Biomedical Engineering Reference
In-Depth Information
interface, and revealed that the ectodomain of GCPII is composed of three
intertwined domains.
77
More detailed information later became available with
publication of a series of moderate- to high-resolution structures.
67,70,78-84
The extracellular portion of GCPII folds into three distinct domains, and
residues from all three domains shape the GCPII specificity pocket and
contribute to substrate recognition (seeFigure3.1).Theproteasedomain,
the fold of which is most closely related to the M28 aminopeptidases, spans
amino acids 57-116 and 352-590 and features a central seven-strand mixed
b-sheet flanked by 10 a-helices. The apical domain (amino acids 117- 351)
is inserted between the first and second b-strand of the protease domain.
The apical domain covers the active site and, together with the protease
domain, shapes the deep substrate binding funnel. The apical domain
also harbors four solvent-exposed, consecutive proline residues (Pro146-
Pro149) that make up a polyproline type-II helix—a signature motif that is
typically implicated in intermolecular contacts via its interaction with SH3
(src-homology 3) modules. The C-terminal domain (dimerization domain;
amino acids 591-750) can be viewed as a four-helix bundle that is engaged in
extensive intermolecular contacts with the second monomer of the GCPII
physiological homodimer.
81
Four inorganic ions, two Zn
21
,Cl
-
,andCa
21
, are present in the GCPII
structure. The two zinc ions, coordinated by the side chains of His377,
Asp387, Asp453, Glu425, and His553, are located between the S1 and S1
0
specificity pockets and are a fundamental part of the active site. In addition
to their structural role, they are indispensable for peptide bond hydrolysis
and are exploited for the design of high-anity GCPII inhibitors. The
chloride anion, positioned in the vicinity of the P1 arginine patch, is likely
involved in productive positioning of positively charged GCPII residues
implicated in substrate/inhibitor interactions. Finally, the calcium ion, at a
distance of 20 A
˚
from the active site, is involved in GCPII dimerization by
stabilizing the loop composed of amino acids 272-279 at the dimerization
interface.
81
Homodimerization of GCPII (believed to be indispensable for
the enzymatic activity) is also aided by the N-linked sugars attached to the
side chain of Asn638, the importance of which is supported by several
biochemical studies.
69,85-87
In addition to the aforementioned structural features, two major flexible
segments have been identified in the GCPII structure: the glutarate sensor
(amino acids Tyr692-Ser704) and the entrance lid (amino acids Trp541-
Gly548). Both are indispensable for proper functioning of the enzyme and can
be exploited for inhibitor design.
70,79,81,84
The glutarate sensor covers an
extensive portion of the S1
0
specificity pocket and can adopt a variety of
conformations with a maximal positional difference of
6
˚
(Ca atoms of
Gly702) for the two extreme positions ('up' and 'down'). It can be inferred that
the 'down' position is elicited by the presence of glutamate(like) residues in the
S1
0
pocket, as its a and g carboxylic groups 'pull down' the sensor via inter-
actions with the side chains of Tyr700 and Lys699, respectively. Conversely,
when the S1
0
B
site is empty, the glutarate sensor adopts the 'up' or several
