(Received for publication, June 23, 1995; and in revised form, October 2, 1995)
From the
The binding modes of three peptidomimetic P-P
butanediamide renin inhibitors have been determined by x-ray
crystallography. The inhibitors are bound with their backbones in an
extended conformation, and their side chains occupying the S
to S
` pockets. A (2-amino-4-thiazolyl)methyl side
chain at the P
position shows stronger hydrogen-bonding and
van der Waals interactions with renin than the His side chain, which is
present in the natural substrate. The ACHPA-
-lactam transition
state analog has similar interactions with renin as the
dihydroxyethylene transition state analog.
The aspartic protease renin plays an important role in the regulation of blood pressure by catalyzing the release of the decapeptide angiotensin I from angiotensinogen(1) . Removal of the two C-terminal residues from angiotensin I, catalyzed by the angiotensin-converting enzyme, produces the physiologically active octapeptide angiotensin II. Inhibitors of ACE have become successful therapeutic antihypertensive agents(1) . However, the angiotensin-converting enzyme inhibitors produce unwanted side effects in treatment and have only a 50% response rate in monotherapy(2) , clearly indicating the need for other therapeutic agents. The inhibition of renin represents a possible alternative for developing successful antihypertensives.
The
cleavage in human angiotensinogen catalyzed by renin occurs between
residues 10 and 11 in the sequence
His-Pro-Phe-His-Leu-Val
-Ile. Compounds
containing a butanediamide backbone at the P
-P
positions (3) are potent peptidomimetic inhibitors of
human renin (Fig. 1). (
)They contain either the
dihydroxyethylene (4) (inhibitors 1 and 2) or the
ACHPA(
)-
-lactam (5) (inhibitor 3) as the
transition state analog occupying the P
and P
`
positions(3) . The His side chain in angiotensinogen at the
P
position is replaced with a (2-amino-4-thiazolyl)methyl
group.
Figure 1:
Chemical structures and potencies of
three P-P
butanediamide renin inhibitors.
IC
values are measured at pH 6.0 using purified
recombinant human renin.
Crystal structures of free and inhibited human renin have
been reported previously at medium
resolution(6, 7, 8) . We recently reported
the crystal structure at 1.8-Å resolution of human renin in
complex with a polyhydroxymonoamide inhibitor(9) . In this
paper we describe the binding modes of the P-P
butanediamide renin inhibitors as determined by x-ray crystallography
and compare their binding interactions with those of other inhibitors (7, 9
At this point reflection data to 1.8-Å resolution became available for human renin in complex with a polyhydroxymonoamide inhibitor, in an isomorphous crystal form(9) . The refined model of renin in complex with 1 was used as the initial model for the refinement at 1.8-Å resolution. This produced a more accurate atomic model for renin (9) . Consequently, the structure refinement of the inhibitor 1 complex was restarted using the 1.8-Å renin model. This 1.8-Å structure of renin was also used as the starting model in the structure refinement of renin in complex with inhibitors 2 and 3. The final atomic models were obtained after one cycle of refinement with the program TNT (Fig. 2)(17) . The refinement statistics of the three structures are summarized in Table 1.
Figure 2:
Final
2F-F
electron density map for inhibitor 1 in complex with renin in the
closed conformation. Reflection data between 20- and 2.4-Å
resolution were used in the calculation. The contour level is at 1 root
mean square deviation above the mean of the electron density
values.
The crystal structures of recombinant human renin in complex
with three different P-P
butanediamide
inhibitors are presented in this paper. The crystallographic data and
the refinement statistics are summarized in Table 1. The
structure of human renin in complex with inhibitor 1 is at the
higher resolution of 2.4 Å, whereas the other two structures are
at medium resolutions of 2.7 and 2.8 Å. The atomic coordinates
have been deposited at the Brookhaven Protein Data Bank.
There are two independent renin complexes in each crystal. As has been observed in our earlier study(9) , these two renin inhibitor complexes adopt different conformations. One has a closed conformation in which residues in the C-terminal domain (198-204, 224-259, and 269-286; pepsin numbering) are closer to the N-terminal domain and the inhibitors, whereas the other renin molecule has an open conformation. Similar conformational differences have also been observed in other aspartic proteinases(18) .
For structural
comparisons, the six renin complexes from the three crystal structures
were superimposed. The renin molecule in complex with inhibitor 1 in the closed conformation was used as the reference in this
superposition. The C positions of residues in the
N-terminal domain of renin were used to calculate the transformation
matrix. This superposition based on the renin portion of the complex
also led to a general overlap of the inhibitors (Fig. 3). The
observed variation in the position of the inhibitors (Fig. 3) is
due partly to the differences among the inhibitors in their
interactions with renin. This variation may also be attributable to the
limited resolution of the current structures. Our earlier study of
renin-inhibitor complexes at high resolution showed much closer overlap
of the inhibitors for the P
, P
, and
P
` residues(9) .
Figure 3:
Stereo diagram showing the overlap of the
three P-P
butanediamide inhibitors as bound to
renin. Inhibitors 1, 2, and 3 as bound to renin in the closed
conformation are shown in white, green, and pink, respectively. The
corresponding inhibitors as bound to renin in the open conformation are
shown in cyan, brown, and purple,
respectively.
The inhibitor molecules are
bound in a groove between the N- and C-terminal domains of renin. The
backbones of the inhibitors are in an extended conformation. The side
chains occupy the S to S
` substrate binding
pockets. The pattern of hydrogen bonding between the polar atoms in the
backbone of the inhibitors and renin is similar to that reported for
other peptidomimetic renin
inhibitors(7, 8, 9) . The absence of a
P
amido nitrogen in the butanediamide backbone results in
the loss of a hydrogen bond to the side chain of
Thr
(7) . The two carbonyl oxygen atoms of the
butanediamide backbone are located at similar positions and maintain
similar hydrogen bonding interactions to renin as the carbonyl oxygen
atoms of the P
and P
residues in a peptide
substrate (see below).
The pyridylethyl group in inhibitor 2 is exposed to solvent and has weak electron density, suggesting it
may also be flexible. The pyridyl ring is folded close to the P phenyl ring of the inhibitor and occupies the S
pocket (Fig. 3). The lack of additional interactions of
this pyridyl group with renin is consistent with the observation that
this compound is as potent as inhibitor 1 (Fig. 1). The
pyridyl group of the inhibitor bound to the renin molecule in the open
conformation is involved in crystal-packing interactions, which may
explain its positional difference from that of the pyridyl ring in the
inhibitor bound to the renin molecule in the closed conformation (Fig. 3).
The plane of the amide group of the P residue is perpendicular to that of the P
residue in
all three inhibitors (Fig. 3). The N-methyl group is
pointed toward the side chain of Tyr
in the S
pocket. In comparison with the structures of renin in complex
with polyhydroxymonoamide inhibitors(9) , which lack a P
group, the side chain of Tyr
rotates by about
20° and 60° across the
and
torsion angles to avoid steric contact with this methyl group (Fig. 4). This change in the conformation of the Tyr
side chain is observed in both the open and the closed
conformations of renin for all three inhibitors. The orthogonality of
the amide planes of the P
and P
residues
projects the methyl group deeper into the S
pocket as
compared with the P
residue in the compound CGP 38`560 (Fig. 5)(7) . A smaller change in the position of the
Tyr
side chain is observed in the latter complex, which
is in the closed conformation.
Figure 4: A, stereo diagram showing the overlap of residues 219-222 and 286-290 of renin in complex with inhibitor 1 (thin solid lines) and the polyhydroxymonoamide inhibitor 4 (thin gray lines)(9) . The corresponding inhibitors are shown in thick solid and gray lines, respectively. B, the chemical structures of inhibitor 4 and CGP 38`560(7) .
Figure 5: Stereo diagram showing the overlap of inhibitor 1 (thick lines), the polyhydroxymonoamide inhibitor 4 (thin lines)(9) , and the inhibitor CGP 38`560 (gray lines)(7) .
In contrast to earlier structure
studies(7, 9) , which showed the phenyl group at the
P position having a common binding orientation (Fig. 5), the P
phenyl groups of the three
inhibitors studied here assume a variety of orientations, due mostly to
changes in the
torsion angle (Fig. 3). The
methyl group on the benzylic carbon of the P
side chain is
projected into the solvent. The side chains in these inhibitors are
connected to a planar amido nitrogen atom rather than a tetrahedral
carbon as in other peptidomimetic inhibitors(7, 9) .
Consequently, the side chain enters the S
binding pocket
from a different direction as compared with the inhibitors with a
tetrahedral carbon (Fig. 5). The amino acid side chains of renin
forming the S
pocket show no significant differences among
the structures.
The P group of the polyhydroxymonoamide
inhibitors in our earlier study (9) is the smaller
cyclopropylmethyl group. A water molecule was observed at the base of
the S
pocket in those structures. In the current
structures, the larger aminothiazole ring fills the S
pocket more fully. The amino group displaces the water molecule
observed in the earlier structures (9) and is hydrogen-bonded
to the side chain hydroxyl group of Ser
and the main
chain carbonyl group of Tyr
(Fig. 4). The sulfur
atom of the aminothiazole ring is surrounded mostly by side chains of
hydrophobic residues (Ala
, Ile
,
Met
, and Leu
). The higher polarizability of
the sulfur atom may probably give rise to stronger van der Waals
interactions with these side chains. The bulkier sulfur atom, and
possibly the additional hydrogen-bonding interactions of the amino
group, also cause a shift in the position of the P
(2-amino-4-thiazolyl)methyl residue as compared with the position
of the P
His residue in the CGP 38`560 complex (Fig. 5).
The change in conformation of the Tyr side chain due to the P
residue of these inhibitors
is coupled with a change in conformation of the His
side
chain, which was observed to be located close to the Tyr
side chain and away from the S
pocket in the earlier
structures(9) . In the renin molecule with the open
conformation, a small movement is observed for the His
side chain to avoid steric contact with the Tyr
side chain. The His
side chain in the new position
still maintains interactions with the side chain of Asp
.
In the renin molecule with the closed conformation, due to the
proximity of residues 243-245, the His
residue
undergoes a large conformational change (including a change in
of 140°). Its side chain is located close to the
S
pocket in the new position, where it interacts with the
ring nitrogen of the thiazolyl group through a water molecule at the
opening of the S
pocket (Fig. 4). Two other polar
atoms, the main chain amido nitrogen of Tyr
and the
carbonyl oxygen of the P
residue, complete the tetrahedral
coordination of this water molecule (Fig. 4). A change in the
position of the Met
side chain is also observed (Fig. 4).
In the structure of renin in complex with the
inhibitor CGP 38`560(7) , the His side chain was
found to be close to the S
pocket, interacting with a water
molecule at the opening of the S
pocket. The hydrogen bond
between this water and the His side chain of the inhibitor, however,
was not observed. A water molecule at the opening of the S
pocket was also observed in our earlier study, where the
His
is away from the S
pocket (Fig. 4)(9) .
The dihydroxyethylene transition state
analog in the P and P
` positions is bound in a
conformation similar to that observed in the earlier study ( Fig. 3and Fig. 4)(9) . The first hydroxyl group
of the diol is located between the catalytic aspartic acid residues 32
and 215. The ACHPA-
-lactam transition state analog in inhibitor 3 occupies similar spatial positions as the dihydroxyethylene
analog (Fig. 3). As predicted from modeling studies(5) ,
the gem-dimethyl group on the lactam ring superimposes with
the isopropyl group of the diol analog, mimicking the Val side chain in
the natural substrate. The carbonyl oxygen atom of the lactam occupies
a position similar to that of the second hydroxyl group of the
dihydroxyethylene transition state analog (Fig. 3).
These
P-P
butanediamide inhibitors are about 40-fold
more potent than the polyhydroxymonoamide inhibitors of
renin(9) . The crystal structures show that the
P
-P
butanediamide inhibitors have stronger
interactions with renin at the P
position and additional
interactions due to the P
residue. These may explain the
increased potency of the inhibitors. The contribution of the P
residue is difficult to evaluate, and it is not clear whether the
two different series of inhibitors have similar interactions with renin
at this position.
The atomic coordinates and structure factors (codes 1BIL and 1BIM) have been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.