is the XKR family of proteins that have been identified as the scramblases involved in the dissipation of phosphatidyl-serine asymmetry in the plasma membrane to signal apoptosis. As such, the molecular details of these proteins has been highly sought after. Through the development of a synthetic nanobody that binds to XKR9 from Rattus norvegicus, the authors solved the full-length structure of this small 43 kDa protein by cryo-EM, with a resolution of 3.66 Å. This structure reveals a novel topology, adding to the growing repertoire of membrane protein folds. In addition, they were able to determine the …
Reviewer #1 (Public Review):
Straub et al., present the first structures of membrane proteins from the XKR family of lipid scramblases. While structures of lipid scramblases from the TMEM16 family have been solved previously, it is the XKR family of proteins that have been identified as the scramblases involved in the dissipation of phosphatidyl-serine asymmetry in the plasma membrane to signal apoptosis. As such, the molecular details of these proteins has been highly sought after. Through the development of a synthetic nanobody that binds to XKR9 from Rattus norvegicus, the authors solved the full-length structure of this small 43 kDa protein by cryo-EM, with a resolution of 3.66 Å. This structure reveals a novel topology, adding to the growing repertoire of membrane protein folds. In addition, they were able to determine the structure of the caspase-3 treated protein at a resolution of 4.3 Å, which cleaves a C-terminal peptide that has been proposed to be involved with scramblase activation. In addition, both structures possess densities that are suggestive of lipids, with densities embedded within the protein core, thus mapping out a putative lipid site or pathway. There has been very little structural information about XKR proteins so far, thus, this work is impactful to the field and pushes forward our ability to investigate a new class of lipid scramblases.
A limitation of this study is that the structures do not clearly inform on the mechanism quite yet. Unfortunately, transport function was not observable in the reconstituted liposomes, and so the connection between structure and function are limited. Certainly, it can be challenging to reconstitute function from purified proteins, but given that the previous studies of this protein are based on cellular activity, such as the rescue of PS scrambling of XKR8 knockouts by XKR9 and mutant constructs (Suzuki et al., JBC 2014), it is still not clear whether this protein provides the basic unit for transport or whether other components are required. With this, it is unclear whether the caspase cleaved protein informs on a mechanistically active structure. Thus, the paper needs to be clarified by focusing on the novel structure, potential lipid pathways and the difference in the caspase treated vs. full-length structures without speculating on the molecular mechanism.
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Reviewer #2 (Public Review):
In this report by Straub and colleagues, they describe cryo-EM structures of a rat ortholog of XKR9 in full-length and caspase-9 activated states. The structure is a technical achievement due to the small size of XKR9 and provides a first view into this family of proteins, of which three members, XKR4, XKR8, and XKR9, participate in lipid scrambling. The structures are determined in complex with a synthetic monobody, resulting in an interpretable density map. To begin to understand the role of caspase cleavage in activation, a structure is determined following caspase activation. Notably, no changes could be detected in the cleaved form and it thus remains unclear how caspase activates XKR9 or how activated XKR9 mediates lipid scrambling. Overall, these results will be of broad interest and will likely serve …
Reviewer #2 (Public Review):
In this report by Straub and colleagues, they describe cryo-EM structures of a rat ortholog of XKR9 in full-length and caspase-9 activated states. The structure is a technical achievement due to the small size of XKR9 and provides a first view into this family of proteins, of which three members, XKR4, XKR8, and XKR9, participate in lipid scrambling. The structures are determined in complex with a synthetic monobody, resulting in an interpretable density map. To begin to understand the role of caspase cleavage in activation, a structure is determined following caspase activation. Notably, no changes could be detected in the cleaved form and it thus remains unclear how caspase activates XKR9 or how activated XKR9 mediates lipid scrambling. Overall, these results will be of broad interest and will likely serve as a foundation for future studies into this interesting family of proteins.
-
Reviewer #3 (Public Review):
This is a characteristically high quality report from the Dutzler lab of the atomic structure (via cryo EM, using synthetic single chain antibody for size enhancement) of a class of membrane proteins that was identified as being important for the exposure of the signaling lipid phosphatidylserine at the surface of apoptotic cells. These Xk-related proteins were proposed as caspase-activated lipid scramblases. The Dutzler paper reveals the structure of the Xkr9 homolog but the data do not allow conclusions about scramblase activity. No activity was detected and the protein in its two very similar conformations before and after caspase treatment offers no obvious clue as to its function. Nevertheless this is an important first step in this nascent field.
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