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PDBsum entry 2fln
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Replication/DNA
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PDB id
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2fln
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References listed in PDB file
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Key reference
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Title
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An incoming nucleotide imposes an anti to syn conformational change on the templating purine in the human DNA polymerase-Iota active site.
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Authors
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D.T.Nair,
R.E.Johnson,
L.Prakash,
S.Prakash,
A.K.Aggarwal.
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Ref.
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Structure, 2006,
14,
749-755.
[DOI no: ]
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PubMed id
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Abstract
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Substrate-induced conformational change of the protein is the linchpin of
enzymatic reactions. Replicative DNA polymerases, for example, convert from an
open to a closed conformation in response to dNTP binding. Human DNA
polymerase-iota (hPoliota), a member of the Y family of DNA polymerases, differs
strikingly from other polymerases in its much higher proficiency and fidelity
for nucleotide incorporation opposite template purines than opposite template
pyrimidines. We present here a crystallographic analysis of hPoliota binary
complexes, which together with the ternary complexes show that, contrary to
replicative DNA polymerases, the DNA, and not the polymerase, undergoes the
primary substrate-induced conformational change. The incoming dNTP "pushes"
templates A and G from the anti to the syn conformation dictated by a rigid
hPoliota active site. Together, the structures posit a mechanism for template
selection wherein dNTP binding induces a conformational switch in template
purines for productive Hoogsteen base pairing.
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Figure 3.
Figure 3. Comparison of the hPolι Active Site in Binary and
Ternary Complexes
(A) Top: close-up views of the hPolι
active site region in hPolι[A] binary (left) and hPolι[A.dTTP]
ternary (right) complexes. The fingers and palm domains and the
PAD are shown in yellow, blue, and green, respectively. The DNA
is colored gray, and the template dA and incoming dTTP are
shown in red. The putative Mg^2+ ions in the ternary complex
are shown in dark blue. The catalytic residues (D34, D126, and
E127), the residues apposed close to template dA (Q59, K60,
L62, V64, L78, S307, K309, and R347), and incoming dTTP (Y39,
T65, Y68, R71, and K214) are highlighted and labeled. Note that
template dA is in the anti conformation in the binary complex
but flips to the syn conformation in the ternary complex. Note
also that some of the amino acids, including Leu62, Val64, and
Arg71, change conformation in response to dTTP binding. Bottom:
simulated annealing F[o] − F[c] omit maps (contoured at
3.0σ) showing template dA in the anti conformation in the
binary complex (left) and in the syn conformation in the
ternary complex (right). In the ternary complex, dA makes a
Hoogsteen base pair with incoming dTTP, which remains in the
anti conformation.
(B) Top: close-up views of the hPolι
active site region in hPolι[G] binary (left) and
hPolι[G.dCTP] ternary (right) complexes. Note that template dG
is in the anti conformation in the binary complex but flips to
the syn conformation in the ternary complex. Bottom: simulated
annealing F[o] − F[c] omit maps (contoured at 3.0σ) showing
template dG in the anti conformation in the binary complex
(left) and in the syn conformation in the ternary complex
(right). In the ternary complex, dG makes a Hoogsteen base pair
with incoming dCTP, which remains in the anti conformation and
is likely protonated. Figure 3. Comparison of the hPolι
Active Site in Binary and Ternary Complexes(A) Top: close-up
views of the hPolι active site region in hPolι[A] binary
(left) and hPolι[A.DTTP] ternary (right) complexes. The fingers
and palm domains and the PAD are shown in yellow, blue, and
green, respectively. The DNA is colored gray, and the template
dA and incoming dTTP are shown in red. The putative Mg^2+ ions
in the ternary complex are shown in dark blue. The catalytic
residues (D34, D126, and E127), the residues apposed close to
template dA (Q59, K60, L62, V64, L78, S307, K309, and R347), and
incoming dTTP (Y39, T65, Y68, R71, and K214) are highlighted and
labeled. Note that template dA is in the anti conformation in
the binary complex but flips to the syn conformation in the
ternary complex. Note also that some of the amino acids,
including Leu62, Val64, and Arg71, change conformation in
response to dTTP binding. Bottom: simulated annealing F[o] −
F[c] omit maps (contoured at 3.0σ) showing template dA in the
anti conformation in the binary complex (left) and in the syn
conformation in the ternary complex (right). In the ternary
complex, dA makes a Hoogsteen base pair with incoming dTTP,
which remains in the anti conformation.(B) Top: close-up views
of the hPolι active site region in hPolι[G] binary (left) and
hPolι[G.DCTP] ternary (right) complexes. Note that template dG
is in the anti conformation in the binary complex but flips to
the syn conformation in the ternary complex. Bottom: simulated
annealing F[o] − F[c] omit maps (contoured at 3.0σ) showing
template dG in the anti conformation in the binary complex
(left) and in the syn conformation in the ternary complex
(right). In the ternary complex, dG makes a Hoogsteen base pair
with incoming dCTP, which remains in the anti conformation and
is likely protonated.
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Figure 4.
Figure 4. anti versus syn Conformation
Templates dA and dG
are in the anti (binary) conformation in the absence of
incoming dNTP (left), but they rotate about their glycosidic
bond to the syn (ternary) conformation in response to dNTP
binding (right). Figure 4. anti versus syn
ConformationTemplates dA and dG are in the anti (binary)
conformation in the absence of incoming dNTP (left), but they
rotate about their glycosidic bond to the syn (ternary)
conformation in response to dNTP binding (right).
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The above figures are
reprinted
by permission from Cell Press:
Structure
(2006,
14,
749-755)
copyright 2006.
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