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PDBsum entry 3fld
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References listed in PDB file
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Key reference
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Title
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A novel fold in the trai relaxase-Helicase c-Terminal domain is essential for conjugative DNA transfer.
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Authors
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L.M.Guogas,
S.A.Kennedy,
J.H.Lee,
M.R.Redinbo.
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Ref.
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J Mol Biol, 2009,
386,
554-568.
[DOI no: ]
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PubMed id
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Abstract
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TraI relaxase-helicase is the central catalytic component of the multiprotein
relaxosome complex responsible for conjugative DNA transfer (CDT) between
bacterial cells. CDT is a primary mechanism for the lateral propagation of
microbial genetic material, including the spread of antibiotic resistance genes.
The 2.4-A resolution crystal structure of the C-terminal domain of the
multifunctional Escherichia coli F (fertility) plasmid TraI protein is
presented, and specific structural regions essential for CDT are identified. The
crystal structure reveals a novel fold composed of a 28-residue N-terminal
alpha-domain connected by a proline-rich loop to a compact alpha/beta-domain.
Both the globular nature of the alpha/beta-domain and the presence as well as
rigidity of the proline-rich loop are required for DNA transfer and
single-stranded DNA binding. Taken together, these data establish the specific
structural features of this noncatalytic domain that are essential to DNA
conjugation.
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Figure 6.
Fig. 6. (a) CDT efficiencies of E. coli strains containing
specifically designed TraI-CT mutations. (b) “pmut”
indicates the mutation of prolines 1518, 1523, and 1525
simultaneously to glycine, while “Δloop” indicates the
removal of the entire proline-rich loop. (c) The h1/s1 variant
is the mutations of V1478, E1482, and F1485 to alanine on helix
1 and the mutation of I1541 to alanine and that of G1540 to
glutamic acid on strand 2. (Helix 1 is shown here in orange to
indicate the domain-swapped interaction.) (d) The h3/s2 (helix
3/sheet 2) mutants replace L1574, Q1575, and V1603 all with
alanine.
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Figure 7.
Fig. 7. Binding of ssDNA by the TraI-CT measured by
fluorescence anisotropy. TraI 1476–1756 at 75 and 150 mM NaCl
is indicated by continuous and dashed blue lines (K[d] = 2.9 μM
and K[d] = 7.7 μM), respectively. TraI 1476–1756 with a
deletion of the proline-rich loop at 75 and 150 mM NaCl is
indicated by continuous and dashed red lines (K[d] > 17.1 μM
and K[d] > 15.9 μM), respectively. TraI 1476–1756 with
mutations of prolines 1518, 1523, and 1525 to glycine at 75 and
150 mM NaCl is indicated by continuous and dashed green lines
(K[d] > 13.9 μM and K[d] > 18.2 μM), respectively, while the
binding of 1476–1630 at 150 mM NaCl is indicated in black
(K[d] > 22.6 μM).
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2009,
386,
554-568)
copyright 2009.
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