Importance of the amino-acid composition of the shutter region of
plasminogen activator inhibitor-1 for its transitions to latent and
substrate forms
Martin Hansen, Marta N. Busse and Peter A. Andreasen
Laboratory of Cellular Protein Science, Department of Molecular and Structural Biology, University of Aarhus, Denmark
The serpins are of general protein chemical interest due to
their ability to undergo a large conformational change
consisting of the insertion of the reactive centre loop (RCL)
as strand 4 of the central b sheet A. To make space for the
incoming RCL, the ‘shutter region’ opens by the b strands
3A and 5A sliding apart over the underlying a helix B. Loop
insertion occurs during the formation of complexes of
serpins with their target serine proteinases and during
latency transition. This type of loop insertion is unique to
plasminogen activator inhibitor-1 (PAI-1). We report here
that amino-acid substitutions in a buried cluster of three
residues forming a hydrogen bonding network in the shutter
region drastically accelerate a PAI-1 latency transition; that
the rate was in all cases normalized by the PAI-1 binding
protein vitronectin; and that substitution of an adjacent b
strand 5A Lys residue, believed to anchor b strand 5A to
other secondary structural elements, had differential effects
on the rates of latency transition in the absence and the
presence of vitronectin, respectively. An overlapping, but
not identical set of substitutions resulted in an increased
tendency to substrate behaviour of PAI-1 at reaction with its
target proteinases. These findings show that vitronectin
regulates the movements of the RCL through conformation-
al changes of the shutter region and b strand 5A, are in
agreement with RCL insertion proceeding by different
routes during latency transition and complex formation, and

residue by
an ester bond [4–6]. The subsequent RCL insertion into
b sheet A therefore results in an < 7-nm translocation of
the proteinase from the position of its initial encounter with
the RCL to the other pole of the serpin [7– 10]. The
translocation results in distortion of the proteinase [11] and
inactivation of the enzymatic machinery [10]. Delayed RCL
insertion results in hydrolysis of the ester bond, the serpin
thus behaving as an ordinary substrate [12]. The stabil-
ization caused by RCL insertion also underlies the unique
conversion of active PAI-1 to the latent state, in which the
N-terminal part of the intact RCL is inserted as b strand 4A
without cleavage of any peptide bonds, and the C-terminal
part is stretched along the surface of the molecule [13]
(Fig. 1).
In order to make space for the incoming new strand
during RCL insertion, a fragment of the structure consisting
of b strands 1A, 2A, 3A, and a helix F (the small serpin
fragment) must slide away from the rest of the structure (the
large serpin fragment). During the b sheet opening, the
region around a helices D and E forms a flexible joint, and
b strands 3A and 5A slide apart in a shutter-like manner over
the underlying a helix B [14]. The central part of b strands
3A and 5A and the N-terminal part of a helix B is therefore
referred to as the shutter region [2]. By high resolution X-ray
crystal structure analysis of the native form of the serpin
plasminogen activator inhibitor-2 (PAI-2) and the P
1
–P
1

1
-antitrypsin template numbering scheme [1,3]). Sequence
alignments of 219 serpins showed that residue 53 is a Ser in
92% of the cases; residue 56 is a Ser in 74% of the cases;
residue 186 an Asn in 87% of the cases; and residue 334 a
His in 80% of the cases [3]. In addition, residue 54 is a Pro in
89% of the cases. The importance of the identity of the
residues present in these and adjacent positions are
supported by the clustering of disease-causing mutations
in the shutter region [16,17].
PAI-1 differs from most other serpins with respect to the
identity of the residues in the buried cluster in the shutter
region, having a Gly in position 56 and a Gln in position
334 (Fig. 1). This composition of amino acids in
positions 53/56/334 is present in only 5% of the serpins,
for example PN-1, RASP-1, TSA2004, and the viral serpins
SPI-1, M2L, and H14-B [3]. A few previous studies have
addressed the importance of the shutter region for the
movements of the RCL in PAI-1. Berkenpas et al. [18]
demonstrated that Ser and Thr substitutions of Pro54
delayed latency transition. We showed that a Q334H
substitution accelerated latency transition [19]. We also
implicated the region of b strand 5A overlying the buried
cluster in RCL movements by demonstrating that increased
proteolytic susceptibility of the peptide bonds Gln331–
Ala332, Ala332–Leu333, and Lys335–Val336 accom-
panied a transition to substrate behaviour in detergent-
containing buffers at low temperatures [20,21]; and that a
K335A substitution potentiated activity-neutralization of
PAI-1 by some monoclonal antibodies [22]. Substitutions of

b strand 1C in active PAI-1 and RCL inserted
as b strand 4A in latent PAI-1. The P
1
Arg is
displayed as a stick. The lower panel shows the
three-dimensional structure of the shutter
region of active PAI-1 (left) and latent PAI-1
(right). The molecules were rotated < 908
around a horizontal axis compared to the top
panel. The colour code for secondary structure
elements are as in the top panel. Presented
amino-acid residues are: green, shutter region
residues Ser53, Gly56, and Gln334; grey,
Asn186 in b strand 3A; yellow, Lys335; purple,
potential interaction partners for Lys335, i.e.
Glu294 in b strand 6A and the backbone of
Asn171 in the a helix F/b strand 3A loop.
Note:
SWISSPDB VIEWER uses the same
signature for a helices and the short 3
10
-helix
found in the a helix F/b strand 3A loop of
active PAI-1.
q FEBS 2001 PAI-1 shutter region mutations (Eur. J. Biochem. 268) 6275
MATERIALS AND METHODS
PAI-1
In order to generate recombinant wild-type and mutated
PAI-1, PAI-1 cDNA [27] was cloned into the expression
vector pcDNA3.1(–) (Invitrogen) by use of standard

O added to a total of 1752 mL),
248 mL2
M CaCl
2
, and 2 mL 42 mM Hepes, pH 7.05,
274 m
M NaCl, 10 mM KCl, 1.5 mM Na
2
HPO
4
,11mM
D
-(þ )-glucose. After 1–2 min, this mixture was added
dropwise to the cell medium and carefully distributed. Fresh
medium without fetal bovine serum and chloroquine was
added after 9–11 h of incubation. The conditioned medium
was harvested after 48 and 96 h. Nontransfected or mock
transfected HEK293T cells were shown not to express either
PAI-1 or uPA by standard ELISA with monoclonal and
polyclonal antibodies as capture and detection antibodies,
respectively. Recombinant PAI-1 variants were purified
from serum-free conditioned medium of the transfected
cells by immunoaffinity chromatography in one step
[29,30]. After purification, the variants were dialysed
against NaCl/P
i
(0.01 M NaH
2
PO
4

The specific inhibitory activity of the reactivated PAI-1
variants was measured by titration against uPA in a direct
peptidyl anilide assay at 37 8C, in the presence or absence of
a slight excess of vitronectin over PAI-1 [30]. A twofold
dilution series of PAI-1, with or without vitronectin, was
made immediately after refolding, to avoid loss of activity
due to fast latency transition. The dilution series of
denatured and refolded PAI-1 (0–20 mg·mL
21
, 0–370 nM)
were quickly (in less than 1 min) mixed with an equal
volume (100 mL) of 0.25 mg·mL
21
(4.3 nM)uPA,0.1M
Tris, pH 8.1, 1% BSA or 0.2% Triton X-100. The final
concentrations of uPA was 0.125 mg·mL
21
(2.15 nM), of
PAI-1 in the range 0–10 mg·mL
21
(0–185 nM), and of
vitronectin in the range 0 –15 mg·mL
21
(0–200 nM). Upon
completion of the uPA inhibition reaction (. 5 min), the
remaining uPA activity in the reaction mixture was
determined by use of
L-5-oxopropyl-glycyl-L-arginine-
p-nitroanilide (S-2444), a chromogenic peptidyl anilide
substrate for uPA. The amount of active PAI-1, and thus the

micrograms of monoclonal murine anti-(PAI-1) IgG from
hybridoma clone 2 [31], coupled to Sepharose-4B, was
transferred to Ultrafreew-MC 0.22-mm filter units for
6276 M. Hansen et al. (Eur. J. Biochem. 268) q FEBS 2001
centrifugal filtration (Millipore, USA) and washed twice
with 0.1
M Tris, pH 8.1. The sample (PAI-1, LMW-uPA,
BSA) was then added and incubated for at least 30 min
followed by four washes with 0.1
M Tris, 1 M NaCl, pH 8.1,
and one wash with 0.1
M Tris, pH 8.1. PAI-1 was eluted by
incubation with 400 mL3
M ammoniumthiocyanat
(NH
4
SCN) for at least 30 min at 37 8C before centrifugation
at 16 500 g for 10 min. The samples were precipitated
with trichloroacetic acid, and subjected to 6–16% gradient
SDS/PAGE.
Determination of second order rate constants for the
reaction between PAI-1 and uPA
The second order rate constants were determined as
described previously [32]. The calculation of the second
order rate constants is based on the assumption that the
concentration of active PAI-1 is unchanged during the assay.
As most of these variants have significantly shorter
functional half-lives (see below) than wild-type, the
calculated second order rate constants for the variants
were expected to be somewhat lower than their real values.

r
67 000), murine IgG (M
r
150 000), and
b-galactosidase (M
r
540 000) are indicated by arrows above the
profiles.
Table 1. Specific inhibitory activity of PAI-1 variants towards uPA. The most common amino-acid composition of the buried polar cluster
(positions 53/56/334) in serpins is S/S/H. The composition S/S/Q is identical to that of alaserpin, S/G/H is identical to that of CP-9, A/G/H is identical
to that of heparin cofactor II, while the S/A/S composition is present in angiotensinogen [1,3]. The investigated residues according to the PAI-1
numbering (1Ser-Ala-Val-His-His-) are 37/40/324/325 [27]. Means ^ SD (numbers of assays are indicated). *, Significantly different from wild-type
(P , 0.005). †, Significantly different from the value without vitronectin (P , 0.005).
PAI-1 variant
Composition of
positions 53/56/334
PAI-1 activity
(% of theoretical max)
Vitronectin effect
(fold increase)– Vitronectin þ Vitronectin
Wild-type S/G/Q 87.1 ^ 22.2 (20) 113.7 ^ 28.0 (10) † 1.3
K335A S/G/Q 114.9 ^ 21.5 (5) 129.5 ^ 21.1 (3) 1.1
S53A A/G/Q 71.6 ^ 9.4 (8) 84.9 ^ 5.9 (3) 1.2
S53A/K335A A/G/Q 84.3 ^ 15.5 (4) 91.0 ^ 13.3 (3) 1.1
G56A S/A/Q 59.6 ^ 7.8 (6) * 56.1 ^ 6.3 (3) * 0.9
G56S S/S/Q 73.1 ^ 10.6 (5) 127.1 ^ 6.2 (3) † 1.7
G56S/K335A S/S/Q 120.0 ^ 1.6 (3) 129.6 ^ 2.5 (3) † 1.1
Q334A S/G/A 58.1 ^ 7.5 (6) * 73.1 ^ 5.7 (3) 1.3
Q334A/K335A S/G/A 62.8 ^ 4.7 (4) 57.8 ^ 5.3 (4) * 0.9
Q334H S/G/H 61.9 ^ 13.5 (6) 90.7 ^ 5.8 (3) 1.5

was routinely reactivated using SDS, as some of the variants
had a very fast latency transition (see below) and would
therefore lose all activity during the dialysis used for
refolding after guanidinium chloride denaturation. The
specific inhibitory activities of most PAI-1 variants, when
denatured with SDS and refolded in BSA-containing buffer,
were 60– 80% of the theoretical maximum, and thus
indistinguishable from recombinant PAI-1 wild-type
(Table 1). However, the recombinant variants G56A,
Q334A, S53A/Q334H, G56A/Q334S, and G56S/Q334H
showed a small, but statistically significant (P , 0.005)
reduction in specific inhibitory activity as compared to wild-
type. All variants except the variant G56A/Q334S had a
second order rate constant differing less than 2.5-fold from
that of wild-type (data not shown). The second order rate
constant for G56A/Q334S was 3.8-fold lower than that of
the wild-type, but this can be ascribed to a fast decrease in
inhibitory activity of this variant during the experiment (see
below). Vitronectin caused a small, but statistically
significant increase of the specific inhibitory activity of
the PAI-1 wild-type and the variants G56S, S53A/Q334H,
G56A/Q334S, G56S/Q334H, and G56S/K335A. Interest-
ingly, the specific inhibitory activity of G56A/Q334S was
slightly decreased by vitronectin.
Latency transition of PAI-1 wild-type and PAI-1 variants in
the absence and the presence of vitronectin
To estimate the functional stability of the variants, their
specific inhibitory activities were measured after different
times of incubation at 37 8C. The rate of activity loss was
determined in the absence or the presence of vitronectin.

towards that of PAI-1 wild-type (Table 2). Surprisingly, the
stabilizing effect of vitronectin was abolished by the K335A
substitution. None of the variants with this substitution had
longer half-lives in the presence of vitronectin, and with
most of them, vitronectin even accelerated the activity loss.
Hence, while the K335A substitution had a stabilizing
effect in the absence of vitronectin, it had a destabilizing
effect in the presence of vitronectin. Importantly, the
K335A substitution did not result in altered affinity of PAI-1
to vitronectin (T. Wind, & P.A. Andreasen, Department of
Molecular and Structural Biology, Aarhus University,
Denmark, personal communication).
Although the assays were routinely performed in a buffer
of 0.1
M Tris, pH 8.1, similar results were obtained with a
buffer of 0.1
M Tris, pH 7.4. In addition, when examining
the results obtained with SDS-activated PAI-1 vs.
guanidinium chloride-activated PAI-1 with wild-type and
two of the most stable variants (S53A and K335A), no
distinguishable difference was observed.
In order to ensure that the loss of activity during the
incubations at 37 8C was due to latency transition, PAI-1,
that had been incubated for various time periods at 37 8C,
was reacted with an excess of LMW-uPA, and the reaction
products were analysed by SDS/PAGE. Representative
experiments are shown in Fig. 4. Nonincubated wild-type
reacted to form the expected < 80 000-Da LMW-uPA–
PAI-1 complex. A fraction of wild-type reacted in a
substrate-manner, giving rise to the < 50 000-Da

substrate form, we replaced the BSA in the assay buffer with
0.2% Triton X-100. The variants with a S53A, Q334A, or
Q334S substitution all had significantly reduced specific
inhibitory activity in Triton X-100 containing buffer at
37 8C as compared to PAI-1 wild-type (Table 3). Analysis of
Table 2. Stability of specific inhibitory activity of PAI-1 variants at 37 8C. Means, SDs, and numbers of experiments are indicated. *, Significantly
different from wild-type (P , 0.005). †, Significantly different from the value without vitronectin (P , 0.005). ‡, Significantly different from the
corresponding variants without the K335A substitution (P , 0.02).
PAI-1 variant
Functional half-lives
(min)
Vitronectin effect
(fold increase)– Vitronectin þ Vitronectin
Wild-type 54.7 ^ 13.5 (16) 63.4 ^ 11.6 (10) 1.2
K335A 76.9 ^ 11.6 (4) *‡ 35.3 ^ 6.7 (3) *†‡ 0.5
S53A 64.0 ^ 8.6 (5) 100.7 ^ 9.4 (3) *† 1.6
S53A/K335A 85.1 ^ 20.5 (4) * 32.0 ^ 10.0 (3) *†‡ 0.4
G56A 26.1 ^ 4.4 (4) * 36.5 ^ 6.8 (3) * 1.4
G56S 19.7 ^ 1.7 (4) * 54.9 ^ 4.1 (3) † 2.8
G56S/K335A 25.5 ^ 6.3 (3) * 12.3 ^ 2.1 (3) *‡ 0.5
Q334A 10.9 ^ 1.5 (4) * 39.9 ^ 10.2 (3) *† 3.7
Q334A/K335A 12.9 ^ 2.9 (3) * 14.1 ^ 0.9 (3) *‡ 1.1
Q334H 10.9 ^ 1.4 (4) * 52.3 ^ 6.0 (3) † 4.8
Q334H/K335A 32.1 ^ 2.7 (5) *‡ 29.4 ^ 1.2 (3) *‡ 0.9
Q334S 23.4 ^ 2.5 (5) * 75.2 ^ 15.8 (3) † 3.2
S53A/Q334H 18.5 ^ 3.2 (7) * 78.3 ^ 12.5 (3) † 4.2
G56A/Q334S 9.7 ^ 1.6 (4) * 72.9 ^ 17.7 (3) † 7.5
G56S/Q334H 6.1 ^ 1.5 (7) * 62.4 ^ 15.6 (4) † 10.2
G56S/Q334H/K335A 27.8 ^ 2.6 (5) *‡ 34.3 ^ 2.3 (3) *‡ 1.2
q FEBS 2001 PAI-1 shutter region mutations (Eur. J. Biochem. 268) 6279

must imply the passage of the intact RCL through the ‘gate
region’, which is situated between (a) the turn between b
strands 3C and 4C (residues 204–219) and (b) the turn
between b strands 3B and a helix G (residues 257–259)
[13,38,39] (Fig. 1). Because of steric reasons, it is not very
likely that the RCL can surround the turn between b strands
3C and 4C without having a completely stretched-out
conformation. Only after the RCL has passed this turn can
the final insertion into b sheet A proceed [39]. Considering
that RCL insertion into b sheet A is several orders of
magnitude faster during complex formation than during
latency transition, it seems reasonable to presume that the
passage of the RCL through the gate region is rate limiting
for latency transition. This presumption is supported by the
observation that substitutions of basic residues in the turn
between b strands 3C and 4C with acidic residues accelerate
latency transition [40,41]. On this basis, we reach the
conclusion that the substitutions in the shutter region affect
the rate of latency transition by affecting the rate of passage
of the RCL through the gate region. Based on the amino-
acid sequence of the RCL and b strand 5A being directly
continuous, it may be proposed that movements of the RCL
during passage through the gate region are coupled to
movements of b strand 5A and therefore sensitive to the
interactions of b strand 5Awith the underlying structure. An
alternative, but with the presently available information, a
less likely explanation is that passage of the RCL through
the gate region is rapid and reversible, and that it is the b
sheet A opening and the final insertion of RCL as b strand
4A that is rate limiting for latency transition.

Table 3. Effect of 0.2% Triton X-100 on specific inhibitory activity
of PAI-1 at 37 8C. The specific inhibitory activity of each variant is
given as a fraction of the specific inhibitory activity of the same variant
in 1% BSA. Means, SDs, and numbers of experiments are indicated. *,
Significantly different from wild-type (P , 0.005).
PAI-1 variant Specific inhibitory activity
Wild-type 0.87 ^ 0.12 (7)
K335A 0.95 ^ 0.09 (3)
S53A 0.20 ^ 0.02 (3)*
S53A/K335A 0.17 ^ 0.02 (3)*
G56A 0.65 ^ 0.11 (3)
G56S 1.17 ^ 0.16 (3)
G56S/K335A 1.06 ^ 0.03 (3)
Q334A 0.12 ^ 0.01 (3)*
Q334A/K335A 0.18 ^ 0.03 (3)*
Q334H 0.61 ^ 0.08 (3)*
Q334H/K335A 0.77 ^ 0.01 (3)
Q334S 0.21 ^ 0.05 (3)*
S53A/Q334H 0.13 ^ 0.01 (3)*
G56A/Q334S 0.10 ^ 0.02 (3)*
G56S/Q334H 0.45 ^ 0.03 (3)*
G56S/Q334H/K335A 0.74 ^ 0.09 (3)
6280 M. Hansen et al. (Eur. J. Biochem. 268) q FEBS 2001
the conformation of the shutter region. Second, the rate of
strand insertion during latency transition may be affected
not only through a change in the conformation of the active
form, but also by a change in the conformation of a
transition state with an unknown three-dimensional
structure. Nevertheless, it seems reasonable to conclude
that the hydrogen bonds from the side chain of S53A have

over-ruling the effect of the local hydrogen bonding network
of the residues in positions 53, 56, and 334.
The K335A substitution delayed the latency transition
when introduced in some of the variants, and most strongly
when introduced into the very unstable variants Q334H and
G56S/Q334H. The side chain of Lys335 points away from
the buried cluster in positions 53/56/334 (Fig. 1). On the
basis of the available three-dimensional structures, several
intramolecular interactions of Lys335 may be suggested.
The possible interactions include a connection to the loop
between a helix F and b strand 3A by a hydrogen bond to
the carbonyl oxygen atom of the backbone of Asn171
[19,22], by hydrophobic interactions with residues in that
loop [23], or by participation in formation of a chloride
binding site together with residues in that loop and Lys337
[44]. The possible interactions also include a salt bridge to
Glu294 in b strand 6A (Fig. 1). It therefore seems likely that
the constraints caused by the interactions of Lys335
contribute to maintaining the RCL in a state with a
relatively facilitated passage through the gate region during
latency transition, via an effect on the conformation of
b strand 5A and of the buried cluster in positions 53, 56, and
334.
In contrast, in the presence of vitronectin, the K335A
substitution caused a twofold to fivefold acceleration of
latency transition compared to wild-type. In fact, vitronectin
did not delay latency transition of any of the variants
harbouring the K335A substitution. On the basis of the
opposite effects of the K335A substitution in the absence
and presence of vitronectin, we propose that the

between the side chains, resulting in a delay in strand
insertion during reaction with the target proteinase. On the
other hand, the Triton X-100-induced substrate behaviour
did not seem to implicate the interactions of the Lys335 side
chain, in contrast to antibody-induced substrate behaviour
that was potentiated by the K335A substitution [19,22]. The
observation of latency transition and complex formation
being affected differently by mutations in the shutter region
and b strand 5A is in agreement with RCL insertion
following different routes in the two cases.
PAI-1 is a potential target for antithrombotic [45] and
anticancer therapy [46,47]. The biochemical mechanism of
action of a few PAI-1 neutralisers has been characterized,
including monoclonal antibodies and organochemical
compounds. These compounds neutralize PAI-1 either by
steric hindrance, by inducing conversion to the latent state,
by inducing substrate behaviour, and/or by inducing
conversion to inert polymers [48– 52]. The present results
prompt further studies into the role of the shutter region and
b strand 5A in PAI-1 in conformational changes leading to
neutralization.
ACKNOWLEDGEMENTS
Dr Kees Rodenburg is thanked for fruitful discussions in the early phase
of this work. Dr Claus Oxvig is acknowledged for providing the
HEK293T cell line. This work was supported financially by the Danish
Cancer Society, the Danish Research Agency, the Danish Heart
Foundation, the NOVO-Nordisk Foundation, and the Danish Cancer
Foundation.
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