Structural Characterization of Arabidopsis thaliana NAP1-Related Protein 2 (AtNRP2) and Comparison with its Homolog AtNRP1.

Kumar Kumar,Kumar Singh Kumar Singh,Chandrakant Bobde Chandrakant Bobde,Vasudevan Vasudevan

doi:10.3390/molecules24122258

Kumar Kumar, Kumar Singh Kumar Singh + Show 2 more

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https://doi.org/10.3390/molecules24122258

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Abstract

Nucleosome Assembly Protein (NAP) is a highly conserved family of histone chaperones present in yeast, animals, and plants. Unlike other organisms, plants possess an additional class of proteins in its NAP family, known as the NAP1-related proteins or NRP. Arabidopsis thaliana possesses two NRP isoforms, namely AtNRP1 and AtNRP2, that share 87% sequence identity. Both AtNRP1 and AtNRP2 get expressed in all the plant tissues. Most works in the past, including structural studies, have focused on AtNRP1. We wanted to do a comparative study of the two proteins to find why the plant would have two very similar proteins and whether there is any difference between the two for their structure and function as histone chaperones. Here we report the crystal structure of AtNRP2 and a comparative analysis of its structural architecture with other NAP family proteins. The crystal structure of AtNRP2 shows it to be a homodimer, with its fold similar to that of other structurally characterized NAP family proteins. Although AtNRP1 and AtNRP2 have a similar fold, upon structural superposition, we find an offset in the dimerization helix of the two proteins. We evaluated the stability, oligomerization status, and histone chaperoning properties of the two proteins, for a comparison. The thermal melting experiments suggest that AtNRP2 is more stable than AtNRP1 at higher temperatures. In addition, electrophoretic mobility shift assay and isothermal titration calorimetry experiments suggest histone binding ability of AtNRP2 is higher than that of AtNRP1. Overall, these results provide insights about the specific function and relevance of AtNRP2 in plants through structural and biophysical studies.

Highlights

The nuclear processes like DNA replication, transcription, DNA repair, and DNA recombination require a highly regulated disassembly and assembly of nucleosomes, which form the basic repeating units of chromatin
We find that AtNRP2 and AtNRP1 are quite stable at physiologically relevant higher temperatures, suggesting them to be crucial during hyperthermal stress conditions
We find that binding affinity to histone H2A-H2B is significantly higher for AtNRP2 than for AtNRP1. We show that both AtNRP1 and AtNRP2 are capable of nucleosome assembly activity in vitro and act as histone chaperones

Summary

Introduction

The nuclear processes like DNA replication, transcription, DNA repair, and DNA recombination require a highly regulated disassembly and assembly of nucleosomes, which form the basic repeating units of chromatin. In this context, chromatin remodeling is a crucial step, and it is brought about by a variety of histone modifying enzymes, and ATP-dependent chromatin remodeling factors [1] in coordination with a heterogeneous group of ATP-independent chromatin remodelers known as the histone chaperones [2,3,4].

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