Long-range intramolecular allostery and regulation in the dynein-like AAA protein Mdn1

Abstract

Mdn1 is an essential mechanoenzyme that uses the energy from ATP hydrolysis to physically reshape and remodel, and thus mature, the 60S subunit of the ribosome. This massive (>500 kDa) protein has an N-terminal AAA (ATPase associated with diverse cellular activities) ring, which, like dynein, has six ATPase sites. The AAA ring is followed by large (>2,000 aa) linking domains that include an ∼500-aa disordered (D/E-rich) region, and a C-terminal substrate-binding MIDAS domain. Recent models suggest that intramolecular docking of the MIDAS domain onto the AAA ring is required for Mdn1 to transmit force to its ribosomal substrates, but it is not currently understood what role the linking domains play, or why tethering the MIDAS domain to the AAA ring is required for protein function. Here, we use chemical probes, single-particle electron microscopy, and native mass spectrometry to study the AAA and MIDAS domains separately or in combination. We find that Mdn1 lacking the D/E-rich and MIDAS domains retains ATP and chemical probe binding activities. Free MIDAS domain can bind to the AAA ring of this construct in a stereo-specific bimolecular interaction, and, interestingly, this binding reduces ATPase activity. Whereas intramolecular MIDAS docking appears to require a treatment with a chemical inhibitor or preribosome binding, bimolecular MIDAS docking does not. Hence, tethering the MIDAS domain to the AAA ring serves to prevent, rather than promote, MIDAS docking in the absence of inducing signals.

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

“…Consistent with this idea, Brownian dynamics simulations have shown that docking of the MIDAS domain onto the AAA ring stretches the unstructured portion of the linker such that 1-2 pN of tension is generated (Mickolajczyk et al, 2020). This tension would be transmitted along the MIDAS-UBL bond axis, and its magnitude may even be enhanced by ATP-dependent motions propagated from the AAA ring (Chen et al, 2018;Ulbrich et al, 2009).…”

“…Mdn1 approaches the pre-60S particle and binds via its AAA ring, at which point the MIDAS domain both docks onto the AAA ring and binds the UBL-domain substrate (Chen et al, 2018;Kater et al, 2020;Sosnowski et al, 2018). In this MIDAS-docked state, Mdn1's unstructured linker is stretched, building 1-2 pN of tension that is propagated along the MIDAS-UBL bond axis (Mickolajczyk et al, 2020). While the directionality of Mdn1 force generation has not been determined, one possibility is that ATPase activity in the AAA ring adds more tension to the unstructured linker, which would be propagated to the MIDAS-UBL interaction (Figure 4G), and thereby activate the MIDAS-UBL catch bond.…”

“…To investigate the regulation of Mdn1 binding to its UBL domain-containing pre-ribosomal substrate Rsa4, we first sought to characterize binding in vitro. The Mdn1 MIDAS domain (amino acids 4381-4717) with an N-terminal SNAP tag was expressed in E. coli as previously (Figure 1A) (Mickolajczyk et al, 2020). Both wild type MIDAS domain (MIDAS-WT) and MIDAS domain harboring a point mutation known to disrupt UBL domain binding (MIDAS-Y4666R) were generated (Ahmed et al, 2019).…”

“…The SNAP-MIDAS construct, as reported previously (Mickolajczyk et al, 2020), was generated by subcloning Mdn1 aa4381-4717 into pSNAP-tag(T7)-2 (NEB N9181S) downstream of the SNAP tag. A N-terminal 6xHis tag and a Tobacco Etch Virus (TEV) protease site were added upstream of the SNAP tag.…”

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

“…Although a UBL domain is structurally distant from extracellular integrin ligands, it is possible that Mdn1 may similarly use mechanoregulation for its function. Consistent with this idea, Brownian dynamics simulations have shown that docking of the MIDAS domain onto the AAA ring stretches the unstructured portion of the linker such that 1–2 pN of tension is generated ( Mickolajczyk et al, 2020 ). This tension would be transmitted along the MIDAS-UBL bond axis, and its magnitude may even be enhanced by ATP-dependent motions propagated from the AAA ring ( Chen et al, 2018 ; Ulbrich et al, 2009 ).…”