X-ray structure of full-length human RuvB-Like 2 \u2013 mechanistic insights into coupling between ATP binding and mechanical action

Sara T N Silva,José A Brito,James Doutch,José-María Carazo,Christine Ebel,José L Carrascosa,Tiago M Bandeiras,Mark D Tully,Pedro M Matias,Rocío Arranz,Carlos Óscar S Sorzano

doi:10.1038/s41598-018-31997-z

Abstract

RuvB-Like transcription factors function in cell cycle regulation, development and human disease, such as cancer and heart hyperplasia. The mechanisms that regulate adenosine triphosphate (ATP)-dependent activity, oligomerization and post-translational modifications in this family of enzymes are yet unknown. We present the first crystallographic structure of full-length human RuvBL2 which provides novel insights into its mechanistic action and biology. The ring-shaped hexameric RuvBL2 structure presented here resolves for the first time the mobile domain II of the human protein, which is responsible for protein-protein interactions and ATPase activity regulation. Structural analysis suggests how ATP binding may lead to domain II motion through interactions with conserved N-terminal loop histidine residues. Furthermore, a comparison between hsRuvBL1 and 2 shows differences in surface charge distribution that may account for previously described differences in regulation. Analytical ultracentrifugation and cryo electron microscopy analyses performed on hsRuvBL2 highlight an oligomer plasticity that possibly reflects different physiological conformations of the protein in the cell, as well as that single-stranded DNA (ssDNA) can promote the oligomerization of monomeric hsRuvBL2. Based on these findings, we propose a mechanism for ATP binding and domain II conformational change coupling.

Highlights

RuvB-Like (RuvBL) proteins are found in the domains Archaea and Eukarya, including humans and fungi, where they exert roles in transcription regulation, DNA damage repair, cell cycle control, stress adaptation and disease[1,2,3]
The hexameric arrangement of hsRuvBL2 in solution was confirmed by small angle X-ray scattering (SAXS - see below) coupled to size exclusion chromatography, which unequivocally showed the formation of a complex with a mean radius of gyration of ca. 52 Å throughout the elution peak (Supplementary Fig. S1)
The collected results suggest a putative mechanism whereby binding of a nucleotide to the binding pocket promotes the stabilization of the external part of domain II against the ATPase core, by eliciting conformational changes of the N-terminal loop

Summary

Introduction

RuvB-Like (RuvBL) proteins are found in the domains Archaea and Eukarya, including humans and fungi, where they exert roles in transcription regulation, DNA damage repair, cell cycle control, stress adaptation and disease[1,2,3]. Expression of the metastasis suppressor KAI1 is regulated by binding of either Tip60:RuvBL1 or β-catenin:RuvBL2SUMO to its promoter These mutually exclusive processes are directly correlated with RuvBL2 SUMOylation state, and lead respectively to upregulation or repression of KAI1 expression[15]. Both RuvBL1−/− and RuvBL2−/− mice are embryonic lethal[16,17], suggesting a pivotal role for these proteins at the onset of development. Available atomic resolution structures of RuvB-Like proteins include the human RuvBL1 hexamer[18], the human RuvBL2 hexameric ATPase core[19], the RuvBL1:RuvBL2 complex from the thermophilic fungus Chaetomium thermophilum[20,21], and the human RuvBL1ΔDII:RuvBL2ΔDII dodecameric complex[22] In the latter, the mobile domain II was removed for crystallization purposes. We further tackle the interference of purification tags on the oligomerization state of the protein, which has for long been an open question in human RuvBLs, and shed some light on the mode of ssDNA binding

Methods

Results

Conclusion