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PDBsum entry 3bhx
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References listed in PDB file
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Key reference
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Title
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Structural basis of interactions between human glutamate carboxypeptidase ii and its substrate analogs.
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Authors
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C.Barinka,
K.Hlouchova,
M.Rovenska,
P.Majer,
M.Dauter,
N.Hin,
Y.S.Ko,
T.Tsukamoto,
B.S.Slusher,
J.Konvalinka,
J.Lubkowski.
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Ref.
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J Mol Biol, 2008,
376,
1438-1450.
[DOI no: ]
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PubMed id
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Abstract
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Human glutamate carboxypeptidase II (GCPII) is involved in neuronal signal
transduction and intestinal folate absorption by means of the hydrolysis of its
two natural substrates, N-acetyl-aspartyl-glutamate and
folyl-poly-gamma-glutamates, respectively. During the past years, tremendous
efforts have been made toward the structural analysis of GCPII. Crystal
structures of GCPII in complex with various ligands have provided insight into
the binding of these ligands, particularly to the S1' site of the enzyme. In
this article, we have extended structural characterization of GCPII to its S1
site by using dipeptide-based inhibitors that interact with both S1 and S1'
sites of the enzyme. To this end, we have determined crystal structures of human
GCPII in complex with phosphapeptide analogs of folyl-gamma-glutamate,
aspartyl-glutamate, and gamma-glutamyl-glutamate, refined at 1.50, 1.60, and
1.67 A resolution, respectively. The S1 pocket of GCPII could be accurately
defined and analyzed for the first time, and the data indicate the importance of
Asn519, Arg463, Arg534, and Arg536 for recognition of the penultimate (i.e., P1)
substrate residues. Direct interactions between the positively charged
guanidinium groups of Arg534 and Arg536 and a P1 moiety of a substrate/inhibitor
provide mechanistic explanation of GCPII preference for acidic dipeptides.
Additionally, observed conformational flexibility of the Arg463 and Arg536 side
chains likely regulates GCPII affinity toward different inhibitors and modulates
GCPII substrate specificity. The biochemical experiments assessing the
hydrolysis of several GCPII substrate derivatives modified at the P1 position,
also included in this report, further complement and extend conclusions derived
from the structural analysis. The data described here form an a solid foundation
for the structurally aided design of novel low-molecular-weight GCPII inhibitors
and imaging agents.
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Figure 2.
Fig. 2. The substrate-binding cavity of GCPII can be closed
by the ‘entrance lid.’ (a) Superposition of the ‘entrance
lids’ from rhGCPII/MPE and rhGCPII/SPE complexes. The model of
rhGCPII/MPE is shown in cartoon representation and colored gray.
The ‘entrance lid,’ formed by the amino acids
Trp541–Gly548, is painted red and blue for the open (observed
in the rhGCPII/MPE complex) and closed (taken from the
superimposed structure of the rhGCPII/SPE complex)
conformations, respectively. The active-site-bound SPE
inhibitor is represented by sticks, and the active-site Zn^2+
ions are represented by magenta spheres. (b and c) A close-up
view of the ‘entrance lid’ in open/closed conformation. The
protein is represented by its molecular surface, with the
‘entrance lid’ colored red or blue for the open
(rhGCPII/MPE, b) and closed (rhGCPII/SPE, c) conformations,
respectively. The molecule of MPE, visible in the current
projection, is represented by sticks (b), while a molecule of
SPE is buried by the ‘entrance lid’ (c). A surface defined
by the active-site zinc ions is colored magenta.
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Figure 3.
Fig. 3. The electron density maps of the substrate-binding
cavity of human GCPII in complex with SPE (a) and EPE (b). The
protein residues are shown in ball-and-stick representation,
while Zn^2+ and Cl^− ions are depicted as blue and yellow
spheres, respectively. The F[o] − F[c] electron density
omit maps around inhibitor molecules are contoured at the 3
σ level (green), and the 2F[o] − F[c] electron density maps
are contoured at the 1σ level (blue). The picture was generated
using MolScript^28 and BobScript^29 and rendered with PovRay.
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The above figures are
reprinted
by permission from Elsevier:
J Mol Biol
(2008,
376,
1438-1450)
copyright 2008.
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