Structural basis for cytoplasmic dynein-1 regulation by Lis1
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Evaluation Summary:
LIS1 is a key regulator of the microtubule motor cytoplasmic dynein. Here the authors use yeast proteins and streptavidin-coated grids to solve the first high-resolution (3.1Å) structure of the dynein-Lis1 complex. The two beta-propellors in a Lis1 dimer make contact with different sites on a single dynein motor domain. Mutagenesis shows both sites are important for yeast and human dynein and uncover how they modulate motor function.
(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 #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
The lissencephaly 1 gene, LIS1 , is mutated in patients with the neurodevelopmental disease lissencephaly. The Lis1 protein is conserved from fungi to mammals and is a key regulator of cytoplasmic dynein-1, the major minus-end-directed microtubule motor in many eukaryotes. Lis1 is the only dynein regulator known to bind directly to dynein’s motor domain, and by doing so alters dynein’s mechanochemistry. Lis1 is required for the formation of fully active dynein complexes, which also contain essential cofactors: dynactin and an activating adaptor. Here, we report the first high-resolution structure of the yeast dynein–Lis1 complex. Our 3.1 Å structure reveals, in molecular detail, the major contacts between dynein and Lis1 and between Lis1’s ß-propellers. Structure-guided mutations in Lis1 and dynein show that these contacts are required for Lis1’s ability to form fully active human dynein complexes and to regulate yeast dynein’s mechanochemistry and in vivo function.
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Evaluation Summary:
LIS1 is a key regulator of the microtubule motor cytoplasmic dynein. Here the authors use yeast proteins and streptavidin-coated grids to solve the first high-resolution (3.1Å) structure of the dynein-Lis1 complex. The two beta-propellors in a Lis1 dimer make contact with different sites on a single dynein motor domain. Mutagenesis shows both sites are important for yeast and human dynein and uncover how they modulate motor function.
(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 #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
LIS1 is a key dynein regulator and details about its mechanism of action have captivated many in the field. In this manuscript, the authors reported the first high-resolution (3.1Å) structure of the dynein-Lis1 complex using yeast proteins including the dynein motor domain and LIS1 dimer. This high-resolution structure allows the authors to reveal new details of interaction interfaces, specifically the LIS1-binding site on AAA5, the LIS1-binding site on the stalk as well as the contact site between the two LIS1 propellers (one binds the ring and the other binds stalk). The authors made specific mutations (in LIS1 or dynein) to investigate the significance of these interactions. Finally, they also made mutations in human dynein that affect LIS1 binding to the same sites (ring and stalk) and investigated the …
Reviewer #1 (Public Review):
LIS1 is a key dynein regulator and details about its mechanism of action have captivated many in the field. In this manuscript, the authors reported the first high-resolution (3.1Å) structure of the dynein-Lis1 complex using yeast proteins including the dynein motor domain and LIS1 dimer. This high-resolution structure allows the authors to reveal new details of interaction interfaces, specifically the LIS1-binding site on AAA5, the LIS1-binding site on the stalk as well as the contact site between the two LIS1 propellers (one binds the ring and the other binds stalk). The authors made specific mutations (in LIS1 or dynein) to investigate the significance of these interactions. Finally, they also made mutations in human dynein that affect LIS1 binding to the same sites (ring and stalk) and investigated the functional significance of the involved amino acids in cargo-adapter- and dynactin-mediated dynein activation/motility assay. Their results suggest that these sites are important for the formation of active human dynein complexes.
Major Strengths of the methods and results:
Overall, the authors have done a very impressive and beautiful series of experiments. Solving such a high-resolution structure of the dynein-LIS1 complex is a great accomplishment and will be highly impactful. Another major strength is the combination of in vivo and in vitro functional analyses on the significance of the amino acids at the contact sites. The functional study was done by using multiple assays both in vitro (binding curve and effect on in vitro velocity) and in vivo (nuclear positioning and dynein localization in budding yeast). This combination is very powerful, as it allows the authors to reveal separation-of-function mutations. One mutation affecting the interaction between LIS1 and the dynein stalk is particularly significant (please see my detailed comments).
Major weaknesses of the methods and results:
In vitro assays were done to test whether the mutations affect LIS1's ability to induce a tight dynein-microtubule binding or an inhibition of dynein motility in the presence of ATP, while Marzo, et al., 2020 NCB has shown that yeast LIS1 enhances dynein processivity if salt concentration is increased for the in vitro motility assay.
More detailed comments:
1. Currently, the model that the authors proposed emphasizes the idea that the LIS1-induced tight dynein-microtubule binding enhances the microtubule-plus-end accumulation of dynein, which in turn promotes active complex formation. This is inconsistent with experimental findings: In the budding yeast, the microtubule-binding domain is not required for the plus-end accumulation of dynein (Lammer and Markus 2015). In mammalian cells (as well as filamentous fungi), dynactin is needed for the plus-end accumulation of dynein (Xiang et al., 2000; Zhang et al., 2003, 2008, Lenz et al., 2006; Egan et al., 2012, Splinter et al., 2012., Yao et al., 2012) and this is also true in vitro in reconstituted systems (Duellburg et al., 2014; Baumbach et al., 2017; Jha et al., 2017). In addition, it is unclear how LIS1 would affect the mechanochemistry of dynein to induce a tight binding of dynein to microtubules (aside from inducing the open state).
2. Data were presented to argue for an inconsistency with a "tethering" model in which LIS1 tethers dynein to microtubules non-specifically. This was done to argue against the idea proposed by Marzo et al., 2020 that the Pac1/LIS1-caused speed reduction of dynein is due to the non-specific binding of Pac1/LIS1 to microtubules. My problems with the argument are as follows: Marzo et al showed that Pac1/LIS1 does not need to bind dynein to cause a speed reduction, since dynein complexed with Pac1 or not complexed with Pac1 gets the same kind of speed reduction. Aside from that, the dimeric LIS1 (LIS1 WT) does seem to lower the velocity more dramatically (Fig 3C), and the argument that dynein-binding and microtubule-binding cannot possibly use the same site of LIS1 only works when a monomer is considered. In this context, while the current study clearly shows the importance of propeller-propeller interaction, LIS1 monomer was shown to be effective in promoting active human dynein complex formation in vitro, suggesting that monomers at a high concentration may still lead to propeller interaction (Htet et al., 2020).
3. Lis1S248Q and Lis1F185D, I189D, R494A are capable of inducing tight microtubule binding in the presence of ATP but mutants carrying these mutations show a clear nuclear-positioning phenotype in yeast. This at least argues against the tight MT-binding being the only key effect of LIS1 in vivo. What would be the other key effects? "The weakening of dynein's interactions with microtubules when AAA3 is bound to ATP" does not seem to agree with the genetic data from Aspergillus that the wB-AAA3 mutation allows LIS1 function to be bypassed (Qiu et al., 2021 bioRxiv). It seems more likely that LIS1's effect on promoting the open dynein state is relevant in vivo as this was shown in both yeast and Aspergillus (Qiu et al., 2019; Marzo et al., 2020). These two mutants that the authors made are significant, and it would be very interesting to see if the open dynein (phi mutant) would suppress their nuclear-positioning defect.
4. The detailed analysis performed by the authors has revealed Lis1S248Q (affecting the stalk interaction) as a mutation that affects dynein function (cortical interaction and nuclear distribution) but does not affect the plus-end dynein accumulation. I would like to emphasize more explicitly the importance of this separation-of-function mutant of Pac1/LIS1 (to my knowledge, this is the first such separation-of-function mutant of Pac1/LIS1). The cortical interaction of dynein-dynactin with Num1 depends on the plus-end accumulation of dynein in budding yeast, and thus, it depends on Pac1/LIS1 (Lee et al., 2003; Sheeman et al., 2003; Markus and Lee 2011). Pac1/LIS1 recruits dynein to the plus end most likely because Pac1/LIS1 binds directly to the microtubule plus-end-tracking protein Bik1/Clip170 (Sheeman et al., 2003; Coquelle et al., 2002; Lin et al., 2001; Perez et al 1999; Markus et al., 2012). Due to the requirement of Pac1/LIS1 for the plus-end dynein accumulation, it was not easy to dissect the specific role of Pac1/LIS1 in cargo adapter-mediated dynein activation in budding yeast, although the coiled-coil domains of the cortical adapter Num1 also activates dynein to cause its relocation from the plus end to the minus ends (Lammers and Markus 2015). In Aspergillus nidulans and ustilago maydis, LIS1 is not required for the plus-end accumulation of dynein (Zhang et al., 2002, 2003, Lenz et al., 2006, Egan et al., 2012), which makes it more straightforward to find a role of LIS1 in cargo adaptor-mediated dynein activation in A. nidulans (Qiu et al., 2019). In this context, I believe that this new Lis1S248Q mutant will become a wonderful tool for the field because it will allow new assays (both in vivo and in vitro) to be performed to further study the function of LIS1.
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Reviewer #2 (Public Review):
The main goal of the current study was to better define interaction sites between LIS1 and the microtubule (MT) motor cytoplasmic dynein 1 (dynein). Dynein exists in a closed, autoinhibited form and an open form capable of binding to activating factors dynactin and various cargo adaptors. These "DDA complexes" are processive, while mammalian dynein on its own is not. Cell-based studies indicated that LIS1 stimulates dynein, but work from this group and others indicated that LIS1 inhibits dynein by inducing a tight MT bound state. New data shows LIS1 binds to the open form of dynein and prevents it from switching back to the closed form, catalyzing the formation of processive DDA complexes and preventing conversion to the autoinhibited state. LIS1 also recruits 2 dynein motors into the complex, leading to …
Reviewer #2 (Public Review):
The main goal of the current study was to better define interaction sites between LIS1 and the microtubule (MT) motor cytoplasmic dynein 1 (dynein). Dynein exists in a closed, autoinhibited form and an open form capable of binding to activating factors dynactin and various cargo adaptors. These "DDA complexes" are processive, while mammalian dynein on its own is not. Cell-based studies indicated that LIS1 stimulates dynein, but work from this group and others indicated that LIS1 inhibits dynein by inducing a tight MT bound state. New data shows LIS1 binds to the open form of dynein and prevents it from switching back to the closed form, catalyzing the formation of processive DDA complexes and preventing conversion to the autoinhibited state. LIS1 also recruits 2 dynein motors into the complex, leading to more force generation and increased speeds and run lengths. The current thinking about LIS1's ability to stall dynein by inducing a tight MT-bound state is that binding at MT plus ends allows it to recruit plus-end-localized dynactin to the open dynein complex .
In previous work this group identified two PAC1 (budding yeast LIS1) interaction sites in dynein heavy chain which they call site "ring" and site "stalk". However, the resolution was not sufficient to build models to predict the interaction sites at finer resolution. In the current manuscript the resolution of Cryo-EM data was increased by biotinylating dynein and using streptavidin affinity grids, which was able to reduce dynein's strong preferred orientation on the EM grids and allowed it to be tethered to the grid in random orientations. They identified sites of contact between LIS1 and dynein and between the two LIS1 b-propellers that, when mutated, prevent LIS1 from being able to induce the formation of activated dynein complexes in yeast, and reduced LIS1's capacity to stimulate human DDA complexes. This paper clearly adds to our understanding of the precise sites of dynein - LIS1 interactions and has several strengths, but there are a few concepts that need clarification.
Strengths
• This paper represents a huge body of work and the sites identified by their approach will guide future studies from many labs.
• The work revealed a previously unknown additional contact site for LIS1 in dynein's AAA5 and used a yeast assay for dynein activity to show this contact site is important for LIS1 to regulate dynein in vivo.
• They provide evidence that the interaction between LIS1 b-propellers is required for LIS1 regulation at site "stalk".
• The work supports a model in which the NUM1 cortical protein in yeast can act as a cargo adaptor for dynein/dynactin complex.
• They show that mutations in residues they identified as important in regulating yeast dynein are also important for human dynein.Weaknesses
The following points were not clear to me as a general reader and it would have been helpful if they were clarified
• Is it possible that the mechanism of driving orientation of the sample on the grid changes potential interactions or selects for particular interactions?
• It is not clear why the decision was made to generate a model with ATPs in AAA1, AAA2, and AAA3. Also, ADP was apparently bound to AAA4 in their complexes. How was this determined, and why was this the case? Does LIS1 impact ATP hydrolysis at AAA4?
• Binding affinities in Figure 2 , 3 and 7 all appear to utilize monomeric dynein. Is there any data regarding if or how these values would change using the dynein holoenzyme?
• In FIG 3 PAC1/LIS1 is predicted not to interact directly with MTs, in part because monomeric PAC1/LIS1 also slowed dynein. If it did so by crosslinking dynein and MTs, then dynein's stalk angle would need to be ~ 15˚, which is rarely seen, at least with tail-truncated monomeric dynein (Can et al, 2019). Have stalk angles on MTs been measured in the presence of either monomeric or dimeric LIS1? -
Reviewer #3 (Public Review):
This manuscript reports a high resolution (~3.1 Å) cryo-EM structure of the yeast dynein-Lis1 complex. The resolution of the EM map allowed for the determination of additional dynein-Lis1 contacts that are important for Lis1's regulation of dynein. Specifically, the molecular interactions between the Lis1 ß-propellers and its two binding sites on dynein were described and probed. The authors propose a model in which Lis1 does not tether dynein to microtubules. Taken together, the authors leverage their high-resolution structure of the dynein-Lis1 complex to provide insights into how Lis1 modulates yeast dynein function. Mutations in human dynein at the same sites disrupt Lis1-mediated dynein activation.
This manuscript is extremely well written and we appreciated the effort made by the authors in laying out …
Reviewer #3 (Public Review):
This manuscript reports a high resolution (~3.1 Å) cryo-EM structure of the yeast dynein-Lis1 complex. The resolution of the EM map allowed for the determination of additional dynein-Lis1 contacts that are important for Lis1's regulation of dynein. Specifically, the molecular interactions between the Lis1 ß-propellers and its two binding sites on dynein were described and probed. The authors propose a model in which Lis1 does not tether dynein to microtubules. Taken together, the authors leverage their high-resolution structure of the dynein-Lis1 complex to provide insights into how Lis1 modulates yeast dynein function. Mutations in human dynein at the same sites disrupt Lis1-mediated dynein activation.
This manuscript is extremely well written and we appreciated the effort made by the authors in laying out the manuscript and figures, as well as the introduction. The authors have also incorporated previously published data and possible models into their analyses. Technically, this is a very difficult sample for cryo EM, with severe preferred orientation, and the use of specialized streptavidin affinity grids was key in facilitating the high resolution structure. The conclusions are supported by the cryo EM and functional data presented, and the model is consistent with the data.
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