F-93: SSV1 DNA Binding
Protein
Emilee Kaser and Liz Narducci '05
Contents:
I. Introduction
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 vicious 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.
II. Basic Structure
The F-93 exists in solution as a homodimer consisting
of two identical asymmetric monomers
. Each
monomer is composed of a single polypeptide chain that codes for 93 amino acid
residues. The secondary structure of each monomer consists mainly of four
alpha-helices and two beta-sheets
. The
single polypeptide chain runs through helix 1 to helix 2 to helix 3. These helices
are followed by beta-sheets 1 and 2. The beta-sheets
are connected by a reverse turn to form the flanking wing
of the DNA binding domain
.
These sheets generally provide interactions with the backbone of the DNA The helix-loop-helix
motif common to DNA binding proteins and major groove interactions corresponds
to helicies 1 and 3 of each F-93 monomer, with helix 3 acting as the recognition
helix that likely binds the major groove of target DNA
. A C-terminal extension gives rise to the fourth helix. Interactions between
helix 4, the N-terminus, and the first half of helix 1 form the dimer
interface of F-93
.
III. F-93 Homologs
Because, the protein structure of SSV1 F-93 was engineered,
the function 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, extensions
at the N- or C-terminus often contribute to the formation of a dimer interface.
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.
IV. DNA Binding
Please wait ten seconds for molecule.
For considering how F-93 may actually bind DNA,
it is necessary 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 plosive 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
plosive potential for putative DNA binding at the surface of the
dimer interface
.
V. Future
Research
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 constitutively 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.
VI. References
Kraft, Paul et
al. "Crystal Structure of F-93 from Sulfolobus Spindle Shaped Virus 1,
a Winged-Helix DNA Binding Protein." Journal of Virology 78 (2004): 11544-11550.
Prangishvili,
David et al. "Viruses of the extremely thermophilic archeaon Sulfolobus."
Trends in Microbiolgy 9 (2001): 39-43. 7 Dec. 2005.
Stedman, Kenneth
M et al. "Relationships between fuselloviruses infecting the extremely
thermophilic archeaon Sulfolobus: SSV1 and SSV2." Research in Microbiology
154 (2003): 295-301. 7 Dec. 2005.
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