HU histone-like DNA-binding protein from Thermus thermophilus: structural and evolutionary analyses

Abstract

The histone-like DNA-binding proteins (HU) serve as model molecules for protein thermostability studies, as they function in different bacteria that grow in a wide range of temperatures and show sequence diversity under a common fold. In this work, we report the cloning of the hutth gene from Thermus thermophilus, the purification and crystallization of the recombinant HUTth protein, as well as its X-ray structure determination at 1.7 Å. Detailed structural and thermodynamic analyses were performed towards the understanding of the thermostability mechanism. The interaction of HUTth protein with plasmid DNA in solution has been determined for the first time with MST. Sequence conservation of an exclusively thermophilic order like Thermales, when compared to a predominantly mesophilic order (Deinococcales), should be subject, to some extent, to thermostability-related evolutionary pressure. This hypothesis was used to guide our bioinformatics and evolutionary studies. We discuss the impact of thermostability adaptation on the structure of HU proteins, based on the detailed evolutionary analysis of the Deinococcus-Thermus phylum, where HUTth belongs. Furthermore, we propose a novel method of engineering thermostable proteins, by combining consensus-based design with ancestral sequence reconstruction. Finally, through the structure of HUTth, we are able to examine the validity of these predictions. Our approach represents a significant advancement, as it explores for the first time the potential of ancestral sequence reconstruction in the divergence between a thermophilic and a mainly mesophilic taxon, combined with consensus-based engineering.

NMR elucidation of monomer–dimer transition and conformational heterogeneity in histone‐like DNA binding protein of Helicobacter pylori

“…Thus, we believe that the exhibited dynamic equilibrium between monomer and dimeric states of Hup aided by conformational flexibility of their DNA binding C‐terminal arms provides a level of exquisite control to regulate rapid and accurate functioning of cell cycle activities or modulating gene expression in response to changing external conditions . The HU are ubiquitously found in bacteria that grow in a wide range of temperature (cold to hot) conditions and temperature‐induced structural changes, especially in DNA binding proteins, may play a role in modulating gene expression crucial for their survival under varying temperature conditions. The temperature dependent studies revealed that Hup exhibits dynamic equilibrium between its M‐ and D‐conformations (Figure ), and this equilibrium shifts more towards M–conformation at near physiological temperature.…”

CaC3H14encoding a tandem CCCH zinc finger protein is directly targeted by CaWRKY40 and positively regulates the response of pepper to inoculation byRalstonia solanacearum

“…The protein expression of the two proteins and their purification were carried out following the method of Papageorgiou et al . ().…”

“…For the overproduction of CaWRKY40-GFP, the pET-11a-CaWRKY40-GFP plasmid was used to transform the Escherichia coli expression strain BL21(DE3). The protein expression of the two proteins and their purification were carried out following the method of Papageorgiou et al (2016).…”

“…The protein and DNA were mixed and balanced for 10 min, after which the samples were loaded into a hydrophilic capillary for DNA-protein interaction measurement in a monolith NT.115 5 instrument (NanoTemper Technologies GmbH, Munich, Germany) using 50% IR laser power and an LED excitation source with k 5 470 nm at ambient temperature. The NanoTemperAnalysis 1.2.20 software was used to fit the data and determine the apparent K d values (Papageorgiou et al, 2016;Zillner et al, 2012).…”

Identification and functional characterization of Histone‐like DNA‐binding protein in Nocardia seriolae (NsHLP) involved in cell apoptosis

“…Similar with pathogenic Nocardia, Mycobacterium smegmatis can survive for long periods during persistent infections and escape from the intracellular killing of phagocytes, and a class of histone-like DNA-binding proteins (HLPs), the mycobacterial DNA-binding protein 1 (MDP1), were revealed to involved in this progress (Enany et al, 2017). HLPs are basic bacterial proteins, which are analogues to eukaryotic histones (Li, Arnold-Schulz-Gahmen, & Kellenberger, 1999;Papageorgiou et al, 2016;Ravinder & Goyal, 2017). The function of HLPs is associated with the nucleoid, and they play important roles in the maintenance of DNA architecture, such as DNA replication, recombination and transcription (Anuchin, Goncharenko, Demidenok, & Kaprelyants, 2011).…”