(Received for publication, September 13, 1995; and in revised form, October 13, 1995)
From the
Inhibitors that belong to the serine protease inhibitor or
serpin family have reactive centers that constitute a mobile loop with
P1-P1` residues acting as a bait for cognate protease. Current
hypotheses are conflicting as to whether the native serpin-protease
complex is a tetrahedral intermediate with an intact inhibitor or an
acyl-enzyme complex with a cleaved inhibitor P1-P1` peptide bond. Here
we show that the P1` residue of the plasminogen activator inhibitor
type 1 mutant (P1` Cys) became more accessible to radiolabeling in
complex with urokinase-type plasminogen activator (uPA) compared with
its complex with catalytically inactive anhydro-uPA, indicating that
complex formation with cognate protease leads to a conformational
change whereby the P1` residue becomes more accessible. Analysis of
chemically blocked NH termini of serpin-protease complexes
revealed that the P1-P1` peptide bonds of three different serpins are
cleaved in the native complex with their cognate protease. Complex
formation and reactive center cleavage were found to be rapid and
coordinated events suggesting that cleavage of the reactive center loop
and the subsequent loop insertion induce the conformational changes
required to lock the serpin-protease complex.
The serine protease inhibitor, or serpin ()superfamily, is a family of structurally related proteins
that controls many physiological reactions and includes most of the
protease inhibitors in
blood(1, 2, 3, 4) . The serpins have
a unique inhibitory specificity, but they share a common molecular
architecture based on a dominant five-stranded A
-sheet(5) . In intact serpins, the reactive center
constitutes an exposed mobile loop with the P1-P1` residues acting as a
bait for the target protease(2, 6) . In cleaved forms
of serpins the P1 and P1` residues are located on opposite ends of the
molecule indicating that reactive center loop cleavage results in
insertion of the reactive center loop into the A
-sheet of the
molecule(5) . Structural data for native serpins complexed to
protease are lacking, and the mechanisms by which serpins inhibit their
target proteases remain unclear. Existing
hypotheses(4, 7, 8, 9, 10, 11, 12) assume
that serpins contain an exposed reactive center loop but vary in the
way they predict how the reactive center loop is held in an inhibitory
conformation. To acquire a canonical shape necessary for effective
inhibition, the reactive center loop appears to partially insert itself
into the gap between strands 3 and 5 of the A
-sheet(2, 10, 13) . This insertion seems
to be essential for inhibitor function but may not be required for
protease recognition(14) . After docking with enzyme, the
reactive center loop is presumed to insert further into the A
-sheet forming a structure that fits the substrate binding site of
the target protease and thereby achieving tight binding and
inhibition(3, 15) .
Analysis of serpin-protease
complexes by SDS-PAGE reveals the presence of a complex stabilized by
an acyl-ester linkage between the carbonyl of the P1 residue of the
cleaved serpin and the active site serine of the protease. However,
since denaturation may alter the stability of the complex and shift the
reaction toward cleavage this does not prove the existence of a cleaved
serpin in the native serpin-protease complex. Consistent with this
hypothesis, the complex between -antiplasmin
(
-AP) and non-cognate protease trypsin could
dissociate to give active inhibitor and enzyme(7) , and a
tetrahedral intermediate formed during complex formation between
porcine pancreatic elastase (PPE) and human
-antitrypsin (
-AT) was detected by
NMR(16) . Based on these studies it has been concluded that
complex formation stops at the tetrahedral intermediate stage with a
non-cleaved inhibitor and that the acyl-ester linkage detected after
treatment of complexes with SDS is an
artifact(4, 7, 16) . However, other data
suggest that the inhibitor is cleaved in the native serpin-protease
complex. Thus serpin-protease complexes have a biological activity
similar to cleaved serpin(8, 13) , and attempts to
detect intact antichymotrypsin after dissociation of the complex with
chymotrypsin were unsuccessful (17) .
By using fluorescence spectroscopy, we and others have shown that the reactive center of PAI-1 undergoes conformational changes following the interaction with target proteases(18, 19, 20, 21) . Our time-resolved fluorescence spectroscopy studies revealed that the orientational restriction of a fluorescent probe attached to the P1` residue decreased following complex formation with plasminogen activators (PAs) whereas it increased following complex formation with proteolytically inactive anhydro-uPA, indicating that the P1` residue became more flexible after complex formation with active protease. Although these data suggest that the P1-P1` bond of PAI-1 is cleaved in the complex with PAs(19, 20) , fluorescence studies only provide indirect evidence. In this study we provide direct evidence that the reactive center loop in PAI-1 as well as in two other serpins is cleaved in the native complex with their cognate proteases.
Figure 1:
Time course of complex
formation between C-labeled P1` Cys and uPA.
[
C]Iodoacetamide-labeled P1` Cys was mixed with
uPA, and at different time points complex formation was stopped. The
samples were analyzed by SDS-PAGE (3-20%) followed by Western
blotting with polyclonal antibodies against PAI-1 (A) or
autoradiography (B). Lane 1, radiolabeled P1` Cys
incubated with inactivated uPA; lanes 2-6, [
C]P1` Cys was mixed with active uPA for
different times. The minor 80-kDa band shown in lane 1 represents dimers of P1` Cys. Arrows indicate the
mobility of prestained molecular weight
standards.
If reactive
center cleavage had already occurred in the native serpin-protease
complex it is likely to result in a conformational rearrangement, which
would not occur in the native complex with a catalytically inactive
protease(5, 20) . Since the P1` residue following
reactive center cleavage constitutes the new NH-terminal
end of the PCF, the increased flexibility of the P1` residue in the
complex with active protease, as detected by fluorescence
spectroscopy(19, 20) , could indicate that PCF has
been released from the active center pocket of PA, thereby becoming
more accessible. To study the accessibility of the P1` residue of PAI-1
in complexes with uPA and catalytically inactive anhydro-uPA, complexes
between Sepharose-bound uPA or anhydro-uPA and P1` Cys were formed.
After complex formation the P1` cysteine residues were specifically
labeled with [
C]iodoacetamide. As shown in Fig. 2, inset, only intact radiolabeled inhibitor was
released from the complex with anhydro-uPA (lane 1) whereas
radiolabeled PCF was released from the complex with uPA (lane
2). Fig. 2demonstrates that the labeling of the P1` Cys
residue was 3-4 times higher for the complex with uPA than with
anhydro-uPA indicating that complex formation with uPA leads to a
conformational change of the reactive center whereby P1` becomes more
accessible for labeling.
Figure 2:
Labeling of the cysteine residue of P1`
Cys in complex with immobilized uPA and anhydro-uPA. Complexes between
P1` Cys and immobilized uPA or anhydro-uPA were labeled with
[C]iodoacetamide under physiological conditions.
At different time points an excess of the radioactive ligand was
removed, the complexes were dissociated, and the radioactivity in the
eluates was quantified. The curves represent the mean ±
S.D. from three independent experiments and are expressed as counts/min
incorporated per µg of bound P1` Cys.
, eluate from P1`
Cys-uPA;
, eluate from P1` Cys-anhydro-uPA. Inset,
50-µl samples of the eluates were analyzed by SDS-PAGE
(3-20%) followed by autoradiography. Lane 1, intact
inhibitor eluted from the immobilized [
C]P1`
Cys-anhydro-uPA complex; lane 2,
C-labeled PCF
eluted from the immobilized [
C]P1` Cys-uPA
complex.
In conclusion, our data reveal that three different serpins are cleaved in their native complexes with cognate proteases, suggesting that complex formation stops at a stage after reactive center cleavage. In the case of PAI-1, we show that complex formation and reactive center cleavage are rapid and coordinated events. Our data therefore support models where serpins act as suicide inhibitors, and the complex is arrested as an acyl-enzyme intermediate or possibly, following addition of a water molecule, as a second tetrahedral intermediate. Taking our findings into account, we suggest the following simple scheme for the reaction of a serpin (I), with a target protease (E).
On-line formulae not verified for accuracy
In this scheme EI is an initial encounter (Michaelis)
complex, and [EI] is a very short lived
tetrahedral intermediate that is rapidly converted to EI*, an
acyl-enzyme intermediate containing cleaved inhibitor. The increased
flexibility (20) and accessibility (Fig. 2) of the
NH
-terminal end of the PCF following reactive center
cleavage suggests that it may be released from the reactive center
pocket. If this conformational change creates room for a water molecule
to enter, the reaction could continue to a second tetrahedral
intermediate, EI*
. If this second tetrahedral
intermediate is stable (28, 29) it may represent the
tetrahedral intermediate detected by Matheson et
al.(16) . The reaction between serpin and protease may
therefore very much resemble the reaction between protease and
substrate with the exception that the deacylation and release of the
cleaved inhibitor are greatly retarded. Previous studies indicate that
a mobile reactive center is required for inhibitory function and that
hindrance of mobility converts the inhibitor to a
substrate(9, 14, 30) . Taken together with
our present finding, i.e. that serpins are cleaved in their
native complexes with target proteases, these data suggest that
cleavage of the reactive center loop by protease and the subsequent
loop insertion induce the conformational changes required to lock the
inhibitor-protease complex. This model is compatible with our
previously proposed model for serpin function(12) .
Note Added in Proof-Since the submission of this paper similar data, indicating that the serpin-protease complex is in the acyl-enzyme intermediate, have been published by Lawrence, D. A., Ginsburg, D., Day, D. E., Berkenpas, M. B., Verhamme, I. M., Kvassman, J.-O., and Shore, J. D.(1995) J. Biol. Chem.270, 25309-25312.