Structural Studies of E73 from a Hyperthermophilic Archaeal Virus Identify the “RH3” Domain, an Elaborated Ribbon–Helix–Helix Motif Involved in DNA Recognition

Abstract

Hyperthermophilic archaeal viruses, including Sulfolobus spindle-shaped viruses (SSVs) such as SSV-1 and SSV-Ragged Hills, exhibit remarkable morphology and genetic diversity. However, they remain poorly understood, in part because their genomes exhibit limited or unrecognizable sequence similarity to genes with known function. Here we report structural and functional studies of E73, a 73-residue homodimeric protein encoded within the SSV-Ragged Hills genome. Despite lacking significant sequence similarity, the nuclear magnetic resonance (NMR) structure reveals clear similarity to ribbon–helix–helix (RHH) domains present in numerous proteins involved in transcriptional regulation. In vitro double-stranded DNA (dsDNA) binding experiments confirm the ability of E73 to bind dsDNA in a nonspecific manner with micromolar affinity, and characterization of the K11E variant confirms the location of the predicted DNA binding surface. E73 is distinct, however, from known RHH domains. The RHH motif is elaborated upon by the insertion of a third helix that is tightly integrated into the structural domain, giving rise to the “RH3” fold. Within the homodimer, this helix results in the formation of a conserved, symmetric cleft distal to the DNA binding surface, where it may mediate protein–protein interactions or contribute to the high thermal stability of E73. Analysis of backbone amide dynamics by NMR provides evidence of a rigid core, fast picosecond to nanosecond time scale NH bond vector motions for residues located within the antiparallel β-sheet region of the proposed DNA-binding surface, and slower microsecond to millisecond time scale motions for residues in the α1−α2 loop. The roles of E73 and its SSV homologues in the viral life cycle are discussed.

Structural entanglements in protein complexes

“…This cavity is distal to the canonical DNA binding site and it is positively charged. It also forms a potential ligand binding site 45 . Fig.…”

Structure and Function of AvtR, a Novel Transcriptional Regulator from a Hyperthermophilic Archaeal Lipothrixvirus

“…Their role in the regulation of the virus infectious cycle remains largely unknown. The only examples of well-studied RHHs in an archaeal context are protein ORF56, encoded by the Sulfolobus islandicus plasmid pRN1 (22,25,(47)(48)(49), …”

“…the regulator SvtR, encoded by the rudivirus SIRV (5), and the recently described protein E73, encoded by the virus SSV-RH (22). ORF56 regulates the plasmid copy number, SvtR is involved in the regulation of the lytic cycle of the rudiviruses, and E73 has been suggested to target elements of the host genome.…”

“…The structures of 21 RHH proteins are presently available (5,(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). Among them, the methionine repressor MetJ (24), the regulator of plasmid copy number CopG (11,(27)(28)(29), and the bacte-riophage P22 Mnt and Arc repressors (21,30) could be considered the prototypical RHH proteins.…”

Unravelling the Role of the F55 Regulator in the Transition from Lysogeny to UV Induction of Sulfolobus Spindle-Shaped Virus 1

“…The lack of functional characterization of proteins encoded by the fuselloviral genomes has limited the dissection of fundamental processes, such as (i) virion uptake, assembly and release, (ii) transcriptional regulation, (iii) genome replication, and (iv) lysogeny/ induction switch. Indeed, with the exception of a few proteins (16)(17)(18)(19)(20)(21)(22)(23), structural and functional annotations in main databases are not available for fuselloviral gene products (2,22).…”