Structural characterization of a novel KH-domain containing plant chloroplast endonuclease

Ashok K Rout,Vandana Raghvan,Himanshu Singh,R Minda,Saurabh Gautam,Sunita Patel,Basuthkar J Rao,Kandala V R Chary

doi:10.1038/s41598-018-31142-w

Abstract

Chlamydomonas reinhardtii is a single celled alga that undergoes apoptosis in response to UV-C irradiation. UVI31+, a novel UV-inducible DNA endonuclease in C. reinhardtii, which normally localizes near cell wall and pyrenoid regions, gets redistributed into punctate foci within the whole chloroplast, away from the pyrenoid, upon UV-stress. Solution NMR structure of the first putative UV inducible endonuclease UVI31+ revealed an α1–β1–β2–α2–α3–β3 fold similar to BolA and type II KH-domain ubiquitous protein families. Three α−helices of UVI31+ constitute one side of the protein surface, which are packed to the other side, made of three-stranded β–sheet, with intervening hydrophobic residues. A twenty-three residues long polypeptide stretch (D54-H76) connecting β1 and β2 strands is found to be highly flexible. Interestingly, UVI31+ recognizes the DNA primarily through its β–sheet. We propose that the catalytic triad residues involving Ser114, His95 and Thr116 facilitate DNA endonuclease activity of UVI31+. Further, decreased endonuclease activity of the S114A mutant is consistent with the direct participation of Ser114 in the catalysis. This study provides the first structural description of a plant chloroplast endonuclease that is regulated by UV-stress response.

Highlights

Chlamydomonas reinhardtii is a single celled alga that swims with its two flagella and undergoes apoptosis in response to UV-C irradiation[1]
The UVI31+ was purified using affinity chromatography followed by size-exclusion chromatography and analyzed by SDS-PAGE, mass spectroscopy (MALDI-TOF) and dynamic light scattering (DLS) experiments
We have determined NMR-derived 3D structure of the UV inducible gene product, UVI31+ protein, from Chlamydomonas reinhardtii, which possesses a α1–β1–β2–α2–α3–β3 fold, similar to the fold found in BolA protein family and K Homology (KH) domain type II proteins

Summary

Introduction

Chlamydomonas reinhardtii is a single celled alga that swims with its two flagella and undergoes apoptosis in response to UV-C irradiation[1]. The alga shows classical hallmarks of animal cell apoptosis and can be used as a model system for studying its molecular mechanism in a plant-like environment Several candidate molecules such as apoptosis protease activating factor (APAF), a caspase-3 like protein, and a defender against apoptotic death (DAD1) were studied in C. reinhardtii, and found to exhibit a distinct activation pattern correlating with the onset of death upon UV irradiation[1,2]. The observed UVI31+ induction upon UV-stress, increased UV resistance upon overexpression in E. coli, and the localization near pyrenoid and chloroplast regions suggest a plausible role of UVI31+ in DNA repair[6,9]. Given its dynamic cellular localization changes as a function of various stress treatments (UV and dark incubation) and associated endonuclease activity, seeking the structural biology of UVI31+ as an interesting plant protein became highly imminent. The study provides the structural basis for DNA recognition and endonuclease catalysis by a plant protein UVI31+

Methods

Results

Conclusion