From the Department of Structural Biology,
Hauptman-Woodward Medical Research Institute,
Buffalo, New York 14203, the § Department of Molecular
and Cellular Biophysics, Roswell Park Cancer Institute,
Buffalo, New York 14263, the ** Department of Clinical Pharmaceutics,
Faculty of Pharmaceutical Sciences, Nihon University, Narashinodai,
7-7-1 Funabashi-shi, Chiba 274-8555, and the
Department of Biochemistry, Faculty of
Pharmaceutical Sciences, Hoshi University, 2-4-41 Ebara,
Shinagawa-ku, Tokyo 142-8501, Japan
Received for publication, January 19, 2001, and in revised form, March 1, 2001
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ABSTRACT |
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Porcine testicular carbonyl reductase
(PTCR) belongs to the short chain dehydrogenases/reductases (SDR)
superfamily and catalyzes the NADPH-dependent reduction of
ketones on steroids and prostaglandins. The enzyme shares nearly 85%
sequence identity with the NADPH-dependent human
15-hydroxyprostaglandin dehydrogenase/carbonyl reductase. The tertiary
structure of the enzyme at 2.3 Å reveals a fold characteristic of the
SDR superfamily that uses a Tyr-Lys-Ser triad as catalytic residues,
but exhibits neither the functional homotetramer nor the homodimer that
distinguish all SDRs. It is the first known monomeric structure in the
SDR superfamily. In PTCR, which is also active as a monomer, a
41-residue insertion immediately before the catalytic Tyr describes an
all-helix subdomain that packs against interfacial helices, eliminating
the four-helix bundle interface conserved in the superfamily. An
additional anti-parallel strand in the PTCR structure also blocks the
other strand-mediated interface. These novel structural features
provide the basis for the scaffolding of one catalytic site within a
single molecule of the enzyme.
The short chain dehydrogenases/reductases
(SDR)1 catalyze critical
steps of activation and inactivation of steroids, vitamins, prostaglandins, and other bioactive molecules by oxidation and reduction of hydroxyl and carbonyl groups, respectively (1). A
member of the SDR superfamily, the multifunctional enzyme porcine testicular carbonyl reductase (PTCR)/3 PTCR is highly homologous to NADP(H)-dependent human and
rat CRs, except for its 13 additional amino acid residues at the C
terminus (2). The sequence identity of PTCR with the human CR, also
known as 15-hydroxyprostaglandin dehydrogenase/9-ketoprostaglandin reductase, is about 85% (2). Alignment of amino acid sequences of PTCR
and human CR with those of other well known members of SDR superfamily,
such as bacterial 3 The other distinguishing feature of all SDRs is that the functional
units are either homotetramers or homodimers (8, 9, 12-15).
Surprisingly, the active units of both PTCR and human CR were reported
to be monomeric (5, 16). Although more than half of the 15 or so
crystal structures of SDRs to date exhibit the same tetrameric
quaternary structure, only the four-helix bundle or the Q axis
interface that involves the largest surface area of association (8) is
conserved among SDRs. The other surface of close association of
monomers in tetrameric SDRs is the so-called P axis interface involving
an anti-parallel pair of strands and a pair of helices (8) that has
been observed only once in a dimeric SDR (17). The interfacial
four-helix bundle that borders two catalytic sites and buries
hydrophobic surfaces of helices is predicted to be important for the
integrity of the active site clefts (18, 19). Here we present the first crystal structure of a monomeric SDR and explain how the functional determinants of oligomeric SDRs could be retained within a monomer of
PTCR having the basic SDR fold.
Purification, Crystallization, and Data Collection--
The
enzyme was purified as described (5). Crystallization and data
collection details have also been published (20). Briefly, the enzyme
was crystallized from a solution of 36-37% saturated ammonium sulfate
in 10 mM MES buffer, pH 6.0. Crystals of diffraction size
were grown by macroseeding. The space group is
P43212 and the unit cell parameters are
a = b = 58.53 Å and c = 165.64 Å with one molecule (288 amino acid residues) in the asymmetric unit. Data collection was carried out on a Rigaku R axis II
image plate detector at ambient temperature and processed with the
software package DENZO (21). Data from three crystals between
resolution range 55.2 and 2.30 Å were merged to yield a data set of
10792 unique reflections (F > 0) with an average of
about three observations per reflection. The overall
Rmerge was 0.066 on the intensities. The data
were 80% complete overall and 52% complete in the resolution shell
between 2.50 and 2.30 Å.
Structure Solution--
The structure was solved by the single
isomorphous replacement and anomalous scattering method, combined with
phases from a molecular replacement solution. Two platinum
compounds, potassium chloroplatinate (PtCl4) and
di-µ-iodobis(ethylenediamine)diplatinum(II) nitrate (PIP), were used
to prepare heavy atom derivative crystals, both yielding the same major
platinum-binding site. A total of 4351 reflections to 3.00 Å was
phased using the PIP derivative, and 3564 reflections to 3.20 Å were
phased using the PtCl4 derivative. The latter data set also contained
anomalous signal for 1660 reflections to 3.40 Å. The centric
R value and the phasing power for the PIP and PtCl4
derivatives were 0.56 and 1.64 and 0.63 and 1.41, respectively. The
phasing power for the anomalous data was 1.92. The software package
PHASES (22) was used for calculating and combining phases.
The molecular replacement solution was obtained using a search model,
which was a superposition of equally weighted structures of
3 Model Building and Refinement--
Model building was carried
out on a Silicon Graphics R10000 Indigo2 work
station using a figure of merit weighted Fo map, phased with the combined experimental phases, and the software CHAIN
(24). Initially, 235 of 288 amino acids were fit in the electron
density map, which also unequivocally showed the NADP density. The
model was subjected to 26 cycles of refinement and rebuilding
processes, using XPLOR (25) and CNS (23) routines. The resolution was
gradually extended to 2.30 Å. The C-terminal tail section was built
during the latter part of the refinement. Various omit maps with
calculated phases were computed to resolve regions of ambiguity. Except
for a few residues in highly exposed regions of the molecule, such as
Glu-261, Lys-219, and Lys-238, side chains were in good agreement with
their electron densities. A sulfate ion and 58 well ordered water
molecules were introduced in the last three cycles of refinement and rebuilding.
A summary of refinement results is provided in Table
I. The analysis of the molecular geometry
was performed with PROCHECK (26), CNS (23), and CCP4 (27) packages. The
illustrations were made with SETOR (28). The atomic coordinates have
been deposited with the Protein Data Bank (code 1HU4).
The Tertiary Structure of PTCR, an Overview of SDRs--
All 288 amino acid residues of one polypeptide chain of PTCR (excluding the
N-terminal methionine) were located from electron density maps. Amino
acid residues are numbered 1-288 starting with the Ser at position 2 in the cDNA sequence (2). A ribbon diagram of the tertiary
structure of PTCR is shown in Fig.
1a. A schematic description of
the structure is provided in Fig. 1b. The basic SDR fold
includes a seven-stranded parallel
The view in Fig. 1a is along the central
Fig. 1c is a superposition of monomers of crystal structures
of all four steroid dehydrogenases belonging to the SDR superfamily (bacterial 3
The other important observation from this superposition is the
diversity of the three-dimensional structures of the regions primarily
contributed by the C-terminal halves of polypeptide chains. This
region, which constitutes the outer walls of the substrate-binding
cleft of the active site, provides specific interactions that are
critical to the selectivity of substrates and to the mechanism of
molecular recognition by the enzyme (19, 29). Although this region is
well developed for 17
The entrance to the active site in PTCR is more open than any other
HSD. This is a direct consequence of a shortened three-residue linkage
between
The Lys-238 side chain in human placental CR is autocatalytically
modified to carboxyethyllysine (30). This side chain in PTCR is in a
highly exposed region, and its electron density is not discernible
beyond the C The Quaternary Structure of SDRs and How a PTCR Monomer Mimics
It--
The Q axis dimer interface involving the four-helix bundle and
the P axis interface involving anti-parallel association of
The all-helix subdomain of the 41-amino acid insertion region obstructs
access to Why Is PTCR NADPH-specific?--
Although no coenzyme was added in
the crystallization medium, a molecule of NADP(H) was bound to the
protein molecule. The coenzyme is in the extended conformation as
observed for all other NAD(P)(H) bound to SDRs (8, 12-15, 29). The
distance between C-2 of the nicotinamide and C-6 of the adenine
ring, a parameter used to compare the degree of extension of coenzymes,
is 14.65 Å, well within the range for SDRs. The
nicotinamide ring is in syn conformation, with the
4-pro-S hydride facing the catalytic site, typical of all
SDRs. The coenzyme molecule and its electron density are shown in Fig.
3a, along with side chains in
the vicinity. Puckering of the nicotinamide ring was not discernible at
the working resolution, and we used parameters of an oxidized coenzyme to restrain its geometry during refinement. Hydrogen bonding and charge
interactions between NADPH and neighboring amino acid residues are
drawn schematically in Fig. 3b.
The high specificity of the enzyme for reduced
Catalytic side chains Tyr-193 and Lys-197 and the main chain carbonyl
group of Asn-89 have contacts with the 2'- and 3'-hydroxyls of the
nicotinamide ribose moiety, as shown in Fig. 3. Not shown in the figure
is a short contact (3.1 Å) between the main chain carbonyl oxygen of
Gly-228 of PTCR, conserved in all steroid dehydrogenases and many other
SDRs, and C-4 of the nicotinamide ring. This interaction and the
orientation of carbonyl with respect to the nicotinamide ring have been
proposed to have an important role in driving the hydride transfer from
the coenzyme to the substrate (19).
Crystal Packing--
The most interesting intermolecular
interaction among crystallographically related monomers of PTCR is the
formation of a crystallographic dimer by the packing of two loops
between
The conformation of this loop is considerably different in PTCR than in
other SDRs (Fig. 4b). The similarity of the conformations of
the loops in 3
The other interesting aspect of this packing interaction is the
formation of a salt bridge between the free C-terminal of one molecule
and the Arg-37 side chain of the other, as shown in Fig. 4c.
This is the same Arg side chain that is involved in a specific
interaction with the 2'-phosphate group of the coenzyme. Furthermore,
the extended loop described above (residues 92-101) approaches the
tail region of the symmetry-related monomer, producing the following
intermolecular stacking interactions among side chains: Ile-92 with
Ala-288, Phe-94 with Val-286, and Ala-288, Pro-99 with Pro-284, and
Pro-101 with Tyr-283.
Thus the two most extended and isolated regions of the PTCR molecule
are stabilized in the crystal by intermolecular interactions with the
same regions of a symmetry-related molecule. Such contacts between two
molecules can only be present in the crystal, as the functional unit of
PTCR was shown to be a monomer in solution from the gel filtration data
(5).
Concluding Remarks--
Previously determined structures of
oligomeric SDRs demonstrated how multiple copies of polypeptides could
be assembled to build functional enzymes with multiple independent
active sites. When allosterism or co-operativity is not required for
function, multimeric assembly may not be an efficient utilization of
biological resources. The PTCR structure demonstrates how alteration of
the protein by substitution and insertion can alleviate the problem of
interdependence among protein products, leading to efficient scaffolding of one fully functional active site within an independent molecule. It is possible that monomeric SDRs like PTCR have appeared later in the evolutionary pathway than their oligomeric counterparts.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
/
, 20
-hydroxysteroid dehydrogenase (20
-HSD), catalyzes the NADPH-dependent
reduction of ketones on androgens, progestins, and prostaglandins, as
well as aldehydes and ketones on a large number of xenobiotics (2, 3).
The high level of 20
-HSD activity in the enzyme is demonstrated by
the reduction of 20-carbonyl groups of C21-steroids, such
as conversion of 17
-hydroxyprogesterone to
17
,20
-dihydroxy-4-pregnen-3-one, which is present in pig testes
during the neonatal stage (4, 5). Purified PTCR also shows vigorous
3
- and 3
-HSD activities with 5
-androstan-17
-ol-3-one
(5
-dihydrotestosterone) as a substrate (6).
,20
-HSD (7, 8), human 17
-HSD type 1 (9),
human 11
-HSD (10), and Drosophila alcohol dehydrogenase
(11), suggests that these CRs have a 41-residue insertion at a
strategic location, before the conserved Tyr-X-X-X-Lys motif. Nearly all SDRs are known to utilize a Tyr-Lys-Ser triad as
catalytic residues (1). The Tyr hydroxyl has been proposed to be the
proton donor in an electrophilic attack on the substrate carbonyl in a
reduction reaction (8).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
,20
-HSD (8), 17
-HSD1 (9), mouse lung carbonyl reductase (13),
and 7
-HSD (14). A large number of rotation search solutions were
computed using compact (without loop regions) and extended (all loops
through the catalytic triad present) search models. The optimal
solution was obtained with the CNS program suite (23) and the
extended search model with data between 15.00 and 3.50 Å, against an
E2E2 synthesis. The translation function search
was conducted in both P43212 and
P41212 space groups. Phases from the most
likely solutions were computed and used to phase a difference Fourier
synthesis with the PIP derivative and native data to 3.00 Å. The
correct translation function solution in the space group
P43212 produced the largest peak at the
platinum-binding site, having a height about three times the
background peaks. The heavy atom hand and the choice of origin were
fixed by this peak position. Phases calculated from the model were
combined with single isomorphous replacement and anomalous scattering
phases using the PHASES package. In all, 4698 reflections were phased
to 3.00 Å, with a combined figure of merit of 0.809, a correlation
coefficient of 0.792, and an Rinverse value of
0.266.
Summary of refinement results
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-sheet (
A to
G) flanked by
three parallel helices on each side (
B,
C,
G and
D,
E,
F). The segment
A to
F is a doubly wound
/
motif, with
alternating
-strands and
-helices. Whereas the
A to
F
segment constitutes the classic "Rossmann fold" associated with the
binding of the coenzyme NADPH, the
D to
G segment, in addition to
being partially within the Rossmann fold, governs quaternary
association and substrate binding. The two longest helices
E and
F form the interfacial four-helix bundle, typical of dimeric and
tetrameric forms.
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Fig. 1.
Tertiary structures of PTCR and other
members of the SDR superfamily. a, stereo ribbon diagram of
the PTCR structure derived from the present study looking down the
central -sheet into the catalytic cleft. The
-helices are drawn
in rose pink and the
-strands in green. Also
shown are catalytic residues Tyr-193, Lys-197, and Ser-139 and the
bound coenzyme NADP. The atoms are color-coded as follows: carbon,
gray-white; nitrogen, blue; oxygen,
red; and sulfur, yellow. The residue numbers
along the polypeptide chain are also shown. b, a schematic
diagram of the PTCR structure showing the nomenclature of secondary
structure elements and the overall fold. The standard nomenclature of
SDRs has been followed. The structural elements bear the numbers of
their terminal residues. The helices
F'-1,
F'-2,
F'-3, and
F'-4 make up the new subdomain in PTCR. c, superposition
of backbones of crystal structures of 3
,20
-HSD
(green), 17
-HSD1 (blue), 7
-HSD
(magenta), and PTCR (yellow) shown in stereo in
an orientation similar to the one in a. Also shown are the
catalytic residues Tyr, Lys, and Ser (in their respective backbone
colors) and NADP (red) bound to PTCR. The general locations
of termini are marked with N and C. Various
general features of these structures are indicated by
arrows.
-sheet of the
PTCR molecule, overlooking the active site delineated by the coenzyme NADP and catalytic residues Tyr-193, Lys-197, and Ser-139. As shown in
Fig. 1, a and b, the basic SDR fold described
above is well preserved in PTCR, except for two important differences. First, the 41-residue insertion before Tyr-193 describes four helices
F'-1 (residues 140-148),
F'-2 (residues 151-158),
F'-3 (residues 164-179), and
F'-4 (residues 182-186). The first turn of
F'-1 coincides with the helical turn immediately following the
catalytic serine (Ser-139 in PTCR) present in nearly all SDRs. This
all-helix subdomain folds away from the active site cleft, toward the
dimer interface of other SDRs. Second, after a tight turn the
polypeptide following
G describes an additional strand
H
(residues 272-275) that is anti-parallel to
G. The last 13 C-terminal residues form an extended tail-like structure that approaches the active site of a 2-fold symmetry-related molecule. The
terminal carboxyl group forms a salt bridge with the Arg-37 side chain
of the second molecule. A similar eighth anti-parallel strand is
present only in one other member of the SDR family, dihydropteridine
reductase (12).
,20
-HSD, human 17
-HSD1, Escherichia
coli 7
-HSD (14), and PTCR). The viewing direction in this
figure is roughly the same as in Fig. 1a. 3
,20
-HSD and
7
-HSD have homotetrameric structures, whereas 17
-HSD1 is
homodimeric (Fig. 2). The structural diversity among monomers of all members of the SDR family is well represented by this group. The overlap of the Rossmann fold and the
catalytic triad among these four enzymes, excluding the 41-residue insertion in PTCR, is striking. Furthermore, two long helices
E and
F from 3
,20
-HSD, 7
-HSD, and 17
-HSD1 that make up the four-helix bundle dimer interface superimpose remarkably well with the
ones from PTCR, which are not involved in dimer formation.
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Fig. 2.
Ribbon diagrams of homotetrameric
3 ,20
-HSD illustrating
the Q axis interface, the so-called four-helix bundle (a pair of
E and
F helices)
(a), and the P axis interface (a pair of strands
G and helices
G)
(b). The same four-helix bundle interface is also
shown in the ribbon diagram of homodimeric 17
-HSD1 in
c. The views are down the molecular 2-fold rotation axes in
all three figures. a and c, active sites across
the four-helix bundle are highlighted by bound inhibitors and/or
coenzyme. A close-up view of the stacking of new subdomain helices
(
F'-2 and
F'-3) of monomeric PTCR against
E and
F is shown
in d.
-HSD1 having additional structural elements
from an insertion between
F and
G, and from an extended C
terminus that surrounds the catalytic cleft, fewer residues from
similar regions of 3
,20
-HSD and 7
-HSD are present in the area.
In 3
,20
-HSD, a segment of the polypeptide chain from the loop
between
F and
G (Met-184 to Pro-206) and C-terminal residues
Trp-243 to Gln-255 border the active site cleft (8). Similarly, in
7
-HSD, residues Ala-191 to Pro-214 between
F and
G and
C-terminal residues Gly-250 to Asn-255 line the active site (14). Thus,
the outer wall structures in 3
,20
-HSD and 7
-HSD differ
significantly from that of 17
-HSD1. These differences in the
architecture of active sites probably explain why 17
-HSD1 has high
specificity for estrone as a substrate, whereas the other two enzymes
are not known to be substrate-specific. Nonetheless, all three enzymes
possess the so-called substrate entry-loop between
F and
G, which
tightens on the substrate upon its entry into the active site providing
additional substrate-specific interactions (8, 14, 19, 29).
F and
G, connecting Gly-236 and Pro-240 (corresponding to
residues Thr-190 and Val 210, respectively, in 3
,20
-HSD). Consequently, the substrate-entry loop is almost non-existent in PTCR.
Moreover, only residues Glu-281 to Ala-288 from the C-terminal tail are
in the vicinity of the active site. The number of residues that border
the substrate-binding cleft is thus only 15 in PTCR, as opposed to 37 in 3
,20
-HSD, 30 in 7
-HSD, and 60 in 17
-HSD1. The openness
of the substrate-binding cleft is consistent with the observed
promiscuity in substrate specificity of PTCR.
atom owing probably to dynamic disorders.
G
strands and
G helices in the prototypical tetrameric 3
,20
-HSD are shown in Fig. 2, a and b. The four-helix
bundle, conserved in dimeric SDRs such as 17
-HSD1 shown in Fig.
2c, conceals most of the hydrophobic surfaces that are at
dimer interfaces (8). Efforts to disrupt the Q axis interface by
mutations resulted in inactive and insoluble monomers (18).
Furthermore, the four-helix bundle is considered critical to function,
since disruption of the helical bundle interferes with the integrity of
the two active sites it borders, as shown in Fig. 2, a and
c. The P axis interface does not involve significant
hydrophobic interactions (8) and is less critical to the structural
integrity of the active site.
E and
F. Helices
F'-1 and
F'-2 and the Trp-276
side chain from the C-terminal end of the new strand
H surround
F
such that four of its seven turns are shielded from solvent exposure.
The four and a half turn helix
F'-3, the longest in the subdomain,
partially shields
F and packs over five of the seven turns of
E,
as shown in Fig. 2d. Together,
F'-2,
F'-3, and
F'-4
occupy the position of symmetry-related helices
E and
F in
3
,20
-HSD, 17
-HSD1, and 7
-HSD, blocking the interface and
preventing dimer formation. However, as shown in a close-up view of the
area in Fig. 2d, this arrangement does not completely shield
the interface leaving
E and
F partially exposed. Selective substitutions of residues on these helices in PTCR render the exposed
surface more hydrophilic than in oligomeric SDRs, as shown in Fig.
2d (substitutions from 3
,20
-HSD to PTCR are as
follows: Phe-116 to Arg-118, Thr-121 to Glu-123, Val-168 to Arg-209,
Gly-171 to Arg-212, and Thr-172 to Glu-213). All four helices of the
insertion domain are amphipathic; their hydrophobic surfaces face the
outer hydrophobic surfaces of
E and
F, and their hydrophilic
surfaces are exposed to the solvent, as shown for
F'-3 in Fig.
2d. Interestingly, the inner hydrophobic surface of
F'-3
contains Met-171 which packs against Met-110, in close proximity to the
Met-115 side chain, both from
E. The S
atom of Met-171 is at a
distance of 3.76 Å from S
of Met-110, which is 6.04 Å from
S
of Met-115. This partially exposed "methionine trap" is
responsible for the high affinity binding of platinum ions (mono- and
di-platinate) to the crystalline protein that resulted in the only
useful isomorphous derivatives for solving the phase problem (see under
"Experimental Procedures").
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Fig. 3.
a, a stereo view of the bound NADP
molecule, within its final (2Fo Fc) electron density map. The map is contoured at
1
. Side chains in the surrounding neighborhood, including the ones
that have direct contact with the coenzyme, are shown. The color codes
for atoms are the same as in Fig. 1. Three-letter codes are used to
identify amino acids. W denotes a water molecule. All the
direct contacts with NADP are shown schematically in b, and
distances are given in Å. Residues that are in the immediate
surroundings but do not have any direct contact are also shown.
-NADPH has been
established. At pH 7, PTCR has the highest affinity for
-NADPH as
evidenced by the lowest Km of 7.2 µM
and the highest Vmax/Km ratio
of 16.2 nmol min
1
mg
1
µM
1 of all coenzymes (6). In
the crystal structure, we find that the negatively charged 2'-phosphate
moiety is surrounded by three positively charged side chains (Arg-37,
Arg-41, and Lys-14), one hydrogen bond forming polar side chain
(Asn-13), and a water molecule. All of these make specific contacts
with the 2'-phosphate group, either through hydrogen bond formation or
electrostatic charge neutralization, as shown in Fig. 3. The Asn-13
side chain carbonyl also accepts a proton from the 3'-hydroxyl of the
adenine ribose. In addition to the presence of charged Arg and Lys
residues near the 2'-phosphate of NADPH noted before (13, 29), specific interactions of the Asn-13 side chain with phosphate and ribose groups
may play an important role in coenzyme selectivity. The location of
Asp-62 near the amide group of the adenine ring is analogous to the
Asp-60 in 3
,20
-HSD (8) and the Asp-68 in 7
-HSD (14). The
carboxamide group of the nicotinamide ring interacts with the main
chain atoms of Val-230, whereas the Thr-232 side chain makes a hydrogen
bond with the bridging pyrophosphate. Interestingly, a sulfhydryl group
from Met-234 is also present in the general vicinity, as has been
observed in other SDR structures (Met-189 in 3
,20
-HSD and Met-193
in 17
-HSD1). Two water molecules are hydrogen-bonded to the phosphates.
D and
E (Ile-92 to Pro-101) about a 2-fold rotation axis.
Because of the openness of the active site cleft, the loops penetrate each other's active site and the Asp-97 side chain from one loop makes
two strong hydrogen bonds to the catalytic residues Tyr-193 and Ser-139
(2.57 and 2.58 Å, respectively) of the other molecule, as shown in
Fig. 4a. Interaction of the
hydroxyl group of the catalytic Tyr with a carboxyl acid group was
previously observed in the structure of the carbenoxolone complex of
3
,20
-HSD (31), in which the hemisuccinate side chain of the
inhibitor formed a strong hydrogen bond with the Tyr-152 hydroxyl. The
main chain amide of Asp-97 makes a hydrogen bond (2.96 Å) with Glu-141
O
2 from a symmetry-related molecule. The 2-fold rotation axis
passes through the center of two Gln-95 side chains, whose O
1 atoms are 3.48 Å apart and make hydrogen bonds (2.84 Å) with the main chain
amides of Leu-96s of symmetry-related molecules. The Leu-96 side chain
stacks against the Ala-93 and Tyr-193 side chains, and Asn-98 side
chain stacks against the Trp-229 side chain.
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Fig. 4.
a, interactions between two PTCR
monomers related by a crystallographic 2-fold rotation axis. The Asp-97
side chain of one monomer forms hydrogen bonds with catalytic Tyr-193
and Ser-139 side chains of the other (distances are 2.57 and 2.58 Å,
respectively). NADP and other side chains have been removed for
clarity. The backbones of two loops between D and
E are shown.
The view is down the 2-fold axis. b, a close-up of the
superposition of a section of this loop for four SDRs, illustrating the
difference in its conformation between PTCR and other SDRs. The
backbone atoms of PTCR are color-coded, whereas those of
3
,20
-HSD, 17
-HSD1, and 7
-HSD are shown in green,
blue, and magenta, respectively. c,
interaction of the free carboxyl end of one monomer with the Arg-37
side chain of the other. The Arg-37 side chain also have specific
contacts with 2'-phosphate of the bound NADP molecule.
,20
-HSD, 17
-HSD1, and 7
-HSD is characterized by the presence of a distorted 310 helical turn (residues
95-99 in 3
,20
-HSD, 98-102 in 17
-HSD1, and 103-106 in
7
-HSD), which is absent in PTCR. The loop adopts an extended
conformation in PTCR. The distances between C
-99 and the
equivalent C
-95 in 3
,20
-HSD, C
-98 in 17
-HSD1, and
C
-103 in 7
-HSD are 12.02, 9.33, and 8.59 Å, respectively. It is
not clear whether this difference in conformation of the loop results
from the interaction of the Asp-97 side chain with catalytic residues
or if this altered conformation leads to dimerization.
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FOOTNOTES |
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* This work was supported in part by Grant DK26546 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1HU4) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ To whom correspondence should be addressed: Hauptman-Woodward Medical Research Institute, 73 High St., Buffalo, NY 14203. Tel.: 716-856-9600 (Ext. 316); Fax: 716-852-6086; E-mail: ghosh@hwi.buffalo.edu.
Present address: Center for Advanced Research in
Biotechnology, Rockville, MD.
Published, JBC Papers in Press, March 8, 2001, DOI 10.1074/jbc.M100538200
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ABBREVIATIONS |
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The abbreviations used are:
SDR, short
chain dehydrogenases/ reductases;
PTCR, porcine testicular carbonyl
reductase;
20-HSD, 20
-hydroxysteroid dehydrogenase;
PIP, di-µ-iodobis(ethylenediamine)diplatinum(II) nitrate;
PtCl4, potassium chloroplatinate;
CR, carbonyl reductase;
MES, 4-morpholineethanesulfonic acid.
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