Viruses can be classified as both living and non-living. Their structure and function are elegantly simple, but they often produce some of the most vicisous and complex diseases in the world. Despite the wealth of information currently available about viruses, much remains to be learned about their lifecycles and pathogenicity.
SSV1 is a virus in the family Fuselloviridae that infects Sulfolobales, a crenarcheota that is found all over the world. Sulfolobales live in extremely high temperature hot springs (80º C) and at a low pH (< 4, i.e. very acidic). As with many archea they represent an interesting biological system for study due to their ability to withstand harsh physical environments. SSV1 is the best studied of the 35 known archeal viruses, and is partially responsible for the approval of Archea as a separate domain of life . After extensive study SSV1 has been found contain 34 open reading frames within its double stranded DNA genome of 15.5 kilobases . Many of the gene products of these ORFs are unknown. One of the few known SSV1 ORF gene products is F-93.
F-93 is an SSV1 protein that is thought to belong to the winged-helix family of DNA binging proteins. The crystal structure of the F-93 protein studied in this model was engineered, rather than isolated from a biological sample. To express and purify the F-93 protein pDEST14/F-93 was changed into BL21(DE3)-RIL using E. coli. Chemical cross-linking analysis produced protein dimeraztion, and F-93 was able to be crystallized through drop vapor diffusion at 4º C. The complete crystal structure was analyzed from diffraction data measured at three-wavelengths (edge, peak, and remote wavelengths), and protein assembly was elucidated through structure determination and refinement techniques. The F-93 protein in this model codes for 93 amino acids along with a C-terminal His6 tag, which totals 99 residues and a calculated mass of 11,789 Daltons. All of the functional analyses are a result of comparisons between F-93 and several homologs.
Because, the protein structure of SSV1 F-93 was engineered, the fuction of F-93 must be inferred by comparing it to sequential and structural homologs. Sequence alignment shows several strictly conserved residues among several F-93 viral homologs that appear to be important for protein structure. Conserved leucine residues are found buried within the hydrophobic core of each F-93 monomer < >. Highly conserved glycine residues are found in connecting loops between helix one and two, as well as between helix 3 and beta-strand 1, and between beta-strands 1 and 2 < >. Finally, a cluster of conserved residues (Lys 65, Leu 67, Leu 69, Lys 72, Gly 73, Lys 74) are found as the peptide leaves strand 2 and enters helix 4 < >. Mapping these conserved residues along with Gly 54 < > to the structure of F-93 gives rise to the formation of a patch of conserved surface area at the N-terminus of helix 4. This surface possibly represents a potential binding site for other transcription-regulating proteins.
These sequential homologies, give rise to important structural homology. F-93 is structurally most similar to the SlyA and MarR sub-families of winged-helix DNA binding proteins, clearly sharing a common structural core with both of these proteins. For F-93 and Enterococcus faecalis SlyA, a VAST search identifies 69 structurally equivalent alpha-carbon atoms < >, while comparison between Escherichia coli MarR and F-93 yields 66 equivalent alpha-carbon positions < >. These commonalities include the fold of the first 3 helices of the F-93 monomer, followed by the anti-parallel beta-strands.
In SlyA and MarR sub-families, extenstions at the N- or C-terminus often contribute to the fromation of a dimer interace. In F-93, this is seen by the C-terminus extension giving rise to beta-sheet 2 and to helix 4 < >. As mentioned above, interactions between Helix 4, the N-terminus, and helix 1 form most of the F-93 dimer interface < >. This dimer interface is composed largely of conserved lysine residues and is substantially hydrophobic < >. The F-93 dimer interface structure is consistent with the large dimer interfaces seen for members of the SlyA and MarR sub-families, and taken together these sequential and structural homologies all suggest F-93 functions as a winged-helix DNA binding protein.
For considering how F-93 may actually bind DNA, it is neccessary to examine another close structural neighbor, the diptheria toxin repressor or DtxR < >. The structure of the DtxR-DNA homodimer complex has been determined and deposited in the Protein Data Bank< >. DtxR acts as a DNA binding protein that shares the core winged-helix structure of F-93 < >, however DtxR has an additional winged-helix giving rise to three DNA binding domains. DtxR places its recognition helices in the major groove of DNA < >, while the wings interact with the DNA backbone. Similar interactions are probable for the F-93 dimer < > by protein-DNA interactions through the alpha helices and beta sheets described above < >. Additionally, F-93 has several positive charges that are clustered around the dimer axis. Two charges come from N-termini and two charges come from Lys-2 < >. This cluster of postive charges has potential to interact with the DNA backbone. Aside from the base-specific interactions in the major groove, electrostatic surface calculations also reveal significant postive potential for putative DNA binding at the surface of the dimer interface < >.
Thus far, research suggests that F-93 binds either viral or host DNA possibly serving the role of a transcription factor. However, despite its structural similarities with known transcription activators and repressors, F-93 may have other functions. Comparison of the structures of F-93, SlyA, and DtxR reveals that the dimer interfaces of SlyA and DtxR are much more complex. Currently, no transcriptional regulators belonging to the winged-helix family are as small as F-93. One of the functions of the large dimer interface in DtxR is that it serves as a binding site for iron, which ultimately regulates the activity of DtxR. The simpler dimer interface in F-93, could suggest that it is constituitively expressed. F-93 also shares structural similarities with the replication terminator protein of Bacillus subtilis, possibly implicating that F-93 aides in replication of the viral genome. However, the structural homology between F-93 and replication proteins is weaker than those seen between the SlyA and MarR families. Future studies will possibly use DNA microarrays among other techniques to examine changes in gene expression relating to the F-93 viral protein, and finally determining protein function.
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