state, designated the occluded, closed, open, and disordered states
[21]. The occluded and closed conformations have also been
observed in NMR experiments [32]. The open conformation has
only been observed in certain crystal forms. The disordered states
described cases in which motion renders the loop unclear or invisible in crystallographic experiments. The four loop conformations
are best characterized by their secondary structure, interactions
with nicotinamide-ribose moiety, and hydrogen bonding with the
F-G and G-H loops. The open conformation displays characteristics between those of the closed and occluded conformations. The
disordered conformation displays characteristics of a time-averaged
exchange of closed and occluded conformations [21].
The conformation of the active site loops depends on the
ligands bound in the substrate and cofactor binding sites. If only
the substrate site is occupied, the enzyme adopts the occluded loop
conformation. Binding of the nicotinamide-ribose moiety of
NADPH within its binding site produces the closed conformation,
in which the Met20 loop is packed against the nicotinamide ring of
NADPH, closing the active site off to the surrounding solvent.
Only the closed conformation allows for the proper positioning
of the NADPH and substrate reactive centers, such that they are in
close enough proximity to facilitate the reaction. Thus, it is apparent that movement of the Met20 loop is directly coordinated with
the stages of the catalytic cycle (Fig. 3) [18, 33].
The occluded and closed conformations differ in structure in
the central portion of the Met20 loop and in the pattern of hydrogen bonds formed between the Met20 loop and the F-G and G-H
loops. In the occluded state, the central region of the Met20 loop
forms a 3 10 -helix, with residues Met16 and Glu17 projecting into
the active site, where they “occlude” the binding site for the
nicotinamide ring moiety of NADPH. The occluded conformation
is stabilized by hydrogen bonding interactions between Asn23
(backbone CO and NH) in the Met20 loop and Ser148 (NH and
Oγ) in the G-H loop. In the closed conformation, residues 16-19
form a β-hairpin structure. Met16 and Glu17 are flipped out of the
active site, thereby allowing nicotinamide binding, while the side
chains of Asn18 and Met20 pack down over the bound substrate
and cofactor. The Asn23/Ser148 hydrogen bonds are disrupted,
and new hydrogen bonds are formed between the backbone NH
and Oδ of Asp122 in the F-G loop and the backbone CO and NH
of Gly15 and Glu17, respectively [21].
Sawaya et al. proposed a detailed structural model for the
conformational changes that occur during the ecDHFR catalytic
cycle [21]. They based their model on the analysis of isomorphous
crystal structures (P2 1 2 1 2 1 ) of different ecDHFR complexes to
ensure that the packing interactions are constant between the different structures in the series and that conformational differences
are due to differences in ligand binding. To investigate the
190
Melanie Goldstein and Nina M. Goodey
[21]. The occluded and closed conformations have also been
observed in NMR experiments [32]. The open conformation has
only been observed in certain crystal forms. The disordered states
described cases in which motion renders the loop unclear or invisible in crystallographic experiments. The four loop conformations
are best characterized by their secondary structure, interactions
with nicotinamide-ribose moiety, and hydrogen bonding with the
F-G and G-H loops. The open conformation displays characteristics between those of the closed and occluded conformations. The
disordered conformation displays characteristics of a time-averaged
exchange of closed and occluded conformations [21].
The conformation of the active site loops depends on the
ligands bound in the substrate and cofactor binding sites. If only
the substrate site is occupied, the enzyme adopts the occluded loop
conformation. Binding of the nicotinamide-ribose moiety of
NADPH within its binding site produces the closed conformation,
in which the Met20 loop is packed against the nicotinamide ring of
NADPH, closing the active site off to the surrounding solvent.
Only the closed conformation allows for the proper positioning
of the NADPH and substrate reactive centers, such that they are in
close enough proximity to facilitate the reaction. Thus, it is apparent that movement of the Met20 loop is directly coordinated with
the stages of the catalytic cycle (Fig. 3) [18, 33].
The occluded and closed conformations differ in structure in
the central portion of the Met20 loop and in the pattern of hydrogen bonds formed between the Met20 loop and the F-G and G-H
loops. In the occluded state, the central region of the Met20 loop
forms a 3 10 -helix, with residues Met16 and Glu17 projecting into
the active site, where they “occlude” the binding site for the
nicotinamide ring moiety of NADPH. The occluded conformation
is stabilized by hydrogen bonding interactions between Asn23
(backbone CO and NH) in the Met20 loop and Ser148 (NH and
Oγ) in the G-H loop. In the closed conformation, residues 16-19
form a β-hairpin structure. Met16 and Glu17 are flipped out of the
active site, thereby allowing nicotinamide binding, while the side
chains of Asn18 and Met20 pack down over the bound substrate
and cofactor. The Asn23/Ser148 hydrogen bonds are disrupted,
and new hydrogen bonds are formed between the backbone NH
and Oδ of Asp122 in the F-G loop and the backbone CO and NH
of Gly15 and Glu17, respectively [21].
Sawaya et al. proposed a detailed structural model for the
conformational changes that occur during the ecDHFR catalytic
cycle [21]. They based their model on the analysis of isomorphous
crystal structures (P2 1 2 1 2 1 ) of different ecDHFR complexes to
ensure that the packing interactions are constant between the different structures in the series and that conformational differences
are due to differences in ligand binding. To investigate the
190
Melanie Goldstein and Nina M. Goodey