Koenig, G. | bioRxiv Science

bioRxiv2022

The Ribosome Maturation Factor Rea1 utilizes nucleotide independent and ATP hydrolysis driven Linker remodelling for the removal of ribosome assembly factors

The ribosome maturation factor Rea1 (or Midasin) catalyses the removal of assembly factors from large ribosomal subunit precursors to promote their export from the nucleus to the cytosol. Rea1 consists of nearly 5000 amino-acid residues and belongs to the AAA+ protein family. It consists of a ring of six AAA+ domains from which the {approx} 1700 amino-acid residue linker emerges that is subdivided into stem, middle and top domains. A flexible and unstructured D/E rich region connects the linker top to a MIDAS (metal ion dependent adhesion site) domain, which is able to bind the assembly factor substrates. Despite its key importance for ribosome maturation, the Rea1 mechanism driving assembly factor removal is still poorly understood. Here we demonstrate that the Rea1 linker is essential for assembly factor removal. It rotates and swings towards the AAA+ ring following a complex remodelling scheme involving nucleotide independent as well as nucleotide dependent steps. ATP-hydrolysis is required to engage the linker with the AAA+ ring and ultimately with the AAA+ ring docked MIDAS domain. The interaction between the linker top and the MIDAS domain allows force transmission for assembly factor removal.

biochemistry↗

bioRxiv2021

A Conformational Transition of Von Willebrand Factor's D'D3 Domain Primes It For Multimerization

Von Willebrand factor (VWF) is a multimeric plasma glycoprotein that is critically involved in hemostasis. Biosynthesis of long VWF concatemers in the endoplasmic reticulum and the (trans-)Golgi is still not fully understood. We use the single-molecule force spectroscopy technique magnetic tweezers to analyze a previously hypothesized conformational change in the DD3 domain crucial for VWF multimerization. We find that the interface formed by submodules C8-3, TIL3, and E3 wrapping around VWD3 can open and expose two previously buried cysteines that are known to be vital for multimerization. By characterizing the conformational change at varying levels of force, we are able to quantify the kinetics of the transition and the stability of the interface. We find a pronounced destabilization of the interface upon lowering the pH from 7.4 to 6.2 and 5.5. This is consistent with initiation of the conformational change that enables VWF multimerization at the DD3 domain by a decrease in pH in the trans-Golgi network and Weibel-Palade bodies. Furthermore, we find a stabilization of the interface in the presence of coagulation factor VIII (FVIII), providing evidence for a previously hypothesized binding site in submodule C8-3. Our findings highlight the critical role of the DD3 domain in VWF biosynthesis and function and we anticipate our methodology to be applicable to study other, similar conformational changes in VWF and beyond.

biophysics↗