(Received for publication, May 23, 1995; and in revised form, August 31, 1995)
From the
The serpin plasminogen activator inhibitor-1 (PAI-1)
spontaneously adopts an inactive or latent conformation by inserting
the N-terminal part of the reactive center loop as strand 4 into the
major -sheet (sheet A). To examine factors that may regulate
reactive loop insertion in PAI-1, we determined the inactivation rate
of the inhibitor in the pH range 4.5-13. Below pH 9, inactivation
led primarily to latent PAI-1, and one predominant effect of pH on the
corresponding rate constant could be observed. Protonation of a group
exhibiting a pK
of 7.6 (25 °C, ionic
strength = 0.15 M) reduced the rate of formation of
latent PAI-1 by a factor of 35, from 0.17 h
at pH 9
to about 0.005 h
below pH 6. The ionization with a
pK
7.6 was found to have no effect on the
rate by which PAI-1 inhibits trypsin and is therefore unlikely to
change the flexibility of the loop or the orientation of the reactive
center. The peptides Ac-TEASSSTA and Ac-TVASSSTA (cf. P14-P7
in the reactive loop of PAI-1) formed stable complexes with PAI-1 and
converted the inhibitor to a substrate for tissue type plasminogen
activator. We found that peptide binding and formation of latent PAI-1
are mutually exclusive events, similarly affected by the
pK
7.6 ionization. This is direct
evidence that external peptides can substitute for strand 4 in
-sheet A of PAI-1 and that the pK
7.6 ionization regulates insertion of complementary,
internal or external, strands into this position. A model that accounts
for the observed pH effects is presented, and the identity of the
ionizing group is discussed based on the structure of latent PAI-1. The
group is tentatively identified as His-143 in helix F, located on top
of sheet A.
Plasminogen activator inhibitor, PAI-1, is a member of the
serpin family of serine proteinase inhibitors and inhibits both tPA ()and urokinase plasminogen activator (1, 2) as well as trypsin(3) . The
serpins(4) , which include most of the inhibitors that regulate
blood coagulation and fibrinolysis, are structurally homogenous and are
distinguished functionally from other types of protein proteinase
inhibitors primarily by the ability to form SDS-stable complexes with
target proteinases. The nature of these complexes and the mechanism by
which they are formed are poorly understood.
Active serpins are
metastable folding intermediates with considerable conformational
strain(5) . They can relax by inserting the N-terminal portion
of the reactive center loop as strand 4 in the major -sheet that
faces one side of the protein (sheet A) and thereby adopt an inactive,
or latent, conformation. This process occurs spontaneously in PAI-1 (6) and has been induced in antithrombin III (5) and
-proteinase inhibitor(7) . Inhibitory serpins
attain an even greater stability after cleavage of the reactive loop,
near or at the susceptible bond, and insertion of the N-terminal
portion into
-sheet A (8, 9, 10) or
after forming complexes with peptides that mimic this part of the
loop(11, 12) . The fact that serpins with a completed
six-stranded sheet A are inactive (13) clearly indicates a role
for loop insertion in the inhibitory mechanism. Data obtained for
-proteinase inhibitor in complex with N-terminal
truncated strand 4A-mimicking peptides were taken to indicate that
insertion of the loop into sheet A down to residue P14 (notation
according to Schechter and Berger(14) ) is necessary and
sufficient for inhibition of trypsin by this serpin(11) . The
concept presented was that with such limited insertion the loop adopts
a more rigid conformation akin to that of the loop in Kunitz type
inhibitors. The consequences of this view are that inhibition is due to
tight binding and that the bound inhibitor can only be cleaved slowly.
The bound reactive center may approach formation of a tetrahedral
intermediate, which could account for the NMR data presented by Travis et al.(15) . Evidence for a fast insertion of the loop
region containing residue P9 upon formation of the tPA
PAI-1
complex was recently obtained in our laboratory from studies of a PAI-1
mutant with a fluorescent probe on P9(16) , and the idea was
put forward that cleavage of the reactive bond may be required to
trigger such a fast, extensive insertion of the loop. This, on the
other hand, is difficult to reconcile with dissociation of
proteinase-serpin complexes into active enzymes and
inhibitors(17) .
Although the fate of the susceptible bond in
the proteinase-serpin complex remains obscure, it has been well
established that in order for serpins to be fully active they must
possess a potential for unhindered insertion of part of the reactive
center loop into -sheet
A(11, 12, 18, 19, 20) .
Consequently, studies of loop insertion leading to latent PAI-1 and
factors that affect this process may yield significant insights into
the mechanism of serpin inhibition. Several studies (21, 22, 23) have shown that one such factor
is pH and that the transformation of active PAI-1 to latent is much
slower at weakly acidic rather than at physiological pH values. The
present investigation was initiated to characterize this pH effect and
the associated proton dissociation equilibrium or equilibria.
Figure 1:
Effects of pH on the rate constant for
inactivation of PAI-1. Data were obtained kinetically () or by
SDS-PAGE (
) as described under ``Experimental
Procedures.'' The inset shows data obtained in the
alkaline pH range. The solid line represents the equation
with the best fit parameter
values k = 0.167 h
, k
= 1132 h
, pK
= 6.041, and pK
=
7.58.
Figure 2: Thermostability analysis of PAI-1. Measurements were performed as described under ``Experimental Procedures.'' Samples of PAI-1 were prepared by incubation of the inhibitor at pH 6 for 16 h (1), pH 9.4 for 6 h (2), pH 10.9 for 2 h (3), and at pH 8.2 for 36 h followed by pH 10.9 for 4 h (4).
where C stands for the total concentration
of X. The ratio in will vary with pH only if the
hydrogen ion has different effects on the reactivity of the two
inhibitors. The values obtained for k
/k
at pH 6.0, 7.0, and
8.1, were 5.7, 6.3, and 6.3 respectively.
Figure S1: Scheme 1.
where the 0 and t subscripts denote the concentration
of active PAI-1 at reaction time 0 and t, respectively, and
the time constant (r) is given by
The laws of probability then predict that in the process of inactivation, latent and peptide-bound PAI-1 are formed according to and , respectively.
It follows from and that the ratio of peptide-bound to latent PAI-1 (R) at any time during the reaction, should be given by
To test the validity of , PAI-1 was incubated with
various concentrations of peptide, and the proportion of cleaved to
latent inhibitor was determined for each concentration by quantitative
analysis of gels, such as the one demonstrated in Fig. 3. The
results are shown in Fig. 4(A and B). The
linear dependence of R on peptide concentration, established
for both peptides, is consistent with and confirms that
peptide binding and formation of the latent inhibitor are competitive
events. The small intercepts observed result from cleavage of some
PAI-1 by tPA in the absence of peptides (Fig. 3, lane
0). The slopes of the regression lines in Fig. 4B,
representing k/k
for
Ac-TVASSSTA at pH 7.0 and 8.1, are 16 and 20
mM
, respectively. If peptide association (k
), in contrast to reactive loop folding (k
), were pH-independent, these slopes should
have differed by a factor of 4. The single line in Fig. 4A was obtained for the association of Ac-TEASSSTA at pH 7.4 and has
a slope of 1.0 mM
. Using the best fit
estimates of k
(the solid line in Fig. 1), k
for Ac-TVASSSTA evaluates to
0.18 and 0.74 M
s
at pH
7.0 and 8.1, respectively, whereas a value of 0.020 M
s
is obtained for
Ac-TEASSSTA at pH 7.4. The effect of pH on R can be seen in Fig. 5and 6. The gel shown in Fig. 5confirms the pH
effects on the rate of formation of inactive PAI-1 reported above and
indicates in a qualitative way that the proportion of peptide-bound to
latent PAI-1 does not vary much with pH and, consequently, that
reactive loop folding and peptide binding exhibit a similar dependence
on pH. This is quantitatively confirmed by the data presented in Fig. 6obtained by analysis of the gel in Fig. 5and by
the data obtained from similar gels developed for both peptides (not
shown).
Figure 3: Effect of peptide concentration on the proportion of peptide-bound (cleaved) to latent PAI-1 demonstrated by SDS-PAGE. PAI-1 (4 µM) was incubated for 5 days at pH 7.4 and 25 °C with increasing concentrations of Ac-TEASSSTA and analyzed by treatment with excess tPA followed by SDS-PAGE.
Figure 4:
Quantitative evaluation of the effect of
peptide concentration on the proportion of peptide-bound (cleaved) to
latent PAI-1. The ratio of cleaved to latent PAI-1, generated by
incubation of the active inhibitor with Ac-TEASSSTA (A) at pH
7.4 and Ac-TVASSSTA (B) at pH 7.0 () and pH 8.1 (
),
was determined from gels like the one shown in Fig. 3and
plotted versus peptide
concentration.
Figure 5: Effects of pH on the ratio of peptide bound (cleaved) to latent PAI-1 demonstrated by SDS-PAGE. PAI-1 (5 µM) was incubated at 25 °C with (+) and without(-) 1 mM Ac-TEASSSTA in buffers ranging in pH from 4.6 to 10.6.
Figure 6:
Quantitative evaluation of the effect of
pH on the proportion of latent to peptide bound (cleaved) PAI-1. The
proportion of latent to cleaved inhibitor, generated after incubation
of the active inhibitor with Ac-TEASSSTA for 6 () and 22 h
(
) were determined from gels like the one shown in Fig. 5.
The dotted line is the proportion expected if peptide
association leading to cleaved PAI-1 were
pH-independent.
Figure 7:
Effects of pH on PAI-1 fluorescence. The
emission spectra were recorded for active PAI-1 at pH 6.0 (solid
line) and 9.4 (dotted line) as described under
``Experimental Procedures.'' The inset shows how the
average emission intensity in the 380-400 nm region increases
with pH; the line represents a protonic dissociation function
with a pK of 7.6. Fluorescence units are arbitrary and the
Y axis of the inset corresponds to 0.025 such
units.
By a systematic study of the effects of pH on the
inactivation rate of PAI-1, we have linked the observed acid
stabilization of the active form of this serpin to protonation of a
single functional group in the protein. To examine whether the
protonated form of the group stabilizes an exposed reactive loop in a
direct manner or indirectly by preventing its binding to sheet A, the
reactivity of PAI-1 and BPTI toward trypsin was compared in the pH
interval over which the rate of loop insertion, leading to latent
PAI-1, exhibits a 15-fold increase. The comparison of the two
inhibitors' rates of reaction with trypsin was performed to mask
the well known effects of pH on the active site of serine proteinases
and to avoid the possible complication of exosite interactions believed
to be involved in formation of the tPAPAI-1 complex(31) .
The absence of a significant effect of pH on the quotient between the
rate constants for inhibition of trypsin by PAI-1 and BPTI in the range
tested indicates that ionization of the pK
7.6
group has no direct influence on the flexibility of the loop or the
exposure of the reactive center in PAI-1. The alternative explanation,
that the reactivity of BPTI is controlled by an internal ionization
that balances the effects of that in PAI-1, lacks support in the vast
literature on BPTI(32) . The most plausible explanation is
therefore that the protonated form of the group stabilizes the active
inhibitor by lowering the affinity of a specific binding site in PAI-1
for the latent reactive center loop.
Peptides with sequences that
match the N-terminal portions of the reactive loops in
-proteinase inhibitor (11) and antithrombin
III (12) form strong complexes with the respective inhibitor
and convert them to substrates for their target proteinases. It has
generally been assumed that this results from binding of the peptides
to the vacant crevice for strand 4A. Bound in this position, the
peptides would prevent insertion of the reactive loop believed to be
necessary for the inhibitory function of the serpin. Recent
publications on the structure of antithrombin (33, 34) show that this serpin has a potential for
peptide binding also to
-sheet C, which indicates that the effects
of peptide binding to serpins must be interpreted with caution. The
present investigation, however, establishes that at least one binding
site for Ac-TVASSSTA and Ac-TEASSSTA in active PAI-1 is occupied by the
loop in latent PAI-1 and, hence, must be located outside
-sheet
C(6) . The crevice for strand 4 in sheet A is the only subsite
for the latent loop that has the structural and chemical
complementarity to the octamer peptides required to account for the
strength of the PAI-1-peptide interactions. The observation that loop
insertion leading to latent PAI-1 and peptide insertion are equally
affected by the pK
7.6 ionization must therefore
be interpreted as an effect of the latter on the access of peptides to
sheet A. Direct stabilization of an extended reactive center loop by
the protonated form of the group would have resulted in a decreased
competition for the strand 4A crevice at low pH and reverted or
abolished the pK
-7.6 effect on association of
external peptides. In summary, we conclude that the octamer loop
analogs used in this study substitute for strand 4A in PAI-1 and that
the pK
7.6 ionization regulates binding of
external and internal peptides to this position.
Electrostatic
repulsion cannot account for the observed effect of pH on association
of the negatively charged octamer peptides (rather, electrostatic
attraction could explain the slightly less pronounced effect of pH,
demonstrated in Fig. 4B and Fig. 6, on peptide
association compared with loop refolding). Our interpretation is that
the data presented in Fig. 1reflect a proton dissociation
equilibrium in PAI-1 linked to the equilibrium between an open (I) and a closed (I
) form of
the inhibitor. The equilibrium constant K
is the
ratio of open to closed inhibitor when the group is protonated, whereas K
is the ratio when the group is unprotonated; K
is the acid dissociation constant for the fully
interacting functional group, whereas K
is the
constant for the noninteracting group (Fig. S2). The protonated
form of the group takes part in an interaction that stabilizes the
closed form of the inhibitor (K
> K
). The interaction results in a reciprocal
decrease in the acid dissociation constant of the functional group from K
to K
. The limiting
concentrations of the open form are given by
Figure S2: Scheme 2.
where C is the total concentration of
active inhibitor. Let k
be the intrinsic rate
constant for loop insertion in the open inhibitor, and the observed
rate constant will be given by
From the data presented in Fig. 1, we have
[I]
/[I
]
= k
/k
= 35. In view of and , this means
that K
< 1/34 and K
K
. The actual position of these equilibria will
determine the relationship between K
and K
in Fig. S1and the experimentally found
constants, K
and K
, presented
in Fig. 1. Clearly, pK
corresponds to the pH at
which [I
]
2
[I
]
. Hence
which, with K
1, simplifies to
the value reported for pK in Fig. 1(6.0)
must be a close estimate of pK
, the pK
of the noninteracting ionizing group.
A pK of 6 is consistent with that of a histidine residue slightly
perturbed by a hydrophobic environment or a neighboring positive
charge. In the search for a candidate among the 13 histidine residues
of human PAI-1, we studied the structure of the latent inhibitor and
focused on histidine residues that could have a direct effect on
binding of peptides to sheet A. This approach is justified by the fact
that apart from the reactive center loop, the differences between
serpins with 5 or 6 strands in sheet A are predominantly observed in
this sheet (33, 34, 35) and its immediate
vicinity. More profound changes may accompany formation of latent PAI-1 (6) but are not likely to be required for insertion of the
octamer peptides. One possible candidate then is His-143 in helix F.
Helix F is part of a loop that connects strand 1A and strand 3A and
covers the lower half of sheet A in all inhibitory serpins. It appears
that the loop containing helix F must move out of the way in order for
the reactive loop to insert completely as strand 4A and that factors
that stabilize the helix should slow down the insertion. In the latent
inhibitor the presumably neutral His-143 (the crystals were grown at pH
8.2(36) ) is seen at hydrogen bond distance to the
-oxygen
of Thr-142 and the carbonyl oxygen of Trp-139, both in helix F. In the
active inhibitor, a positive charge on the histidine in addition to
structural differences may allow the imidazolium ring to rotate into a
different position, where it may stabilize helix F by neutralizing its
dipole field or by forming a hydrogen bond to sheet A. Although there
is no suitable acceptor within distance for the latter bond in latent
PAI-1, it can be seen from the differences between latent PAI-1 and
nonrelaxed serpins (35) that sheet A in the former has expanded
in the region beneath helix F. In the active inhibitor the side chain
oxygen of Thr-94 in strand 2A and one of the nitrogens of His-143 may
very well be within hydrogen bond distance. The spatial relationships
pertinent to this discussion are illustrated by the stereodiagram of
the
-carbon backbone of sheet A and helix F in latent PAI-1
presented in Fig. 8. The side chains of Trp-139 and His-143 in
helix F and Thr-94 in strand 2A are displayed. The sequence of strand
4A, which corresponds to the octamer peptides used in this study, is
represented by the heavy portion of the strand.
Figure 8:
Stereodiagram of latent PAI-1. Side view
of the -carbon backbone of sheet A and helix F in latent PAI-1.
The residues displayed are Trp-139 and His-143 in helix F and Thr-94 in
strand 2A. The sequence of strand 4A homologous to the octamer peptides
used in this study with P14 at the top of the sheet and helix F are
marked by heavy lines.
The identity of the
pK 7.6 group as His-143 seems consistent with the
fluorescence data presented in Fig. 7. The side chain of His-143
is within a distance of 4 Å to the indole ring of Trp-139 in
latent PAI-1, half that between any other His/Trp pair in the
inhibitor. A general stabilization of the sheet A/helix F region in
latent and peptide-bound PAI-1, making it less sensitive to the ionic
state of His-143, may explain the absence of pH effects on the red side
of the emission spectrum of these species.
PAI-1 is the only serpin
known to spontaneously fold its reactive center loop to adopt a latent
conformation at room temperature. Among the 37 serpins listed by
Marshall(37) , PAI-1 and bovine PAI-2 are the only ones with a
histidine residue at position 143 in helix F, and human PAI-1 is unique
in having a threonine at 94 in strand 2A. In fact, 34 of the listed
serpins have either glycine or an aliphatic residue at the latter
position. This combination of side chains in PAI-1, which places polar
groups in the hydrophobic helix F/sheet A interface, may contribute to
a more open sheet A in this serpin compared with that of others and at
the same time provide means for pH regulation of the propensity of
sheet A to bind strand 4. Regardless of the underlying mechanism, we
may speculate about the in vivo significance of the
pH-dependent stability of the active inhibitor. The study by Lindahl et al.(21) suggests that the ionic properties of the
stabilizing group and its effect on active PA1-1 are maintained
at 37 °C. This means that active PAI-1 basically should be about
2.5 times more stable in cells (pH = 6.8) than in plasma (pH
= 7.4). Obstructions to blood flow through active tissues could
lead to a local acidification through accumulation of CO followed by a shift of intermediary metabolism toward
fermentation with a concomitant prolongation of the lifetime of active
PAI-1.