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.

Crenarchaeal viruses are commonly found in hyperthermal acidic environments such as those of Yellow-stone National Park. These remarkable viruses not only exhibit unusual morphologies, but also display extreme genetic diversity. However, little is known about crenarchaeal viral life cycles, virus-host interactions, and their adaptation to hyperthermophilic environments. In an effort to better understand the functions of crenarchaeal viruses and the proteins encoded by their genomes, we have undertaken detailed structural and functional studies of gene products encoded in the open reading frames of Sulfolobus spindle-shaped virus ragged hills. Herein, we report ( 15 N, 13 C, 1 H) resonance assignments of backbone and side chain atoms of a 19.1 kDa homodimeric E73 protein of SSVRH. KeywordsCrenarchaea; Viruses; Hyperthermophiles; NMR resonance assignments; E73 protein; Sulfolobus spindle shaped viruses; Molecular adaptation Biological contextCrenarchaeal viruses are commonly found in hyperthermal acidic environments such as those of Yellowstone National Park. These remarkable viruses not only exhibit unusual morphologies, but also show extreme genetic diversity, leading to the recent recognition of seven new viral families (Prangishvili et al. 2006;Lawrence et al. 2009). The best studied are the Fuselloviridae, which are ubiquitous to acidic hot springs (T > 70°C, 1 < pH < 4) worldwide, and include Sulfolobus spindle-shaped virus 1 (SSV1) and Sulfolobus spindle-shaped virus ragged hills (SSVRH) (Wiedenheft et al. 2004). These viruses are characterized by approximately 60 × 90 nm spindle-or lemon-shaped viral particles with tail fibers that emanate from one end. They contain double stranded circular DNA genomes that encode 34 and 37 open reading frames (ORFs), respectively (Wiedenheft et al. 2004), where the names of the ORF and corresponding protein are derived from the reading frame (A-F) and number of Greater insight into the viral gene products is clearly essential for a deeper comprehension of the SSV1 life cycle and interactions with its Sulfolobus host, and for a better understanding of the biology of crenarchaeal viruses in general. Towards this goal, molecular analyses of the SSV1 viral particle identify five proteins: VP1, VP2, VP3, C792 and D244 (Reiter et al. 1987;Menon et al. 2008). VP1, VP3 and C792 are membrane embedded structural proteins, while VP2 is a packaged DNA binding protein.Importantly, structural studies are uncovering distant evolutionary relationships that are not apparent in the primary sequence of these proteins (Lawrence et al. 2009). Structural annotation now suggests that D-63 is an adaptor protein similar to Repressor of Primer, while F112 and F93 are putative transcription factors (Menon et al. 2008;Lawrence et al. 2009).To assess the validity of the bioinformatics prediction that SSV1 E51 and its SSVRH homolog, E73, are ribbon-helix-helix type DNA binding proteins, and to gain deeper structural and functional insights, we have undertaken biochemical characterization and m...

Summary Anastellin is a small recombinant fragment derived from the extracellular matrix protein fibronectin; it comprises the 1st type III (FN3) domain without the two N-terminal β-strands. It inhibits angiogenesis, tumor growth and metastasis in mouse models and requires endogenous fibronectin for its in vivo anti-angiogenic activity. It binds to fibronectin in vitro and converts the soluble protein to insoluble fibrils that structurally and functionally resemble fibronectin fibrils deposited in the extracellular matrix by cells. Anastellin binds to several FN3 domains in fibronectin, but how it interacts with these domains and why the interactions lead to aggregation of fibronectin is not well understood. In this work, we investigated the interaction between anastellin and the 3rd FN3 domain (3FN3) from fibronectin. We show that anastellin binds with high affinity to a peptide comprising the two N-terminal β-strands from 3FN3, and we present here the structure of the resulting complex. The peptide and anastellin form a composite FN3 domain, with the two N-terminal β-strands from 3FN3 bound in place of the two β-strands that are missing in anastellin. We also demonstrate using disulfide crosslinking that a similar interaction involving the two N-terminal β-strands of 3FN3 occurs when intact 3FN3 binds to anastellin. 3FN3 adopts a compact globular fold in solution, and to interact with anastellin in a manner consistent with our data, it has to open up and expose a β-strand edge that is not accessible in the context of the folded domain.

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