The MIDAS domain of AAA mechanoenzyme Mdn1 forms catch bonds with two different substrates

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.

Combining DNA scaffolds and acoustic force spectroscopy to characterize individual protein bonds

“…J-DNA scaffolds. Similarly to the others nucleic acids-based scaffolds recently developed for single-molecule investigations (15,16,20,22,(27)(28)(29)(30)(31)(32)39), J-DNAs have a forceps-like anatomy (23) (Fig. S1A).…”

“…An additional advantage provided by such constructs is mechanical fingerprinting: upon bond rupture, the two partners must end separated by the length of the leash; events that do not verify this criterion can be easily sorted out and discarded (1,(22)(23)(24). In practice, these leashes may consist in peptidic chains (1,2,4,25,26), single DNA strands (27)(28)(29), or double DNA strands, with (15,22,30) or without nicks (20,31,32). While peptidic linkers require to design and produce a specific fusion protein, nucleic acids based structures offer more modularity when combined with state-of-the-art DNA-protein coupling strategies (20,23,(33)(34)(35)(36)(37)(38).…”

“…While peptidic linkers require to design and produce a specific fusion protein, nucleic acids based structures offer more modularity when combined with state-of-the-art DNA-protein coupling strategies (20,23,(33)(34)(35)(36)(37)(38). As a matter of fact, the latter kind of scaffold has already been used for non-covalent bond characterization on various SMFS setups: OT (22,(27)(28)(29), MT (20,28,31,32), CFM (14,15), and LFC (30).…”

Prey killing without invasion by Bdellovibrio bacteriovorus defective for a MIDAS-family adhesin

“…They initially defined the MIDAS motif as Asp-X-Ser-X-Ser with an associated additional acidic residue. Mickolajaczyk and co-workers further examined the activities and functional processes of MIDAS domain proteins from eukaryotes, working in ribosome biogenesis 9 . They found that MIDAS protein binding time on substrates is enhanced by mild dissociative forces up to approximately 4pN and then that 10x stronger dissociative forces abolish binding.…”

Concurrent remodelling of nucleolar 60S subunit precursors by the Rea1 ATPase and Spb4 RNA helicase

“…In a second maturation step, which goes along with CP formation in the nucleoplasm, Rea1 dislodges assembly factor Rsa4 and thereby primes the 60S precursors for their export to the cytoplasm ( Barrio-Garcia et al, 2016 ; Kater et al, 2020 ; Matsuo et al, 2014 ; Ulbrich et al, 2009 ). The interaction of Rea1 with Ytm1 and Rsa4 is established between a metal-ion-dependent adhesion site (MIDAS) at the C-terminal tip of the Rea1 tail and the ubiquitin-like (UBL) domains of its assembly factor ligands ( Ahmed et al, 2019 ; Bassler et al, 2010 ; Mickolajczyk et al, 2022 ; Ulbrich et al, 2009 ). We previously revealed the architecture of Rea1 and its associated pentameric Rix1-complex, which serves as docking platform for the ATPase on nucleoplasmic pre-60S particles ( Barrio-Garcia et al, 2016 ; Kater et al, 2020 ).…”