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PDBsum entry 1e9d
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Potentiating azt activation: structures of wild-Type and mutant human thymidylate kinase suggest reasons for the mutants' Improved kinetics with the HIV prodrug metabolite aztmp.
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Authors
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N.Ostermann,
A.Lavie,
S.Padiyar,
R.Brundiers,
T.Veit,
J.Reinstein,
R.S.Goody,
M.Konrad,
I.Schlichting.
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Ref.
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J Mol Biol, 2000,
304,
43-53.
[DOI no: ]
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PubMed id
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Abstract
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The 60-fold reduced phosphorylation rate of azidothymidine (AZT) monophosphate
(AZTMP), the partially activated AZT metabolite, by human thymidylate kinase
(TMPK) severely limits the efficacy of this anti-HIV prodrug. Crystal structures
of different TMPK nucleotide complexes indicate that steric hindrance by the
azido group of AZTMP prevents formation of the catalytically active closed
conformation of the P-loop of TMPK. The F105Y mutant and a chimeric mutant that
contains sequences of the human and Escherichia coli enzyme phosphorylate AZTMP
20-fold faster than the wild-type enzyme. The structural basis of the increased
activity is assigned to stabilization of the closed P-loop conformation.
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Figure 3.
Figure 3. The 60-fold reduced catalytic rate with AZTMP in
comparison to TMP suggests that TMPK can nevertheless adopt the
partially closed (i.e. active) conformation despite the presence
of the azido group. Overlay of the bisubstrate inhibitor
complexes with either TP[5]A, shown in pink and modeled as TMP
and ATP, and AZTP[5]A (modeled as AZTMP and ATP) reveal that the
side-chain of Asp15 points away from the 3' substituent in the
case of AZTP[5]A, but makes an interaction with the 3'-hydroxyl
group in the case of TP[5]A. Only the fully closed conformation
was observed for the P-loop in the presence of TP[5]A, while
both the open and closed P-loop conformations were observed in
the presence of AZTP[5]A, again suggesting a higher barrier for
reaching the closed conformation in the presence of the azido
group.
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Figure 4.
Figure 4. The F105Y mutant adopts a partially closed
conformation even in the presence of ADP. (a) Stereoview of the
overlay between the complex of TMP and ADP with wild-type TMPK
and the F105Y mutant, respectively. In the wild-type complex
structure (depicted in pink), seven interconnected water
molecules are observed to stabilize the open conformation. The
presence of the hydroxyl group of Y105 hinders the formation of
such a water structure, thereby destabilizing the open
conformation. In addition, the hydroxyl moiety interacts with
the side-chain of Gln157, which interacts with the amide
nitrogen atom of the P-loop Asp15 (broken lines), resulting in
the stabilization of the closed conformation. (b) To illustrate
the steric clash that would ensue due to the introduced tyrosine
hydroxyl moiety (instead of Phe), the structure observed in the
open TMP-ADP complex is depicted with its interconnecting water
structure, and the tyrosine residue (pink) from the F105Y
structure.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2000,
304,
43-53)
copyright 2000.
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Secondary reference #1
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Title
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Insights into the phosphoryltransfer mechanism of human thymidylate kinase gained from crystal structures of enzyme complexes along the reaction coordinate.
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Authors
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N.Ostermann,
I.Schlichting,
R.Brundiers,
M.Konrad,
J.Reinstein,
T.Veit,
R.S.Goody,
A.Lavie.
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Ref.
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Structure, 2000,
8,
629-642.
[DOI no: ]
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PubMed id
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Figure 4.
Figure 4. Conformational changes of Arg97 and the
phosphoryl groups of TDP to the stable product conformation in
the TDP-ADP bound complex. Overlay of the TMP/TDP-binding site
of the structures of TMPK in complex with TMP, ADP and AlF[3]
(red) and TDP and ADP (yellow). In the complex with bound TDP
and ADP the sidechain of Arg97 rotates (90°) around the bond
between the atoms CG and CD such that it cannot act as a clamp
to bring both nucleotides together for the backward reaction.
The figures were generated using the programs Molscript [28] and
Raster 3D [29].
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The above figure is
reproduced from the cited reference
with permission from Cell Press
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