• Home
  • Search Results
  • Long-range intramolecular allostery and regulation in the dynein-like AAA protein Mdn1.

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

Proceedings of the National Academy of Sciences of the United States of America (2020-07-23)
Keith J Mickolajczyk, Paul Dominic B Olinares, Yiming Niu, Nan Chen, Sara E Warrington, Yusuke Sasaki, Thomas Walz, Brian T Chait, Tarun M Kapoor
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.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Copper(II) sulfate pentahydrate, ACS reagent, ≥98.0%
Sigma-Aldrich
Adenosine 5′-triphosphate disodium salt hydrate, Grade I, ≥99%, from microbial
Sigma-Aldrich
(+)-Sodium L-ascorbate, powder, BioReagent, suitable for cell culture
Sigma-Aldrich
Pyruvate Kinase from rabbit muscle, Type II, ammonium sulfate suspension, 350-600 units/mg protein
Sigma-Aldrich
Phospho(enol)pyruvic acid monopotassium salt, ≥97% (enzymatic)
Sigma-Aldrich
β-Nicotinamide adenine dinucleotide, reduced disodium salt, ~98%, pkg of 2 mg (per vial)