Structural Insights into the Nucleotide-Binding Domains of the P1B-type ATPases HMA6 and HMA8 from Arabidopsis thaliana.

Hubert Mayerhofer,Eva Pebay-Peyroula,Daphné Seigneurin-Berny,Norbert Rolland,Emeline Sautron,Stéphanie Ravaud,Patrice Catty,Oleg Y Dmitriev

doi:10.1371/journal.pone.0165666

Abstract

Copper is a crucial ion in cells, but needs to be closely controlled due to its toxic potential and ability to catalyse the formation of radicals. In chloroplasts, an important step for the proper functioning of the photosynthetic electron transfer chain is the delivery of copper to plastocyanin in the thylakoid lumen. The main route for copper transport to the thylakoid lumen is driven by two PIB-type ATPases, Heavy Metal ATPase 6 (HMA6) and HMA8, located in the inner membrane of the chloroplast envelope and in the thylakoid membrane, respectively. Here, the crystal structures of the nucleotide binding domain of HMA6 and HMA8 from Arabidopsis thaliana are reported at 1.5Å and 1.75Å resolution, respectively, providing the first structural information on plants Cu+-ATPases. The structures reveal a compact domain, with two short helices on both sides of a twisted beta-sheet. A double mutant, aiding in the crystallization, provides a new crystal contact, but also avoids an internal clash highlighting the benefits of construct modifications. Finally, the histidine in the HP motif of the isolated domains, unable to bind ATP, shows a side chain conformation distinct from nucleotide bound structures.

Highlights

Heavy metals, with their unique chemical properties, play an essential role in cellular processes as cofactors, structural stabilizers or redox partners and plants have evolved to maintain physiological concentrations of crucial metal ions
The vector contains an N-terminal His6-tag followed by a Tobacco Etch Virus protease (TEV) cleavage site in case of HMA8N or an GB1 expression and solubility enhancer [19] flanked by a N-terminal His6-tag and a TEV cleavage site in case of HMA6N, with a three amino acid linker (GAS) that remains at the N-terminus of both proteins after TEV cleavage
The structure of HMA8N was solved at 1.75 Å by molecular replacement, using the N-domain of pdb 3A1C as a search model, with two molecules in the asymmetric unit (AU)

Summary

Introduction

With their unique chemical properties, play an essential role in cellular processes as cofactors, structural stabilizers or redox partners and plants have evolved to maintain physiological concentrations of crucial metal ions. Nucleotide-Binding Domains of Plant P1B-type ATPases supported by the Institut Universitaire de France.

Results

Conclusion