Structural and functional studies of Stf76 from the Sulfolobus islandicus plasmid–virus pSSVx: a novel peculiar member of the winged helix–turn–helix transcription factor family

Patrizia Contursi,Salvatore Fusco,Luigi Russo,Roberto Fattorusso,Emilia Pedone,Simonetta Bartolucci,Luciano Pirone,Biancamaria Farina

doi:10.1093/nar/gku215

Abstract

The hybrid plasmid–virus pSSVx from Sulfolobus islandicus presents an open reading frame encoding a 76 amino acid protein, namely Stf76, that does not show significant sequence homology with any protein with known 3D structure. The recombinant protein recognizes specifically two DNA-binding sites located in its own promoter, thus suggesting an auto-regulated role of its expression. Circular dichroism, spectrofluorimetric, light scattering and isothermal titration calorimetry experiments indicated a 2:1 molar ratio (protein:DNA) upon binding to the DNA target containing a single site. Furthermore, the solution structure of Stf76, determined by nuclear magnetic resonance (NMR) using chemical shift Rosetta software, has shown that the protein assumes a winged helix–turn–helix fold. NMR chemical shift perturbation analysis has been performed for the identification of the residues responsible for DNA interaction. In addition, a model of the Stf76–DNA complex has been built using as template a structurally related homolog.

Highlights

Studies on crenarchaeal viruses have shown that they possess unique morphological features and quasi-orphan genome sequences that distinguish them from bacteriophages and eukaryal viruses [1,2,3]
Stf76 was purified to homogeneity in a three-step procedure: the soluble fraction was heated at 70◦C for 20 min taking advantage from the high thermostability of the protein, successively, an affinity and a size-exclusion chromatography allowed to obtain an homogeneous sample with a yield corresponding to ∼10 mg of protein per liter of culture
A novel protein Stf76, encoded by the hybrid plasmid–virus pSSVx and homolog of ORF80, has been functionally and structurally analysed to shed light on the molecular mechanism underpinning its interaction with DNA as well as on its biological function

Summary

Introduction

Studies on crenarchaeal viruses have shown that they possess unique morphological features and quasi-orphan genome sequences that distinguish them from bacteriophages and eukaryal viruses [1,2,3]. Seven families of doublestranded DNA viruses have been identified, among which the Fuselloviridae and Rudiviridae are the most well-studied specimens and represent model systems for detailed studies of archaeal virus biology [5]. These are maintained under laboratory conditions and can be obtained in sufficient yields, which is not the case for many other archaeal viruses [6,7,8,9,10]. A relatively high proportion of archaeal viral sequences is predicted to carry folds associated to transcription factor (TF) [11]. Structural and functional information on archaeal transcription regulators are scarce and only for few of them the 3D structure has been determined [6,8,12,13,14]

Methods

Results

Conclusion