Biomedical Engineering Reference
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intermediate conformations, thus substantially increasing the volume of the S1 0
pocket. 79,81
In its closed conformation, the entrance lid adopts a single turn a-helical
conformation and covers the S1 funnel (see below), shielding the GCPII sub-
strate binding cavity from the external space. Therefore, for an inhibitor (or
substrate or reaction product) to enter the GCPII active site, the lid has to
'open,' exposing the enzyme's interior. The 'closed-open' transition is realized
by the flipping of the peptide bond between Asn540 and Trp541 at one hinge
and the flexibility of Gly548 at the other hinge. While processing the bulkier
substrates (such as pteroate-poly-glutamates) or upon binding of larger inhi-
bitors, the entrance lid remains in the open position to avoid steric clashes with
the substrate/inhibitor moieties. Interestingly, several lines of evidence suggest
that Trp541, one of the residues forming the entrance lid, is involved in
recognition of folates (Navratil et al., unpublished) and can be exploited for the
design of ultra-high anity GCPII inhibitors (see below). 84
Figure 3.1 Homodimer of human GCPII in the middle. One monomer shown in
semitransparent surface representation with individual domains of the
extracellular part colored red (protease domain; amino acids 57-116 and
352-590), yellow (apical domain; amino acids 117-351), and cyan (C-
terminal; amino acids 591-750). N-linked sugar moieties are shown in
gray, and the entrance into the S1 funnel is circled. The second monomer is
in cartoon representation (gray), with inorganic ions shown as blue, green,
and magenta spheres for Zn 21 ,Cl - , and Ca 21 , respectively. Close-ups of
the chloride anion surroundings (A), the active-site zincs (B), and the
calcium loop (C). (D) GCPII homodimer (top view). The S1 funnel and
the S1 0 pocket are delineated by the black line, with the MTX inhibitor
(PDB code 3BI1) shown in ball-and-stick representation.
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