(Received for publication, October 21, 1994; and in revised form, December 9, 1994)
From the
A mutant recombinant plasminogen activator inhibitor 1 (PAI-1)
was created (Ser-338 Cys) in which cysteine was placed at the
P
position of the reactive center loop. Labeling this
mutant with N,N`-dimethyl-N(acetyl)-N`-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)
ethylene diamine (NBD) provided a molecule with a fluorescent probe at
that position. The NBD-labeled mutant was almost as reactive as wild
type but was considerably more stable. Complex formation with tissue or
urokinase type plasminogen activator (tPA or uPA), and cleavage between
P
and P
with a catalytic concentration of
elastase, all resulted in identical 13-nm blue shifts of the peak
fluorescence emission wavelength and 6.2-fold fluorescence
enhancements. Formation of latent PAI showed the same 13-nm spectral
shift with a 6.7-fold fluorescence emission increase, indicating that
the NBD probe is in a slightly more hydophobic milieu. These changes
can be attributed to insertion of the reactive center loop into the
sheet A of the inhibitor in a manner that exposes the NBD probe
to a more hydrophobic milieu. The rate of loop insertion due to tPA
complexation was followed using stopped flow fluorimetry. This rate
showed a hyperbolic dependence on tPA concentration, with a
half-saturation concentration of 0.96 µM and a maximum
rate constant of 3.4 s
. These results demonstrate
experimentally that complexation with proteases is presumably
associated with loop insertion. The identical fluorescence changes
obtained with tPA
PAI-1 and uPA
PAI-1 complexes and
elastase-cleaved PAI-1 strongly suggest that in the stable
protease-PAI-1 complex the reactive center loop is cleaved and inserted
into
sheet A and that this process is central to the inhibition
mechanism.
Plasminogen activator inhibitor-1 (PAI-1) ()is a
member of the serpin class of protease inhibitors, which includes most
of the plasma inhibitors regulating blood coagulation and fibrinolysis.
These inhibitors have extensive homology and form very tight 1:1
complexes with their target proteases(1) . They share a number
of common characteristics. All of the inhibitory proteins have a
reactive bond contained within a loop extending from the protein; upon
cleavage of this bond, the residues of the loop are inserted into the
sheet A at the face of the molecule making it more
stable(2) . This form of the molecule can be created by
cleavage with catalytic concentrations of elastase (3) or
dissociation of the enzyme-inhibitor complex(4) . Cleaved
serpins are no longer able to bind proteases and inhibit
them(2) . Structural studies of
-protease
inhibitor indicate that after cleavage at the
P
-P
` bond of the reactive center loop and
insertion of the P
through P
residues into the
A-
sheet, the P
and P
` residues are
separated by approximately 70 Å(5) .
Until recently,
the model for an active serpin has been based on the crystallographic
structure of ovalbumin, a non-inhibitory member of the serpin
family(6) . Recently, the structures of an active form of
antithrombin dimerized with an inactive form (7) and of the
active form of an antichymotrypsin mutant (8) have been
reported and are consistent with previous concepts of the structure of
active serpin molecules. It has been proposed that loop insertion is
rate-limiting and required for formation of a stable
complex(2) . The hypothesis that the reactive center loop of
serpins is inserted into the sheet A after reaction with
proteases is supported by immunologic evidence with antithrombin III in
which unique epitopes were exposed due to binding to proteases and
cleavage of the reactive bond(9) .
Plasminogen activator
inhibitor 1 (PAI-1) is the primary inhibitor of tPA and uPA, reacting
at diffusion-limited rate constants(10) . It is also reactive
with thrombin, plasmin, and trypsin, with somewhat slower bimolecular
rate constants(11, 12) . An unique feature of PAI-1,
compared with other serpins, is that it spontaneously converts from an
active to a latent form, with a half-life of 3 h at pH 7.4 and 37
°C (17) . This form can be partially reactivated by
denaturation. The structure of the latent form, determined by x-ray
crystallography, indicates that residues P to P
of the reactive center loop are inserted into the
sheet
A(13) . Residues P
-P
` form an
extended loop, which is stretched along the surface of the protein.
Wild type PAI-1 contains no cysteine residues. Thus, the
availability of a recombinant mutant with a sufhydryl group provides an
unique opportunity for reaction with iodoacetamide derivatives of
fluorescent labels and probes. We have reported studies with
fluorescein-labeled P` PAI-1(14) , Strandberg and
Ny (15) have used this approach to label the P
and
P
positions of PAI-1 with NBD, and Gettins et al.(16) labeled the P
position of antithrombin
III with NBD to demonstrate the transfer of a heparin-induced
conformational change to the region of the reactive bond. Fluorescent
probe molecules such as NBD are sensitive to the polarity of the milieu
that surrounds them and respond to a more hydrophobic environment by an
enhanced and blue-shifted fluorescence emission spectrum.
In the
present study, we developed an active PAI-1 mutant with a cysteine
residue at the P position in the reactive center loop,
which was then available for modification with an NBD probe molecule.
This permitted studies of the effects of tPA and uPA binding, formation
of latent PAI-1, and cleavage of the reactive center loop at the
P
-P
position by elastase, on the fluorescence
properties of the label on the P
residue. The changes in
fluorescence spectra and yields, due to these interactions of the PAI-1
molecule, demonstrated that binding with target proteases results in
changes in the milieu of the reactive center loop. Consistent with our
earlier studies(17) , the kinetics of these changes indicate
that loop insertion is a critical component of the inhibitory
mechanism.
Fig. 1shows the
fluorescence emission spectra of free iodoacetamido-NBD (IANBD),
labeled Ser-338 Cys (P
) PAI-1, and the labeled PAI-1
bound to tPA. The free IANBD had an emission peak at 550 nm with
excitation at 480 nm, which shifted to 542 nm when linked to the PAI-1
mutant, accompanied by a 58% enhancement of fluorescence. The
enhancement and blue shift indicate that the probe is in a more
hydrophobic milieu when linked to the protein than when it is free in
solution.
Figure 1:
Altered NBD probe
fluorescence due to local environmental changes. The excitation
wavelength was 480 nm. Figure shows emission spectra of: A,
free IANBD (0.2 µM); B, NBD P PAI-1
(0.2 µM); C, NBD P
PAI-1 (0.2
µM) after addition of r-tc-tPA (0.4
µM).
Figure 2:
Spontaneous conversion of NBD P PAI-1 (0.9 µM) from an active to latent
conformation. Leftaxis (solidcircles), the fluorescence enhancement of NBD probe
emission at 529 nm as a function of time. Right axis (invertedtriangles), the amount of active
labeled PAI-1 remaining was measured by inhibition of
-trypsin by
the labeled PAI-1 as a function of time.
Using the NBD-labeled P PAI-1 mutant,
it was possible to determine the rate of the change in fluorescence
emission due to protease binding. The inset to Fig. 3shows a typical reaction with tPA, followed by stopped
flow fluorimetry. These reactions were performed with various tPA
concentrations under pseudo first order conditions. They fit best to
the equation for two exponentials, with the faster one having 87% of
the amplitude at the lowest tPA concentration (0.3 µM) and
90% at the highest concentration (5 µM). The line through
the data in inset to Fig. 3is from a double
exponential fit. The results presented in Fig. 3show the pseudo
first order rate constants for the fast phase of the interaction as a
function of tPA concentration. The curve fits a hyperbolic dependence
on tPA concentration with a limiting rate of 3.4 s
and a half-saturation concentration of 0.96 µM.
Figure 3: Stopped flow kinetic analysis of the fluorescent signal generated by reaction of the labeled PAI-1 with increasing concentrations of r-tc-tPA. The pseudo first order rate constants measured for the increase in probe fluorescence are plotted as a function of r-tc-tPA concentration. Inset, a typical stopped flow trace at final concentrations of 0.1 µM NBD PAI-1 and 5.0 µM r-tc-tPA.
Replacement of the serine residue at P with
cysteine and derivatization with NBD resulted in rate constants for tPA
and uPA inhibition that were reduced approximately 2-fold compared to
wild type (Table 1), indicating that the substitution has only
modest effects on PAI-1 function. Interestingly, the rate constant for
transformation of active to latent inhibitor for the derivatized
P
mutant was less than half that of wild type. This could
be due to the bulky NBD molecule causing a slower rate of loop
insertion into the
sheet A of the PAI-1. The spectral shift and
enhancement (Fig. 1) due to forming protein-bound NBD,
indicating a more hydrophobic milieu, may be due to interactions with
adjacent residues on the reactive center loop, or with other parts of
the loop. The effect is, however, relatively small compared with the
other NBD effects observed in these studies and suggests that even when
associated with native PAI-1 the probe is likely to be very
solvent-accessible.
The mirror image curves in Fig. 2demonstrate that the process of latent transformation
exactly follows the fluorescence change of the labeled PAI-1. In
addition, the 13-nm spectral shift and greater than 6-fold fluorescence
emission enhancement of the NBD-labeled PAI-1 observed upon this
transition as well as with binding to tPA or uPA and catalytic cleavage
by elastase, are all very similar. We previously demonstrated that
catalytic concentrations of elastase cleave at the
P-P
bond of PAI-1, resulting in a
molecule with physical properties similar to those of latent
PAI-1(3) , in which the reactive center loop is fully inserted
into the
sheet A. The fluorescence changes observed upon
formation of the latent species must result from the increased
hydrophobic milieu surrounding the P
position in
sheet A. These data suggest that the probe on the P
position moves into a nearly identical milieu in cleaved PAI-1 as
in protease-bound or latent inhibitor, associated with the same
conformational change in each case. It should be noted that in the
latent structure of PAI-1,
helix F sits directly above
sheet A and would appear to cover
strands 2, 3, and 4. The
similarity of fluorescence changes due to latency, in which the
reactive loop is known to be inserted(13) , to those caused by
complexation with tPA and uPA or cleavage by elastase, provides
evidence that loop insertion is an essential component of the
inhibitory mechanism. The emission peak wavelength shift due to
formation of latent NBD-PAI-1 is the same as NBD-PAI-1 reacted with tPA
or uPA. However, the 6.7-fold enhancement was somewhat higher than that
observed with PAI-1 which is cleaved or complexed to proteases. This
higher fluorescence yield may be due to a nearly identical position of
the P
probe in the latent form, with a more hydrophobic
milieu. The requirement of insertion of the reactive center loop into
sheet A for inhibition is also consistent with our previous
studies of PAI-1 mutants containing substitutions at the P
position of the loop(18) .
The identical 6.2-fold
fluorescence increases and blue spectral shift due to cleavage by
elastase at the P-P
position, and to binding of
tPA or uPA, indicate that the NBD probe is in a similar hydrophobic
milieu in all three cases. These data suggest that interaction with tPA
or uPA results in insertion of the loop into
sheet A,
presumably similar to the structure of latent PAI-1. The identical
fluorescence changes resulting from tPA and uPA binding also indicate
that these changes derive from alterations in the PAI-1 structure
rather than interaction of the probe with each enzyme.
Since the
large fluorescence enhancement of the NBD label on the Pposition is likely to be measuring insertion of the
reactive center loop, it was of interest to follow the kinetics of this
process. The results in Fig. 3indicate that the rate constant
for loop insertion hyperbolically approaches a limiting rate of 3.4
s
. The minimum mechanism describing this contains
two steps, as shown in .
In this mechanism, E is tPA or uPA, I is PAI-1, and EI* represents the complex after the reactive center loop of PAI-1 has been inserted. The equation for this mechanism would take the following form.
In this equation, K is equal to (k + k
)/k
so that if k
k
, it would be k
/k
, while if k
k
, K would reduce to k
/k
. Regardless of the
rate constants defining K, the rate of inhibition of tPA by
NBD-labeled PAI-1 should be equal to k
/K.
This is indeed true, since the ratio from the data in Fig. 3,
3.4 s
/0.96 µM, equals 3.5
10
M
s
,
which is comparable to the value of 3.5
10
for tPA inhibition determined directly and reported in Table 1. With k
equal to 3.4
s
, the half-life of the reaction is approximately
0.2 s. Most functional assays and kinetic evaluations of PAI-1 use
concentrations in the nanomolar range so that the reaction can be
measured over a time scale of minutes. These conditions are very far
from the PAI-1 concentration necessary for saturation, which would
enable observation of the limiting rate. They are appropriate, however,
for determination of the bimolecular inhibition rate constant, which is
equal to k
/K. The rates of loop insertion
at different tPA concentrations were double exponentials, as the
excellent fit in Fig. 3(inset) shows. Regardless of
the tPA concentration, the rapid phase consisted of approximately 90%
of the total reaction amplitude. We used the fast phase for the plot in Fig. 3, which provided a good hyperbolic fit.
The value of k, 3.4 s
, represents the rate
of insertion of the reactive center loop into
sheet A in the
tPA-PAI-1 complex. This is much faster than the rate constant for
transformation of NBD-labeled PAI-1 to a latent form, which has a
half-life of 23 h at 25 °C. One possible explanation for the rapid
insertion rate due to protease binding could be exosite interaction
with PAI-1, causing a conformational change in the
sheet A that
facilitates loop insertion. Alternatively, formation of the initial EI complex, involving interaction at the
P
-P
` bond, may alter the mobility or
conformation of the loop in a manner that facilitates insertion.
Finally, cleavage of the P
-P
` bond may induce
insertion by removing any constraints on the reactive-center loop. This
mechanism would be compatible with the stable PAI-1-plasminogen
activator complex existing as an acyl enzyme.