predicted to occupy its binding pocket within the active site, in
close proximity to the pterin ring of the substrate. However, in the
occluded D:THF:NADP+ and D:THF:NADPH product complexes, this moiety projects into the solvent. Movement of the
adenosine binding domain relative to the major domain during
the
catalytic
cycle
modulates
the
width
of
the
p-aminobenzoylglutamate (pABG) binding cleft. From the
Michaelis to the transition state analogue complex, they observed
that rotation between the two domains closes the pABG binding
cleft by approximately 0.5 A ˚ . The resulting enhancement of contacts with the pABG moiety may stabilize puckering of C6 of the
pterin ring in the transition state. The domain rotation is further
adjusted by cofactor induced movements of the α B and α C helices,
producing a larger pABG cleft in the product complexes. The
domain rotations are suggested to play a role in transition state
stabilization and NADPH-assisted product release (Fig. 3)
[18, 21]. In this work function was intimately linked to both
macromolecular structure and dynamics by solving crystal structures at different stages of the catalytic cycle by using substrate
analogues. The findings showed that regions distal from the active
site play a role and have inspired experiments to investigate these
regions further. Using the isomorphous structures and GIFMerge,
the authors created a movie that illustrates DHFR’s range of
inferred subdomain and loop movements.
2.3 Species-Specific
Structural Features
of DHFR
2.3.1 Human DHFR
and Comparison
to Bacterial DHFR
The structural differences between vertebrate DHFRs and bacterial
DHFRs are important to the specificity of DHFR inhibitors.
Human DHFR (hDHFR) is a monomeric, 186-amino-acid protein
with a molecular weight of approximately 21.5 kDa. Like ecDHFR,
hDHFR has an eight-stranded β-sheet consisting of seven parallel
strands and a carboxy-terminal antiparallel strand. Five α-helices are
packed against the beta-sheet core, denoted αB, αC, αE, αE
0 , and
αF. The αE
0 helix is perpendicular to αE and may have emerged via a
five-residue insertion mutation in human DHFR relative to bacterial DHFR [34]. In addition, hDHFR has one left-handed, type II
polyproline-like helix, which is not present in ecDHFR. Another
variation from ecDHFR is the presence of a cis-peptide linkage
between residues Arg65 and Pro66. The other cis-peptide linkage
is a conserved structural feature of all DHFRs and is between
residues Gly116 and Gly117, which are located near the nicotinamide binding site. Just as in ecDHFR, the active site cleft in
hDHFR is formed at the junction of the two domains. However,
in the case of hDHFR the larger subdomain is the adenosine
binding domain and the second is the smaller loop domain. The
acidic residue, Glu30, is analogous to Asp27 in ecDHFR [34].
The conformation of vertebrate and human DHFRs is more
rigid than ecDHFR [34]. However, the amino acids required for
catalysis and the general secondary structural features, as well as the
192
Melanie Goldstein and Nina M. Goodey
close proximity to the pterin ring of the substrate. However, in the
occluded D:THF:NADP+ and D:THF:NADPH product complexes, this moiety projects into the solvent. Movement of the
adenosine binding domain relative to the major domain during
the
catalytic
cycle
modulates
the
width
of
the
p-aminobenzoylglutamate (pABG) binding cleft. From the
Michaelis to the transition state analogue complex, they observed
that rotation between the two domains closes the pABG binding
cleft by approximately 0.5 A ˚ . The resulting enhancement of contacts with the pABG moiety may stabilize puckering of C6 of the
pterin ring in the transition state. The domain rotation is further
adjusted by cofactor induced movements of the α B and α C helices,
producing a larger pABG cleft in the product complexes. The
domain rotations are suggested to play a role in transition state
stabilization and NADPH-assisted product release (Fig. 3)
[18, 21]. In this work function was intimately linked to both
macromolecular structure and dynamics by solving crystal structures at different stages of the catalytic cycle by using substrate
analogues. The findings showed that regions distal from the active
site play a role and have inspired experiments to investigate these
regions further. Using the isomorphous structures and GIFMerge,
the authors created a movie that illustrates DHFR’s range of
inferred subdomain and loop movements.
2.3 Species-Specific
Structural Features
of DHFR
2.3.1 Human DHFR
and Comparison
to Bacterial DHFR
The structural differences between vertebrate DHFRs and bacterial
DHFRs are important to the specificity of DHFR inhibitors.
Human DHFR (hDHFR) is a monomeric, 186-amino-acid protein
with a molecular weight of approximately 21.5 kDa. Like ecDHFR,
hDHFR has an eight-stranded β-sheet consisting of seven parallel
strands and a carboxy-terminal antiparallel strand. Five α-helices are
packed against the beta-sheet core, denoted αB, αC, αE, αE
0 , and
αF. The αE
0 helix is perpendicular to αE and may have emerged via a
five-residue insertion mutation in human DHFR relative to bacterial DHFR [34]. In addition, hDHFR has one left-handed, type II
polyproline-like helix, which is not present in ecDHFR. Another
variation from ecDHFR is the presence of a cis-peptide linkage
between residues Arg65 and Pro66. The other cis-peptide linkage
is a conserved structural feature of all DHFRs and is between
residues Gly116 and Gly117, which are located near the nicotinamide binding site. Just as in ecDHFR, the active site cleft in
hDHFR is formed at the junction of the two domains. However,
in the case of hDHFR the larger subdomain is the adenosine
binding domain and the second is the smaller loop domain. The
acidic residue, Glu30, is analogous to Asp27 in ecDHFR [34].
The conformation of vertebrate and human DHFRs is more
rigid than ecDHFR [34]. However, the amino acids required for
catalysis and the general secondary structural features, as well as the
192
Melanie Goldstein and Nina M. Goodey