Structures of human dynein in complex with the lissencephaly 1 protein, LIS1
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eLife assessment
This study presents the cryo-EM structure of the dynein regulator Lis1 bound to human dynein providing important insight into how these two proteins interact. The evidence supporting the claims of the authors is overall convincing though it requires some minor re-analysis. The work will be of interest to researchers working with motor proteins and neurodevelopmental disorders as it helps rationalize how mutations in Lis1 or dynein lead to disease.
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Abstract
The lissencephaly 1 protein, LIS1, is mutated in type-1 lissencephaly and is a key regulator of cytoplasmic dynein-1. At a molecular level, current models propose that LIS1 activates dynein by relieving its autoinhibited form. Previously we reported a 3.1 Å structure of yeast dynein bound to Pac1, the yeast homologue of LIS1, which revealed the details of their interactions (Gillies et al., 2022). Based on this structure, we made mutations that disrupted these interactions and showed that they were required for dynein’s function in vivo in yeast. We also used our yeast dynein-Pac1 structure to design mutations in human dynein to probe the role of LIS1 in promoting the assembly of active dynein complexes. These mutations had relatively mild effects on dynein activation, suggesting that there may be differences in how dynein and Pac1/LIS1 interact between yeast and humans. Here, we report cryo-EM structures of human dynein-LIS1 complexes. Our new structures reveal the differences between the yeast and human systems, provide a blueprint to disrupt the human dynein-LIS1 interactions more accurately, and map type-1 lissencephaly disease mutations, as well as mutations in dynein linked to malformations of cortical development/intellectual disability, in the context of the dynein-LIS1 complex.
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eLife assessment
This study presents the cryo-EM structure of the dynein regulator Lis1 bound to human dynein providing important insight into how these two proteins interact. The evidence supporting the claims of the authors is overall convincing though it requires some minor re-analysis. The work will be of interest to researchers working with motor proteins and neurodevelopmental disorders as it helps rationalize how mutations in Lis1 or dynein lead to disease.
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Reviewer #1 (Public Review):
LIS1 is a key dynein regulator and mutations in LIS1 cause the human brain developmental disease lissencephaly. The authors have previously reported a 3.1Å structure of yeast dynein bound to Pac1 (budding yeast LIS1) (Gillies et al., 2022, Elife). However, mutations they designed using the yeast dynein-PAC1 structure had mild effects on human dynein activation in vitro. Here they reported cryo-EM structures of human dynein-LIS1 complexes. While LIS1 and Pac1 bind to roughly the same sites (ring and stalk) of the dynein motor domains at the level of the 2D class averages, their current 3D cryo-EM structures of human dynein bound to one and two human LIS1 beta-propeller domains (4.0 Å and 4.1 Å resolution respectively) have revealed interesting similarities and differences in the interaction sites. In …
Reviewer #1 (Public Review):
LIS1 is a key dynein regulator and mutations in LIS1 cause the human brain developmental disease lissencephaly. The authors have previously reported a 3.1Å structure of yeast dynein bound to Pac1 (budding yeast LIS1) (Gillies et al., 2022, Elife). However, mutations they designed using the yeast dynein-PAC1 structure had mild effects on human dynein activation in vitro. Here they reported cryo-EM structures of human dynein-LIS1 complexes. While LIS1 and Pac1 bind to roughly the same sites (ring and stalk) of the dynein motor domains at the level of the 2D class averages, their current 3D cryo-EM structures of human dynein bound to one and two human LIS1 beta-propeller domains (4.0 Å and 4.1 Å resolution respectively) have revealed interesting similarities and differences in the interaction sites. In addition, they have provided the locations of missense mutations of LIS1 and dynein that cause lissencephaly and other human brain developmental or neurodegenerative disorders in the context of the human dynein-LIS1 structure. Overall, this first detailed structural analysis on human dynein-LIS1 interaction is well presented and will be important to the dynein field as well as people interested in lissencephaly and/or other neurodevelopmental disorders.
Methods are convincing. I do think it is important to point out that the dynein motor domain rather than full length dynein was used in this study. A relative weakness is the lack of functional analyses on the involved amino acids in the dynein-LIS1 and LIS1-LIS1 interaction interfaces. This is in contrast to the Gillies et al., 2022 paper, in which multiple functional assays were presented. However, knowing that functional assays are much more difficult to perform in human cells than in budding yeast, functional tests can be done in the future after this structural work is published.
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Reviewer #2 (Public Review):
Summary:
Dominant mutations in the gene encoding LIS1 cause lissencephaly, a severe developmental brain disorder. LIS1 regulates the multisubunit microtubule motor, cytoplasmic dynein 1, which can exist in an autoinhibited (closed) form and an activatable (open) form. Dynein is only active when bound to another complex, dynactin, and one of several known cargo adaptors. Because dynactin and cargo adaptors only interact with the open form, the local ratio of open to closed dynein can potentially dictate the proportion of "activatable" motors. The current view is that LIS1 stimulates dynein by reducing the autoinhibited closed form, and by recruiting two dynein motors to the active complex, which is thought to increase speeds and run lengths. LIS1 is highly conserved across animal and fungal species. The …Reviewer #2 (Public Review):
Summary:
Dominant mutations in the gene encoding LIS1 cause lissencephaly, a severe developmental brain disorder. LIS1 regulates the multisubunit microtubule motor, cytoplasmic dynein 1, which can exist in an autoinhibited (closed) form and an activatable (open) form. Dynein is only active when bound to another complex, dynactin, and one of several known cargo adaptors. Because dynactin and cargo adaptors only interact with the open form, the local ratio of open to closed dynein can potentially dictate the proportion of "activatable" motors. The current view is that LIS1 stimulates dynein by reducing the autoinhibited closed form, and by recruiting two dynein motors to the active complex, which is thought to increase speeds and run lengths. LIS1 is highly conserved across animal and fungal species. The budding yeast LIS1 ortholog, called Pac1, is around 43% identical to human LIS1. Both Pac1 and LIS1 regulate dynein but there have been intriguing differences in their effect on dynein processivity in assays with purified proteins. The authors of the current manuscript recently published high resolution cryo-EM studies of Pac1 bound to yeast dynein (Gilles 2022). Based on their models they tested several mutations predicted to impact Pac1 binding to dynein and showed these mutations disrupted the single dynein dependent process in budding yeast, translocation of the mitotic spindle. However, mutations in yeast dynein that impacted LIS1 binding apparently only modestly impacted human dynein, prompting the current study that compares cryo-EM studies of human LIS1 bound to human dynein with the previously published studies using yeast proteins. The work points to subtle differences in how yeast and human LIS1 interact with the stem and loop regions of yeast and human dynein heavy chains and reveal an intriguing difference in the residues predicted to be important for the interaction between the two LIS1 propeller structures in the LIS1 dimer. They also report map known disease causing mutations in LIS1 and dynein on the structures and find three that might impact residues involved in protein-protein interaction.Methods:
This group has been able to use innovative methods to increase the resolution of CryoEM images to 3-4 Å, allowing them to make more accurate predictions about residues involved in protein-protein interactions. They have substantial expertise in the analysis of the resultant data, as demonstrated by past peer review studies. These are very labor-intensive experiments that allow a level of detail not possible with standard biochemical or cell biological analyses.Results:
The studies revealed subtle, but potentially important, differences between the yeast and human proteins.Based on their analyses, the authors predicted specific residues that are likely to be important for human LIS1-dynein interactions with the stem region of dynein (sitestem) and with dyneins AAA domain containing ring (sitering). They also predicted specific residues that are likely to be important for an interaction between the two LIS1 beta-propellers, which in the yeast protein is apparently critical for dynein regulation.
The prediction that K147 could be important in the interaction between beta-propellers is very intriguing, given evidence that a K147A mutation disrupts LIS1 binding to dynein, but not to NDEL1, another interacting protein.
Impact
The predictions set out in this manuscript, if they hold up, could inform the design of tools to study LIS1 in the context of human disease. It seems likely from the data that at least some of the indicated residues will be important for human LIS1/dynein interactions
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Reviewer #3 (Public Review):
The lissencephaly 1 protein, LIS1, is key regulator of cytoplasmic dynein-1. Gillies et al., (2022) had previously reported a 3.1 Å structure of yeast dynein bound to Pac1, the yeast homologue of LIS1. This structure revealed the details of their interactions but mutational studies based on sequence homology indicated that it did not completely represent how Lis1 binds to human dynein. To mitigate this lack of knowledge, in this manuscript, the authors have solved the structure of Lis1 bound to human cytoplasmic dynein-1 using cryo-EM.
The authors solved structures of human dynein bound to one and two LIS1 β-propellers to 4.0 Å and 4.1 Å, respectively. These structures revealed that while the overall structure of dynein's interaction with LIS1/Pac1 is conserved from yeast to humans, there are important …
Reviewer #3 (Public Review):
The lissencephaly 1 protein, LIS1, is key regulator of cytoplasmic dynein-1. Gillies et al., (2022) had previously reported a 3.1 Å structure of yeast dynein bound to Pac1, the yeast homologue of LIS1. This structure revealed the details of their interactions but mutational studies based on sequence homology indicated that it did not completely represent how Lis1 binds to human dynein. To mitigate this lack of knowledge, in this manuscript, the authors have solved the structure of Lis1 bound to human cytoplasmic dynein-1 using cryo-EM.
The authors solved structures of human dynein bound to one and two LIS1 β-propellers to 4.0 Å and 4.1 Å, respectively. These structures revealed that while the overall structure of dynein's interaction with LIS1/Pac1 is conserved from yeast to humans, there are important differences in the specifics of the dynein-LIS1/Pac1 and LIS1/Pac1-LIS1/Pac1 interactions. The authors further suggest residues/interfaces that can be targeted in the future to probe the role of LIS1 in promoting the assembly of active dynein complexes.
This structure is an important piece in the puzzle of how LIS1 activates human dynein. The information on how to better disrupt the human dynein-LIS1 interface and where the human disease-causing mutations lie will be very important for future studies. -
