Intramolecular tension and catch bonds play key roles in the conformational cycle of the dynein-like AAA mechanoenzyme Mdn1

Mdn1 is an essential mechanoenzyme that uses the energy from ATP hydrolysis to physically extract the assembly factors Rsa4 and Ytm1 from the pre-60S subunit during ribosome biogenesis. This massive (>500 kDa) protein has an N-terminal AAA (ATPase associated with various activities) ring, which like dynein has six ATPase sites. The AAA ring is followed by large (>2000 amino acid) linking domains including an ∼500 amino acid disordered (D/E-rich) region, and a C-terminal assembly factor-binding MIDAS domain. Previous structural studies have revealed that when Mdn1 binds to the pre-60S, it docks its MIDAS domain onto its AAA ring and forms a tripartite connection between these two domains and the assembly factor that it is primed to extract. However, it is currently not known how this conformational state is formed and regulated. Here, we use a combination of optical tweezers, bulk biochemistry, and chemical biology approaches to investigate domain interactions and conformational changes in the Mdn1 mechanochemical cycle. We find that the MIDAS domain can bind stably to the AAA ring when they are separated into two different proteins, but that intramolecular tension built up along the linking domains prohibits this interaction in the full-length protein. We further find that the MIDAS domain can form catch bonds - bonds where tension enhances binding affinity - with both Rsa4 and Ytm1. Together, these results suggest a mechanoregulatory mechanism wherein intramolecular tension prevents MIDAS-AAA docking when Mdn1 is off the pre-60S, yet promotes the docked conformation when Mdn1 is on the pre-60S by activating the catch bond.