(Received for publication, April 4, 1995; and in revised form, July 31, 1995)
From the
Complement factor D is a serine protease regulated by a novel
mechanism that depends on conformational changes rather than cleavage
of a zymogen for expression of proteolytic activity. The conformational
changes are presumed to be induced by the single natural substrate,
C3bB, and to result in reversible reorientation of the catalytic center
and of the substrate binding site of factor D, both of which have
atypical conformations. Here we report that replacement of
Ser, Thr
, and Ser
of factor D
(chymotrypsinogen numbering has been used for comparison purposes) with
the corresponding residues of trypsin, Tyr, Ser, and Trp, is sufficient
to induce substantially higher catalytic activity associated with a
typical serine protease alignment of the catalytic triad residues
His
, Asp
, and Ser
. These
results provide a partial structural explanation for the low reactivity
of ``resting-state'' factor D toward synthetic substrates.
Activation of complement leads to expression of important host defense functions and proceeds via pathways that consist of successive enzymatic amplification steps. In the alternative pathway, the first enzymatic reaction is catalyzed by factor D, a serine protease that cleaves factor B only in the context of a complex with C3b, a fragment of the third component of complement, C3(1) . Factor D is unique among serine proteases in that it requires neither enzymatic cleavage for expression of proteolytic activity nor inactivation by an inhibitor for its control. Regulation of factor D activity in blood is apparently attained by a novel mechanism that depends on conformational changes and allows for reversible expression of proteolytic activity(2, 3) . The putative conformational changes are believed to be induced by the natural substrate, C3bB, and to result in reorientation of the catalytic center and of the substrate binding site of factor D, both of which have atypical structures(4) . Functional support for this hypothesis has been provided by the seemingly parodoxical observation that, while the reactivity of factor D with thioester substrates and active site inhibitors is 3-4 orders of magnitude lower than that of typical serine proteases(5, 6) , its proteolytic activity during complement activation is comparable to that of other complement enzymes. Obviously, esterolytic assays assess the resting-state inactive conformation of the enzyme, while complement activation assays the substrate-induced proteolytically active conformation. Structural support for the inactive resting-state conformation hypothesis has been provided by the recently determined crystal structure of factor D(4) .
The two non-crystallographically related molecules, A
and B, present in the triclinic unit cell of factor D have typical
serine protease structural folds. However, they display distinctive
orientations of the side chains of the catalytic triad residues
Asp and His
(chymotrypsinogen numbering (
)has been used throughout this paper), while the
orientation of the third member of the triad, Ser
, is
similar to that of other serine proteases. In all serine proteases of
known structure, the spatial relationships of these three residues are
constant and essential for the formation of a functional unit
responsible for catalytic
activity(7, 8, 9, 10) . By contrast,
in molecule A of factor D, the carboxylate of Asp
is
pointed away from His
and is freely accessible to the
solvent. In molecule B, the imidazolium of His
is oriented
away from Ser
, having assumed the energetically favored trans conformation(4) . Neither of these orientations
would allow expression of catalytic activity, indicating the need for a
realignment of the catalytic triad residues, probably induced by the
single natural substrate, C3b-complexed factor B. A possible structural
explanation for the unusual disposition of Asp
/His
in factor D is provided by the substitution of a Ser for the
bulky aromatic Tyr or Trp residue usually present at position 94 of
serine proteases and also of a Thr for the invariant Ser
(Fig. 1). Both of these residues are among those
identified by Blow et al.(7) as being important for
shielding Asp
from solvent molecules in chymotrypsin. In
addition, a Ser residue that substitutes for the conserved aromatic Trp
or Phe at position 215 may also be a determinant of the unique
conformation of resting-state factor D. Indeed, in molecule B of factor
D, Ser
is positioned between Asp
and
Ser
, preventing His
from assuming the active gauche conformation that is characteristic of serine
proteases. Here we report that replacement of Ser
,
Thr
, and Ser
of factor D with the
corresponding residues of trypsin, Tyr, Ser, and Trp, is sufficient to
induce typical serine protease alignment of the catalytic triad
residues and substantially higher catalytic activity. These results
provide a structural exegesis for the unique conformation of the active
center of resting-state factor D and also for its low reactivity with
synthetic esters. The data also demonstrate the previously unrecognized
importance of residue 94 of serine proteases for catalysis.
Figure 1:
Alignment of partial amino acid
sequences of selected serine proteases. Residues participating in
shielding the catalytic residue Asp from the solvent (7) are shaded. Conserved residues are boxed. Asterisks indicate amino acid residues subjected to
site-directed mutagenesis. Numbers at the top are for
residues of the chymotrypsinogen sequence, while the numbers at the bottom are for the human factor D residues. HFD, human factor D; TRP, bovine pancreas trypsin; CHT, bovine pancreas chymotrypsin; ELA, porcine
pancreas elastase. Sequence data are from (37) , except for the
human factor D sequence, which is taken from (11) .
Four mutant factor D cDNAs, S94Y, S94Y/T214S, S94Y/S215W, and
S94Y/T214S/S215W, were constructed for these studies. The mutant and wt
factor D cDNAs were stably expressed in CHO cells, and the recombinant
proteins were purified to homogeneity. The effects of the mutations on
the catalytic efficiency of resting-state factor D were evaluated by an
esterolytic assay using the thioester substrate Z-Lys-SBz1(12) . Results of multiple experiments are
summarized in Table 2. Previously reported results for the T214S,
S215W, and T214S/S215W mutants of factor D (12) have been
included for comparison. As shown, mutation of Ser to Tyr
in the single S94Y mutant resulted in about 6-fold increase of the
catalytic rate constant (k
). Larger increases in k
were observed when the S94Y mutation was
combined with mutations of Thr
to Ser and of Ser
to Trp. The triple mutant S94Y/T214S/S215W had a k
more than 16-fold higher than wt factor D and
only 3.5-fold lower than trypsin (Table 2). The enhanced k
of all S94Y mutants and of the double
T214S/S215W mutant resulted in increased overall catalytic efficiency
as assessed by the k
/K
ratio, although the K
was affected only
slightly if at all.
The proteolytic activity of mutant factor D was assessed by a sensitive hemolytic assay, and the results (Table 3) were in general agreement with those obtained with the esterolytic assay. The S94Y mutant had higher hemolytic activity than wt factor D, and its activity was synergistically increased in combinations with the S215W and the double T214S/S215W mutations. As expected, all mutants cleaved factor B complexed with cobra venom factor, a C3b analogue of cobra venom, but none cleaved uncomplexed factor B (data not shown).
To investigate the structural correlates
of the increased catalytic activity of mutant factor D, we determined
the crystal structure of the triple mutant S94Y/T214S/S215W. The model
has been refined to an R-factor of 19.8% (Table 1) and
is in agreement with the most favored region of a Ramachandran plot (28) (Fig. 2). Fig. 3shows the electron density
for the mutated residues 94, 214, and 215 in a difference map
calculated by using molecular replacement phases (8.0 to 3.0 Å).
Plots of mean temperature factors (B) per residue and of
real-space density fit per residue (R) are shown in Fig. 4(a and b, respectively). The highest B-factors and worst real-space R-factors are observed
in the segment His-Ile
. In contrast, in
both molecules A and B of native factor D, the B-factors for
this segment were lower than the average values(4) . In typical
serine proteases residue 172 is a highly conserved aromatic (Tyr or
Trp), which is part of a type I
-turn and has been shown to be a
determinant of substrate specificity(29) . Thus, the apparent
flexibility of His
in the mutant may have an effect on
substrate binding. The second highest B-factors were observed
for the Asp
-Val
segment of mutant
factor D. This region is flexible in molecule A, but quite rigid in
molecule B of native factor D. Compared to chymotrypsin or trypsin,
this segment of factor D has 3 or 4 additional residues, respectively.
Figure 2: Ramachandran plot for the refined atomic model of the S94Y/T214S/S215W mutant. Most favored, favored, and additional allowed regions are indicated by progressively lighter shading. Disallowed regions are not shaded. Regions are marked according to the analysis of highly refined structures in the Protein Data Bank. Circles and diamonds represent nonglycine and glycine residues, respectively.
Figure 3:
Stereo representation of (F - F
) map calculated using 20.0 to
2.5-Å resolution data and molecular replacement phases. Side
chains of residues 94, 214, and 215 had been removed in the model used
for molecular replacement. Density is clearly seen for mutated residues
and for deleted solvent molecules.
Figure 4:
Quality of the S94Y/T214S/S215W structure. a, variation of the isotropic B values averaged
(Å) for the main chain (mc) and side chain (sc) atoms of each residue. b, real-space electron
density fit per residue (
R
) computed using
(2F
- F
) exp(i
)
map. The map was calculated using 20.0 to 2.0 Å resolution
data.
Overall, the tertiary structure of the S94Y/T214S/S215W mutant is
very similar to that of native factor D. The root mean square (r.m.s.)
deviations between the coordinates of main chain and side chain atoms
of the mutant and molecules A and B of factor D are shown in Fig. 5(a and b, respectively). Substantial
structural divergence between the mutant and both molecules of native
factor D is observed in two segments. The first includes residues
169-175, which in the mutant form a flexible loop thus making the
observed r.m.s. shifts an unreliable indicator of actual structural
differences. Furthermore, this segment partially overlaps the region
(residues 172-177) with the highest B-factors and worst
real-space R-factors (Fig. 4, a and b). Excluding residues 172-177, the r.m.s. shifts
between the mutant and molecule A (Fig. 5a) are 0.95
Å for main chain atoms and 1.02 Å for side-chain atoms. The
corresponding values for the comparison between the mutant and molecule
B are 0.86 Å and 1.59 Å. By comparison, the r.m.s. shifts
between molecules A and B of native factor D (4) are 0.96
Å for main chain atoms and 1.58 Å for side chain atoms. The
second segment with significant r.m.s. deviations between the mutant
and both molecules of factor D includes residues 214-224.
Residues 214-220 form one of the three walls that line-up the
primary specificity pocket of serine proteases(30) . In
addition, residues 214-216 are part of the S-S
subsite (
)that forms a
-pleated sheet with
residues P
-P
of the
substrate(31, 32, 33) . Two of these
residues, Thr
and Ser
, have been mutated,
which probably accounts at least in part for the observed r.m.s.
deviations. In addition, changes in the orientation of the side chains
of Arg
and Arg
apparently contribute to the
observed r.m.s. shifts. In native factor D Arg
forms a
salt bridge with Asp
at the bottom of the primary
specificity pocket (Fig. 6). This bridge probably contributes to
the low reactivity of resting-state factor D with synthetic substrates,
as it restricts access of the positively charged P
Arg
residue to the negative charge of Asp
. In the mutant,
Arg
seems to be flexible and its side chain points away
from the carboxylate of Asp
and toward the solvent.
Therefore, no salt bridge between this residue and Asp
can form. Instead, Arg
has been reoriented so that
its guanidinium group is H-bonded to Asp
(Fig. 6).
These changes are accompanied by a substantial narrowing of the primary
specificity pocket of the mutant, apparently due to a movement of the
segment formed by residues 214-218 (Fig. 6).
Figure 5: Coordinate shifts between the S94Y/T214S/S215W mutant and native factor D. The r.m.s. deviation (Å) of main chain (mc) and side chain (sc) atoms between the mutant and molecule A (a) and molecule B (b) of native factor D crystallized in the triclinic unit cell(4) . The two molecules are related by a non-crystallographic 2-fold in space group P1.
Figure 6:
Comparison of the primary specificity
pockets of the S94Y/T214S/S215W mutant (black) and molecule B
of native factor D (red). The catalytic residue Ser is shown at the toprightcorner and
Asp
, which in trypsin-like serine proteases forms a salt
bridge with the side chain of the P1 Arg, is shown on the right side of the pocket. The guanidino group of Arg
is
salt-bridged to Asp
in the native structure, but it is
oriented away from Asp
in the mutant. This shift is
compensated by a reorientation of Arg
, the side chain of
which is H-bonded to Asp
in the mutant. The largest
structural differences between the two structures are due to narrowing
of the pocket of the mutant.
Superposition of the mutant on molecule A results in substantial
r.m.s. shifts around the SerTyr substitution (Fig. 5a). Asp
of molecule A is pointed
toward the solvent away from His
at the active center of
factor D. In the mutant, Asp
is forced by the phenyl ring
of Tyr
to assume a position similar to that observed in
typical serine proteases (Fig. 6) and the entire loop joining
residues Val
and Leu
has been rearranged in
a way that resembles the corresponding region of trypsin. The movement
of loop 89-103 is mainly responsible for the observed average
shift of 5.0 Å for main chain atoms and 8.2 Å for side
chain atoms of this segment (Fig. 5a). In molecule B of
factor D, the orientation of Asp
and of the entire loop
89-103 is similar to that of trypsin, which accounts for the lack
of large r.m.s. shifts between molecule B and the mutant (Fig. 5b). However, the imidazolium of
His
, which has an atypical trans orientation in
molecule B of native factor B, assumes the catalytically active gauche orientation in the mutant. This movement is probably
responsible for the r.m.s. shifts observed in this region of the plot
shown in Fig. 5b.
Fig. 7summarizes
graphically the main structural differences between native and mutant
factor D. The catalytic triad residues Asp,
His
, and Ser
of both molecules A and B
present in the triclinic unit cell of native factor D have an atypical
alignment inconsistent with catalysis. In molecule A, Asp
is turned away from His
, having gained access to the
solvent, while in molecule B His
is pointed away from
Ser
and Ser
is occupying the space usually
occupied by His
. In contrast, in S94Y/T214S/S215W the
catalytic triad residues display an orientation very similar to that of
trypsin. This typical serine protease conformation of the catalytic
triad probably accounts for the increased catalytic activity of the
mutant compared to native factor D.
Figure 7:
Active site regions of molecule A (MOLA), and molecule B (MOLB) of native factor D,
bovine trypsin, and factor D S94Y/T214S/S215W mutant (STS).
The models were generated using the Ribbons program(38) . Green is used for carbon, blue for nitrogen, and red for oxygen. The backbone is represented as a tube, and bonds joining the side-chain atoms C onward are
shown as cylinders. Each model shows the orientation of the
catalytic triad and of residues in the immediate vicinity. Bovine
trypsin coordinates were obtained from the Brookhaven Protein Data
Bank.
The present data clearly demonstrate that three residues,
Ser, Thr
, and Ser
, are
responsible for the atypical orientation of the catalytic triad of
native factor D. Replacement of these three residues with those present
in the corresponding positions of trypsin, Tyr
,
Ser
, and Trp
, resulted in a mutant enzyme
with about 20-fold higher reactivity toward the synthetic thioester Z-Lys-SBzl than native factor D. The increased catalytic
efficiency could be accounted for by an increase in k
and could be attributed to structural changes of the catalytic
center. Most importantly, in contrast to native factor D, the side
chains of the catalytic residues Asp
and His
of the S94Y/T214S/S215W mutant have a typical serine protease
orientation very similar to that seen in trypsin (Fig. 7).
Possible lattice effects were considered before concluding that the
observed trypsin-like conformational changes could be attributed
directly to the mutations. The triclinic unit cell of native factor D
contains two non-crystallographically related molecules, whereas the
triple mutant crystallizes in a different space group with one molecule
in the asymmetric unit. Therefore, different lattice forces than those
exercised on either molecule A or B of native factor D should be
expected to impinge on the mutant, possibly contributing to the
observed realignment of key catalytic residues. However, we have
recently crystallized native factor D in space group P2 with one molecule in the asymmetric unit. The structure of this
crystal form of native factor D has been determined by a combination of
isomorphous replacement and molecular replacement methods. The model
has been refined to an R-factor of 18.8% by using 8.0 to 2.0
Å resolution data. The catalytic triad of this molecule has a
conformation similar to that present in molecule B of the triclinic
cell. Similar conformations also have been observed in inhibitor
complexes of factor D. It thus seems unlikely that lattice forces are
major contributors to the observed structural differences between
native and triple mutant factor D.
The spatial relationships of the
three catalytic residues are constant in all serine proteases of known
structure and are stabilized by a network of H
bonds(7, 34) . Specifically, H bonds between the
N1 of His
and the O
2 of Asp
, the
N
2 of His
and the O
of Ser
, and
the O
2 of Asp
and O
of Ser
that
are present in typical serine proteases are also present in the mutant
factor D. In contrast the orientation of the side chains of these
residues in native factor D precludes the formation of these H bonds.
The integrity of the H bonds and the hydrophobic nature of the
environment surrounding the buried Asp
are essential for
the formation of a functional unit responsible for bond formation and
cleavage during
catalysis(7, 8, 9, 10) . Hence, the
proposal for a substrate-induced conformational change of the active
center of factor D to explain the efficient activation of the
alternative complement pathway(2, 3, 5) .
All three mutated residues, Ser, Thr
, and
Ser
, apparently contribute to the unusual conformation of
the catalytic triad of native factor D and the resulting low reactivity
toward small synthetic thioester substrates. However, Ser
seems to be the principal determinant as indicated by the larger
effect of the S94Y mutation on the k
for Z-Lys-SBzl than those obtained for the single T214S and S215W
mutants (Table 2). The introduction of the bulky Tyr residue at
position 94 is probably responsible for forcing Asp
and
His
to assume an active conformation. This is suggested by
the r.m.s. deviation plot (Fig. 5a), which shows
substantial structural differences in the immediate vicinity of the
S94Y substitution when the mutant is superposed on molecule A of factor
D.
A contribution of Thr is clearly indicated by the
synergistic gain in k
(Table 2) observed
when the T214S mutation is combined with the S94Y and S215W mutations.
This effect is probably due to the formation of a H bond between the
side chains of Ser
and Asp
, which helps
stabilize Asp
. A role for the highly conserved aromatic
at position 215 of serine proteases in catalysis has never been probed
before. This residue is part of the S
-S
subsite
in trypsin(33) , which forms a
-pleated sheet with
residues P
-P
of the substrate. In trypsin,
Trp
is involved in the formation of a hydrophobic cluster
that also includes Tyr
, a structural determinant of
substrate specificity(29) . However, in the S94Y/T214S/S215W
mutant loop 172-177 is flexible, precluding a hydrophobic
interaction between His
and Trp
. Instead,
Trp
is oriented in a way favoring hydrophobic
interactions with His
(Fig. 7). Regardless of its
precise structural contribution, the S215W mutation had a significant
effect on catalytic efficiency against Z-Lys-SBzl (Table 2).
An additional interesting structural difference
between S94Y/T214S/S215W and native factor D involves
Arg. In both molecules of factor D, the guanidinium group
of this residue forms a salt bridge with the carboxyl of
Asp
(4) . In trypsin and presumably also in all
trypsin-like serine proteases, the carboxyl of Asp
forms
a salt bridge with the side chain of the P
Arg or Lys
residue of the substrate(35) . This ionic interaction plays a
major role in positioning the scissile bond of the substrate for
hydrolysis. Thus, the Arg
-Asp
bridge of
native D has been considered an important contributor to the low
reactivity of resting-state factor D toward small synthetic
substrates(4, 12) . Furthermore, it has been suggested
that a reorientation of the side chain of Arg
is an
important component of the putative substrate-induced conformational
change that leads to efficient proteolysis of C3b-bound factor B by
factor D(4, 12) . Interestingly, mutation of
Arg
to Gly, which is present in the corresponding
position of trypsin, combined with deletion of Val
, which
is absent from trypsin, resulted in a small increase of esterolytic
activity and an almost complete loss of proteolytic
activity(12) . Indeed, even extensive mutations involving the
substrate binding pocket and associated structural elements, which
yielded a mutant factor D exhibiting 100-fold higher esterolytic
activity than native factor D were associated with extremely low
proteolytic activity attributable to the R218G and V219
mutations (36) . These findings were interpreted to indicate a direct
interaction between Arg
and determinants on the C3bB
complex, the natural substrate of factor D.
The present finding that
the realignment of the catalytic triad residues induced by the
S94Y/T214S/S215W mutations is associated with a reorientation of
Arg away from Asp
indicates a linkage among
the conformations of these elements of the active center. It thus seems
possible that the proposed interaction between Arg
and
negatively charged residue(s) on C3bB not only makes Asp
available to the P
Arg
of factor B, but
also contributes to the realignment of the catalytic triad residues.
Obviously, additional conformational changes particularly of the
substrate binding pocket are necessary for expression of efficient
proteolytic activity. These changes were not induced by the mutation of
the three residues investigated in the present study, as indicated by
the geometry of the primary specificity pocket (Fig. 6) and the
high K
of the triple mutant (Table 2).