The serpins are a large family of proteins which includes most
of the protease inhibitors found in blood, as well as other proteins
with unrelated or unknown functions(1) . Serpins act as
``suicide inhibitors'' that react only once with their
cognate protease, forming an SDS-stable complex. Current models of
serpin structure suggest that, while their overall fold is generally
homologous among family members, the RCL, (
)sometimes
referred to as the strained or stressed loop, is capable of adopting
markedly different conformations relative to the rest of the protein
structure (2, 3, 4, 5) . This
conformational flexibility appears to be necessary for function but can
also lead to inactivation when the loop inserts into the main body of
the inhibitor, becoming the central strand of the major serpin
structural motif,
-sheet A(6, 7, 8) .
This inactive conformation was first observed in RCL-cleaved

-antitrypsin (
AT) (9) and
more recently in the related structure of latent plasminogen activator
inhibitor 1 (PAI-1)(10) . Loop insertion leads to a large
increase in thermal stability, presumably due to reorganization of the
five-stranded
-sheet A from a mixed parallel-antiparallel
arrangement to a six-stranded, predominantly antiparallel,
-sheet (11, 12, 13, 14) . This dramatic
stabilization has led to the suggestion that native inhibitory serpins
may be metastable structures, kinetically trapped in a state of higher
free energy than their most stable thermodynamic state. Such an
energetically unfavorable structure would almost certainly be subject
to negative selection, and thus its retention in all inhibitory serpins
implies that it has been conserved for functional reasons.
Currently, the role of loop mobility in serpin function and the
structure of the serpin-protease complex are
controversial(15, 16, 17, 18, 19, 20, 21, 28) .
In the late 1970s, it was reported that serpins were unlike other tight
binding protease inhibitors and formed covalent ester linkages with
enzymes(15) . However, these conclusions were based on SDS-PAGE
analysis of denatured complexes leaving the nature of the native
complex open to question. Later investigations suggested that the
native serpin-protease complex may be reversible and therefore could
not be covalent but instead might form a Michaelis-like complex similar
to the tight binding inhibitors of the Kunitz and Kazal
families(16) . Finally, a recent NMR study suggests a stable
tetrahedral configuration (19) . To distinguish between these
alternative structural models, we developed methods to monitor the
position of the RCL in the inhibitor-enzyme complex and to determine
the chemical nature of the association between serpins and their target
proteases.
EXPERIMENTAL PROCEDURES
Materials
PAI-1 mutants containing Cys
substitutions at either the P9 position (Ser-338) or the P1` (Met-347) (
)of the RCL were constructed by site-directed mutagenesis
as described(14) . Both mutants were purified and labeled with
the environmentally sensitive probe NBD (Molecular Probes) as
described(21) . The labeled mutants retained full inhibitory
activity toward both urokinase (uPA) and tissue-type plasminogen
activator (tPA), with second order rate constants for inhibition
>10
M
s
in all cases. Recombinant high molecular weight uPA was a
generous gift of Dr. J. Henkin of Abbott Laboratories, and two-chain
tPA was prepared from Activase (Genentech) as described
previously(21) . Porcine pancreatic elastase was from Elastin
Products (Owensville, MO), and human 
AT was from
Athens Research and Technology (Athens, GA).
I-labeled
Bolton-Hunter reagent (22) (monoiodinated) was from DuPont NEN,
and N-hyroxysuccinimide acetic acid (NHS) was from Sigma.
Stopped-flow Fluorescence Analysis
Stopped-flow
fluorimetry was performed on both PAI-1 NBD derivatives as
described(21) . PAI-1 concentrations were 0.25 µM for the P1`-NBD derivative and 0.1 µM for the P9-NBD
derivative. The tPA concentration ranged from 0.15 µM to
6.0 µM.
Bolton-Hunter Labeling
Wild type PAI-1 or
substrate PAI-1 (Thr-333
Arg(7) ) at a concentration of
1 µM was incubated ± 1 µM uPA or tPA
in 50 mM sodium phosphate, pH 7.6, 150 mM NaCl at 23
°C for 30 min, followed by the addition of
I-labeled
Bolton-Hunter reagent to 100 nM. The samples were incubated an
additional 30 min followed by the addition of 1/10 volume of stop
solution (1 M glycine, 50 mM Tris, 150 mM NaCl, pH 7.75) and incubation continued for an additional 30 min.
The labeled proteins were then separated from free
I-Bolton-Hunter by precipitation with 50% saturated
ammonium sulfate for 30 min at 4 °C followed by centrifugation at
14,000
g for 20 min. The supernatant, containing free
I-Bolton-Hunter, was discarded, and the pellets were
washed twice with 50% saturated ammonium sulfate. The pellets were
resuspended in 20 µl of 1% SDS, 10 mM Tris, pH 7.4, 1
mM EDTA, and
250,000 cpm of each sample was subjected to
SDS-PAGE on a 20% homogeneous gel (PhastGel, Pharmacia Biotech Inc.)
followed by staining with Coomassie Brilliant Blue and autoradiography.
Blocking of Free Amino-terminal Residues
Inhibitor
(1 µM) was incubated with enzyme (1 µM) for
30 min at 23 °C in 50 mM sodium phosphate, pH 7.6, 150
mM NaCl, after which NHS was added to either 100 µM or 1 mM. The samples were incubated an additional 30 min
followed by the addition of 1/10 volume of 1 M Tris, pH 7.5,
and continued incubation for 30 min. The Tris was then removed by
ultrafiltration and washing with distilled water in a Prospin column
(Applied Biosystems) prior to automated amino-terminal sequence
analysis using Edman chemistry (Applied Biosystems model 473A).
Pretreatment of PAI-1 or tPA with NHS at both NHS concentrations tested
did not significantly affect the activity of either protein, indicating
that, under these conditions, NHS treatment alone does not result in
protein denaturation.
Molecular Modeling
The model for active PAI-1 has
been described previously(23) . The model for cleaved PAI-1 was
generated using Quanta (Burlington, MA) from the coordinates of latent
PAI-1 (generously provided by Dr. E. Goldsmith) and from the
coordinates of cleaved 
AT obtained from the Brookhaven
data base. The root mean square difference of the C
trace with
latent PAI-1 is <0.04 Å and 2.2 Å with cleaved

AT.
RESULTS AND DISCUSSION
Stopped-flow Fluorescence Analysis
In the first
series of experiments, site-directed mutants of the serpin PAI-1 were
constructed with Cys residues at either the P9 or the P1` position of
the RCL. Each mutant was then labeled with the fluorescent probe NBD.
This probe shows a large enhancement in fluorescence when moved from an
aqueous environment to a hydrophobic milieu. Both mutants were then
reacted with tissue-type plasminogen activator (tPA) in a stopped-flow
fluorimeter, and these results are shown in Fig. 1. Reaction of
the P9-NBD PAI-1 with tPA resulted in a large and rapid enhancement of
the relative fluorescence (Fig. 1) together with a 13 nm blue
spectral shift (data not shown), consistent with our previous data (21) . The extent of this change is nearly identical with that
observed during the transition to the latent conformation (21) and is consistent with insertion of the RCL into
-sheet A and the resultant burying of the P9 residue beneath
-helix F. The observed rates of loop insertion over a range of tPA
concentrations are shown in the inset of Fig. 1. These
data yield a limiting rate constant for this reaction of
4
s
(t
for insertion of
250 ms). In contrast, reaction of the P1`-NBD PAI-1 with tPA
resulted in a 30% decrease in relative fluorescence occurring at
approximately the same rate (Fig. 1). This quench indicates
that, unlike the P9 position of the RCL, the P1` side chain is exposed
to a more hydrophilic environment upon reaction with tPA. Although such
a shift in position could result from a minor conformational change in
the RCL, this explanation seems unlikely given the close association of
the serpin and protease via the directly adjacent P1 Arg residue of
PAI-1 and the S1 subsite of tPA. Alternatively, cleavage of the PAI-1
RCL by tPA between the P1 and P1` residues could permit the NBD
reporter group to move away from the enzyme into a more aqueous
environment. In either case, the limiting rate of the reaction,
calculated from the data in the inset to Fig. 1is
similar to that observed with the P9-NBD mutant (
8 s
versus 4 s
), indicating that changes
at the P1` site must occur either immediately preceding or concurrent
with loop insertion. Since the maximum changes in fluorescence are
stable over time (data not shown), the reaction being monitored in the
stopped flow is proceeding to completion and thus represents formation
of the stable inhibitor-enzyme complex and not a transient or
intermediate reaction.
Figure 1:
Stopped-flow kinetic analysis of the
change in the relative fluorescence of 0.25 µM P1`-NBD
PAI-1 (
) or 0.1 µM P9-NBD PAI-1 (
) reacting
with 2 µM tPA. The inset shows the pseudo-first
order rate constants (k
) for P1`-NBD PAI-1
(
) and P9-NBD PAI-1 (
) determined from the change in NBD
fluorescence versus tPA concentration. (The first 50 ms of
each trace is not shown because of injection
noise.)
Bolton-Hunter Labeling of Free Amines
To
distinguish between cleavage of the P1-P1` peptide bond versus solely a conformational change in the intact RCL, the
PAI-1
PA complex was reacted with the amino-specific
I-Bolton-Hunter reagent(22) . Two different
plasminogen activators were used, tPA and uPA. These experiments were
conducted with trace amounts of
I-Bolton-Hunter reagent
under nondenaturing conditions, followed by treatment of the unreacted
label with glycine and removal prior to SDS-PAGE analysis. This
procedure should report the presence of a cleaved RCL in the complex by
the appearance of a novel labeled peptide fragment of the correct size.
Furthermore, since the unreacted Bolton-Hunter reagent was blocked with
glycine and removed before the samples were denatured by exposure to
SDS, any labeled peptide must have been formed while the complexes were
in their native state. Although all accessible amines could potentially
be labeled, including
-NH
groups of
internal Lys residues, the only position that can incorporate label in
the PAI-1 RCL carboxyl-terminal peptide would be its amino terminus,
since PAI-1 contains no Lys residues in the 33-residue peptide produced
by cleavage of the P1-P1` bond. SDS-PAGE analysis of the labeled
complexes shown in Fig. 2demonstrated a unique
3.0-kDa
band with both PAI-1
PA complexes which was not present with PAI-1
or either PA alone (B). The observed mobility of this novel
peptide is consistent with the predicted molecular mass (3.8 kDa) of
the PAI-1 carboxyl-terminal peptide. As a positive control for cleavage
of the RCL and the labeling efficiency of the C-terminal peptide, we
also tested a mutant PAI-1 that we have previously shown is a pure
substrate for plasminogen activators and is completely cleaved at its
RCL P1-P1` peptide bond(7) . Consistent with previous
observations, the mutant PAI-1 fails to form stable complexes with
either PA and is instead completely cleaved (Fig. 2A,
compare lanes 2 and 3 with lanes 7 and 8). Furthermore, a labeled peptide identical with that
observed with wild type PAI-1
PA complexes is also seen (Fig. 2B, compare lanes 2 and 3 with lanes 7 and 8). This similar efficiency of peptide
labeling for the mutant PAI-1 cleaved by either uPA or tPA compared to
wild type PAI-1 in association with each enzyme indicates that the RCL
within the stable serpin-protease complex is cleaved and suggests that
this cleavage is complete.
Figure 2:
SDS-PAGE analysis of
I-Bolton-Hunter reagent-treated PAI-1 ± uPA or
tPA. A, 20% homogeneous gel stained with Coomassie Blue; lane 1, wild type PAI-1 only; lane 2, wild type PAI-1
+ uPA; lane 3, wild type PAI-1 + tPA; lane
4, uPA only; lane 5, tPA only; lane 6, substrate
PAI-1 only; lane 7, substrate PAI-1 + uPA; lane
8, substrate PAI-1 + tPA. B, autoradiography of the
gel in A. The numbers at the left indicate the
position of molecular mass standard proteins, and the arrow marks the position of the labeled carboxyl-terminal
peptide.
Quantitation of Free Amino-terminal Residues
To
confirm complete cleavage of the PAI-1
PA complex and exclude
substrate behavior by a subset of the inhibitor molecules, the extent
of RCL cleavage was directly quantitated by microsequencing of the
PAI-1
uPA complex. Since complex denaturation during the
sequencing reaction could potentially induce cleavage, the extent of
cleavage in native complexes was determined by a subtractive method.
PAI-1
uPA complexes were first reacted with the amino-specific
reagent NHS under nondenaturing, physiological conditions. This
compound is similar to Bolton-Hunter reagent in its reactivity and
covalently binds to free amines. Treatment of PAI-1
uPA complexes
with NHS should therefore block available amino termini in a
dose-dependent manner. The excess NHS was then reacted with Tris and
removed by ultrafiltration prior to direct amino-terminal sequence
analysis of remaining unreacted amino termini. This analysis is
quantitative, and the relative reactivity of natural amino termini from
both the inhibitor and protease serve as internal controls for NHS
reactivity and sequencing efficiency. The results of this analysis are
shown in Table 1. The yield of RCL peptide amino terminus at each
dose of NHS is very similar to those of the natural PAI-1 and uPA amino
termini. Thus, the RCL peptide amino terminus generated upon complex
formation is as reactive as the natural amino termini and therefore is
very likely to be fully cleaved and exposed, consistent with the
quenched fluorescence of the P1`-NBD PAI-1 and the
I
labeling results. Interestingly, the least NHS-reactive amino terminus
tested is that of the uPA heavy chain (uPAhc, Table 1), a
relatively hydrophobic sequence Ile-Ile-Gly-Gly which is likely to be
oriented toward the interior of the molecule (24) . The latter
observation suggests that this approach is quite sensitive to amino
termini solvent accessibility, further supporting the conclusion that
the PAI-1 RCL must be completely cleaved when in complex with uPA.
Similar results were also obtained with PAI-1
tPA complexes (data
not shown).
To test the general relevance of these observations for
other serpin-protease complexes, the 
AT
elastase
complex was also treated with NHS and subjected to microsequencing. The
results shown in Table 2are similar to the data obtained with
the PAI-1
uPA complexes (Table 1), demonstrating that

AT is also cleaved in its RCL when in complex with
elastase. Taken together with the known stability of these complexes to
SDS-PAGE, these observations strongly suggest that the serpin-protease
complex is trapped in the form of a covalent acyl-enzyme intermediate.
Serpin inhibition appears to be a two-step process with an initial
reversible encounter complex followed by formation of an apparently
irreversible stable complex(21, 25) . (
)It
is likely that the previous studies suggesting complex reversibility
were observing this first step(16) . Peptide bond cleavage by
serine proteases is known to proceed through two tetrahedral
intermediates(26) . Although our data rule out a complex frozen
at the point of the first tetrahedral intermediate(19) , prior
to RCL cleavage, it is possible that the serpin-protease complex is
frozen at the point of the second tetrahedral, following RCL cleavage,
or that it is trapped in a distorted conformation intermediate between
these two forms.
A Model of Serpin Function
Based on these data, we
propose that upon encountering a target protease, serpins bind to the
enzyme forming a Michaelis-like encounter complex. This relationship is
similar to a protease's interaction with its substrate,
progressing to the point of cleavage of the P1-P1` peptide bond and
formation of the first covalent acyl-intermediate. Cleavage is coupled
to a rapid insertion of the RCL into
-sheet A, with exposure of
the P1` residue to solvent and burying of the P9 residue. Fig. 3shows the relative position of the RCL and the P1` and P9
residues in molecular models of active and cleaved PAI-1. Since the
inserting RCL is covalently linked to the enzyme via the active site
Ser, this transition should also affect the protease and significantly
change its position relative to the inhibitor. Such a rapid shift in
the relative positions of the two molecules, centered at the
enzyme's active site, might sufficiently distort the active site
geometry to prevent efficient deacylation and thus trap the complex.
Alternatively, the new position of the acylated protease's active
site may prevent the water necessary for deacylation from entering the
active site. This model is consistent with our previous results
demonstrating that RCL insertion is not required for protease binding
but is necessary for stable inhibition (7) as well as the
observation that only an active enzyme can induce RCL insertion.
We suggest that native serpin structures are kinetically trapped
in a conformation which is not their most stable structure, and that
the stored drive toward a lower energy structure results in trapping of
the protease-inhibitor complex in the acyl-enzyme intermediate form.
Figure 3:
Models of active and RCL-cleaved PAI-1. A, ribbon diagrams of active and cleaved PAI-1. The PAI-1 main
chain is shown in yellow except for the RCL from the P16
(Ser-331) to the P1 residue (Arg-346) which is in white. The
side chain atoms are also shown for the P1` residue (Met-347) in green, and the P9 (Ser-338) in red. B is
identical with A except that all atoms except hydrogen are
shown as space-filling spheres. The colors are as in A.