The MIDAS domain of AAA mechanoenzyme Mdn1 forms catch bonds with two different substrates
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Evaluation Summary:
Mickolajczyk et al. report the development of a new optical tweezers-based unbinding-force assay to characterize the interaction between the MIDAS domain of the mechanoenzyme Mdn1 and the ubiquitin-like (UBL) domain-containing ribosomal proteins Rsa1 and Ytm1. The authors show that the bond between MIDAS and Rsa1/Ytm1 can be best explained by a catch-slip bond behavior. The observations suggest that catch bonding between MIDAS and UBL domains plays a key role in the Mdn1-mediated ribosomal biogenesis. The reported results will be of interesting for the ribosomal and single-molecule biophysics communities and the developed DNA-tether-based optical tweezers assay will be useful for characterizing other molecular bonds.
(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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Abstract
Catch bonds are a form of mechanoregulation wherein protein-ligand interactions are strengthened by the application of dissociative tension. Currently, the best-characterized examples of catch bonds are between single protein-ligand pairs. The essential AAA (ATPase associated with diverse cellular activities) mechanoenzyme Mdn1 drives at least two separate steps in ribosome biogenesis, using its MIDAS domain to extract the ubiquitin-like (UBL) domain-containing proteins Rsa4 and Ytm1 from ribosomal precursors. However, it must subsequently release these assembly factors to reinitiate the enzymatic cycle. The mechanism underlying the switching of the MIDAS-UBL interaction between strongly and weakly bound states is unknown. Here, we use optical tweezers to investigate the force dependence of MIDAS-UBL binding. Parallel experiments with Rsa4 and Ytm1 show that forces up to ~4 pN, matching the magnitude of force produced by AAA proteins similar to Mdn1, enhance the MIDAS domain binding lifetime up to 10-fold, and higher forces accelerate dissociation. Together, our studies indicate that Mdn1’s MIDAS domain can form catch bonds with more than one UBL substrate, and provide insights into how mechanoregulation may contribute to the Mdn1 enzymatic cycle during ribosome biogenesis.
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Evaluation Summary:
Mickolajczyk et al. report the development of a new optical tweezers-based unbinding-force assay to characterize the interaction between the MIDAS domain of the mechanoenzyme Mdn1 and the ubiquitin-like (UBL) domain-containing ribosomal proteins Rsa1 and Ytm1. The authors show that the bond between MIDAS and Rsa1/Ytm1 can be best explained by a catch-slip bond behavior. The observations suggest that catch bonding between MIDAS and UBL domains plays a key role in the Mdn1-mediated ribosomal biogenesis. The reported results will be of interesting for the ribosomal and single-molecule biophysics communities and the developed DNA-tether-based optical tweezers assay will be useful for characterizing other molecular bonds.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the …
Evaluation Summary:
Mickolajczyk et al. report the development of a new optical tweezers-based unbinding-force assay to characterize the interaction between the MIDAS domain of the mechanoenzyme Mdn1 and the ubiquitin-like (UBL) domain-containing ribosomal proteins Rsa1 and Ytm1. The authors show that the bond between MIDAS and Rsa1/Ytm1 can be best explained by a catch-slip bond behavior. The observations suggest that catch bonding between MIDAS and UBL domains plays a key role in the Mdn1-mediated ribosomal biogenesis. The reported results will be of interesting for the ribosomal and single-molecule biophysics communities and the developed DNA-tether-based optical tweezers assay will be useful for characterizing other molecular bonds.
(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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Reviewer #1 (Public Review):
The experiments and analyses presented here seem carefully designed executed for the most part. The manuscript is very well written and conveys a clear and interesting message. Dependence of the binding strength on mechanical force is an intuitive and elegant form of regulation for which the work presented here provides examples. The conclusions should be of broad interest. Some control experiments ruling out artifacts of the attachment strategy would strengthen the conclusions. Likewise, some of the analyses should be better described, and better measurement statistics would improve the precision of the extracted parameters that describe the MIDAS-UBL interaction. The model for how tension is exploited for regulating Mdn1 function is intriguing, but could be better described.
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Reviewer #2 (Public Review):
The manuscript entitled "The MIDAS domain of AAA mechanoenzyme Mdn1 forms catch bonds with two different substrates" by Mickolajczyk reports on optical trap experiments of the Mdn1 MIDAS domain and the UBL domains of two of its known substrates, Rsa4 and Ytm1. The manuscript is clearly written and easy to follow. Using gel filtration shift and microscale thermophoresis studies, the authors demonstrate weak binding affinities between the purified MIDAS domain and the UBL domains of Rsa4 and Ytm1, respectively. This is followed by optical trap experiments that show an increase in bond life time when stronger forces are applied indicative of increased binding affinity. Further increasing the applied force leads to a decrease in bond life time, which reveals the catch-slip bond concept. The authors develop a …
Reviewer #2 (Public Review):
The manuscript entitled "The MIDAS domain of AAA mechanoenzyme Mdn1 forms catch bonds with two different substrates" by Mickolajczyk reports on optical trap experiments of the Mdn1 MIDAS domain and the UBL domains of two of its known substrates, Rsa4 and Ytm1. The manuscript is clearly written and easy to follow. Using gel filtration shift and microscale thermophoresis studies, the authors demonstrate weak binding affinities between the purified MIDAS domain and the UBL domains of Rsa4 and Ytm1, respectively. This is followed by optical trap experiments that show an increase in bond life time when stronger forces are applied indicative of increased binding affinity. Further increasing the applied force leads to a decrease in bond life time, which reveals the catch-slip bond concept. The authors develop a "mechanical circuit model" in order to more precisely quantify the forces involved in the MIDAS-UBL catch-slip bond. All experiments are well designed, include the appropriate controls and the conclusions drawn are justified.
Early work by the Hurt group introduced the MIDAS-UBL catch bond concept based on the sequence homology with integrins (Ulbrich et al., 2009). However, until now no direct experimental proof of this concept has been published. The authors of the current study now demonstrate the catch-slip bond concept using a sophisticated optical trap arrangement. The MIDAS-UBL interaction is a key feature of Mdn1 mediated substrate removal from pre-ribosomal particles and therefore this study provides important insights into the Mdn1 mechanism. For the first time, the forces involved in the MIDAS-UBL interactions are quantified and the force dependency of the bond lifetime is revealed. This study is likely to make an important impact in the field.
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Reviewer #3 (Public Review):
The manuscript by Mickolajczyk et al. reports on the development of a new optical tweezers-based unbinding-force assay to characterize the bond between the MIDAS domain of the mechanoenzyme Mdn1 and the ubiquitin-like (UBL) domain-containing ribosomal proteins Rsa1 and Ytm1. The authors show that while the affinity between the MIDAS domain and Rsa1 and Ytm1 is only weak (≥7 μM) in solution, it increases significantly under an applied load. Using the developed assay, the authors show that the bond between MIDAS and Rsa1/Ytm1 can be best explained by a catch-slip bond behavior. The authors suggest that the catch bonding between MIDAS and UBL domains plays a key role in the Mdn1-mediated ribosomal biogenesis. The reported results are highly interesting for the ribosomal and single-molecule biophysics …
Reviewer #3 (Public Review):
The manuscript by Mickolajczyk et al. reports on the development of a new optical tweezers-based unbinding-force assay to characterize the bond between the MIDAS domain of the mechanoenzyme Mdn1 and the ubiquitin-like (UBL) domain-containing ribosomal proteins Rsa1 and Ytm1. The authors show that while the affinity between the MIDAS domain and Rsa1 and Ytm1 is only weak (≥7 μM) in solution, it increases significantly under an applied load. Using the developed assay, the authors show that the bond between MIDAS and Rsa1/Ytm1 can be best explained by a catch-slip bond behavior. The authors suggest that the catch bonding between MIDAS and UBL domains plays a key role in the Mdn1-mediated ribosomal biogenesis. The reported results are highly interesting for the ribosomal and single-molecule biophysics communities and the developed DNA-tether-based optical tweezers assay will be useful for characterizing other molecular bonds.
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