title: 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.
authors.affiliation: Section of Biochemistry and Molecular Biology, Department of Biology, National and Kapodistrian University of Athens, Panepistimiopolis, 15701, Athens, Greece.|CEITEC-Central European Institute of Technology, Masaryk University, Brno, Czech Republic.
authors.forename: Anna C
authors.initials: AC
authors.lastname: Papageorgiou
authors.affiliation: Section of Biochemistry and Molecular Biology, Department of Biology, National and Kapodistrian University of Athens, Panepistimiopolis, 15701, Athens, Greece.|Unit Biologie Moléculaire du Gène chez les Extrêmophiles, Department of Microbiology, Institut Pasteur, 75015, Paris, France.|Université Paris Diderot, Sorbonne Paris Cité, Paris, France.
authors.forename: Panagiotis S
authors.initials: PS
authors.lastname: Adam
authors.affiliation: Biomolecular Physics Laboratory, INRASTES, National Centre for Scientific Research "Demokritos", 153 10, Aghia Paraskevi, Greece.
authors.forename: Philemon
authors.initials: P
authors.lastname: Stavros
authors.affiliation: Biomolecular Physics Laboratory, INRASTES, National Centre for Scientific Research "Demokritos", 153 10, Aghia Paraskevi, Greece.
authors.forename: George
authors.initials: G
authors.lastname: Nounesis
authors.affiliation: EMBL Hamburg Outstation DESY, Notkestrasse 85, 22603, Hamburg, Germany.
authors.forename: Rob
authors.initials: R
authors.lastname: Meijers
authors.affiliation: Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology-Hellas, N. Plastira 100, 70013, Heraklion, Greece.
authors.forename: Kyriacos
authors.initials: K
authors.lastname: Petratos
authors.affiliation: Section of Biochemistry and Molecular Biology, Department of Biology, National and Kapodistrian University of Athens, Panepistimiopolis, 15701, Athens, Greece. cvorgias@biol.uoa.gr.
authors.forename: Constantinos E
authors.initials: CE
authors.lastname: Vorgias
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017124:Conserved Sequence; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019143:Evolution, Molecular; D006358:Hot Temperature; D011485:Protein Binding; D055550:Protein Stability; D016963:Thermus thermophilus / Q000235:genetics* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins
keywords: Evolution; HUTth; Histone-like DNA-binding proteins; Protein thermostability; X-ray structure