We grafted regions of the hF/s3A-loop from antithrombin III and a1-protease inhibitor onto PAI-1, creating eight variants, and found that one of these rever-sions towards the serpin cons
Trang 1Mutational analysis of plasminogen activator inhibitor-1
Interactions of a-helix F and its neighbouring structural elements regulates
the activity and the rate of latency transition
Troels Wind*, Jan K Jensen, Daniel M Dupont, Paulina Kulig and Peter A Andreasen
Laboratory of Cellular Protein Science, Department of Molecular Biology, Aarhus University, Denmark
The serpin plasminogen activator inhibitor-1 (PAI-1) is a
fast and specific inhibitor of the plasminogen activating
serine proteases tissue-type and urokinase-type plasminogen
activator and, as such, an important regulator in turnover of
extracellular matrix and in fibrinolysis PAI-1 spontaneously
loses its antiproteolytic activity by inserting its reactive centre
loop (RCL) as strand 4 in b-sheet A, thereby converting to
the so-called latent state We have investigated the
import-ance of the amino acid sequence of a-helixF (hF) and the
connecting loop to s3A (hF/s3A-loop) for the rate of latency
transition We grafted regions of the hF/s3A-loop from
antithrombin III and a1-protease inhibitor onto PAI-1,
creating eight variants, and found that one of these
rever-sions towards the serpin consensus decreased the rate of
latency transition We prepared 28 PAI-1 variants with
individual residues in hF and b-sheet A replaced by an alanine We found that mutating serpin consensus residues always had functional consequences whereas mutating nonconserved residues only had so in one case Two variants had low but stable inhibitory activity and a pronounced tendency towards substrate behaviour, suggesting that insertion of the RCL is held back during latency transition as well as during complexformation with target proteases The data presented identify new determinants of PAI-1 latency transition and provide general insight into the characteristic loop–sheet interactions in serpins
Keywords: alignment; conformation; mutational analysis; PAI-1; proteases; serpin
Plasminogen activator inhibitor-1 (PAI-1) is the primary
inhibitor of both urokinase-type and tissue-type
plasmino-gen activator (uPA and tPA, respectively) and as such is an
important regulator of physiological events in which
plasmin-catalysed extracellular proteolysis is involved
PAI-1 belongs to the serine protease inhibitor (serpin)
family whose antiproteolytic activity is governed by their
structural flexibility In the active serpin conformation, the
reactive centre loop (RCL) with the P1–P1¢ bait peptide
bond is surface exposed Formation of the covalent serpin–
protease complexinvolves a Michaelis docking complex,
cleavage of the P1–P1¢ peptide bond, linkage of the active
site Ser of the protease to the carboxyl group of P1by an
ester bond and insertion of the N-terminal part of the RCL
as strand 4 in b-sheet A (s4A) of the serpin Consequently, the protease is trapped in a covalent acyl-enzyme complexin which its reactive site is distorted, as illustrated by the crystal structure of the complexbetween a1-protease inhibitor (a1PI, also referred to as a1-antitrypsin) and trypsin [1] Under some conditions, however, RCL insertion is delayed, resulting in hydrolysis of the ester bond, release of free protease and insertion of the cleaved RCL as s4A This pathway is referred to as substrate behaviour of the serpin Complexformation between serpins and their cognate proteases is fuelled by the thermodynamic properties of the serpin Accordingly, insertion of the RCL as s4A and the ensuing structural rearrangements of the serpin stabilizes the molecule in a so-called relaxed conformation, as opposed to the metastable stressed conformation with the RCL exposed on the surface (reviewed in [2–4])
PAI-1 spontaneously converts into a relaxed conforma-tion at a significant rate without cleavage of the RCL (for a review see [5]) During this structural transformation, referred to as latency transition, the N-terminal part of the intact RCL is inserted as s4A [6] (Fig 1) Latent versions
of the serpins antithrombin III (ATIII) [7], a1-protease inhibitor (a1PI) [8], and a1-antichymotrypsin (a1ACT) [9] have also been isolated, but none of these undergo this transition as readily as PAI-1 The physiological role of PAI-1 latency transition, if any, remains elusive [5] Some PAI-1 variants with single mutations and modest decreases in the rate of latency transition have been obtained through heuristic protein engineering [10,11] while others have been identified by chance [12–15] The variants with the slowest latency transition carry multiple mutations
Correspondence to J K Jensen, Laboratory of Cellular Protein
Science, Department of Molecular Biology, Aarhus University,
Gustav Wieds Vej 10C, 8000 A˚rhus C, Denmark.
Fax: + 45 86123178, Tel.: + 45 89425074,
E-mail: jkj@mb.au.dk
Abbreviations: PAI-1, plasminogen activator inhibitor-1; RCL,
reactive centre loop; a 1 PI, a 1 -protease inhibitor (a 1 -antitrypsin);
a 1 ACT, a 1 -antichymotrypsin; ATIII, antithrombin III;
hF, a-helixF; HMK, heart muscle kinase.
Enzyme: uPA, urokinase-type plasminogen activator (EC 3.4.21.73).
*Present address: Centre for Vascular Research, School of Medical
Sciences, The University of New South Wales,
Sydney NSW 2052, Australia.
(Received 4 December 2002, revised 7 February 2003,
accepted 13 February 2003)
Trang 2and have been obtained by random mutagenesis followed
by screening or selection procedures Berkenpas et al
isolated several PAI-1 variants with increased stability of
which the most stable, referred to in the following as
PAI-1stab, carried four amino acid substitutions (N152H, K156T,
Q321L and M356I) [16] The three-dimensional structure of
PAI-1stabhas been determined and reveals that the
stabil-izing amino acid substitutions N152H and K156T induces a
310-helixspanning residues 155–157 in the loop connecting
a-helixF to b-strand 3A (the hF/s3A-loop, Fig 1) [17–19]
Likewise, Stoop et al isolated a panel of stable PAI-1
variants of which the most stable carried 10 amino acid
substitutions [20] The structure of this variant, however, has
not been determined A prevalent theme among the
stabilizing amino acid substitutions is a reversion towards
the consensus sequence of inhibitory serpins, suggesting that
part of the molecular basis for latency transition can be
found in regions of PAI-1 that deviate from this consensus
[16,20]
Based on the following observations, we speculated that
the amino acid composition of the hF/s3A-loop and hF
plays a role in the stressed-to-relaxed transition of PAI-1:
The hF/s3A-loop must fold away from b-sheet A during the
structural transition to allow insertion of the RCL as s4A [6]
(Fig 1) Stabilizing mutations have been identified in the
hF/s3A-loop [16,20] and the aforementioned 310-helixin
PA1–1stab has been suggested to, at least in part, be
responsible for the increased stability of this variant [17–19]
Some monoclonal antibodies towards hF and the
hF/s3A-loop [21–24] as well as deletion of this region [25] induce substrate behaviour of PAI-1 In addition, Gettins recently hypothesized that a thermodynamically unfavourable dis-location of hF, resulting from insertion of the RCL, provides an energy-reservoir that subsequently fuels the crushing of the protease concomitantly with the return of
hF to its normal position [26]
In the present study, we used structural alignments to define residues in the hF/s3A-loop in PAI-1 that deviate from the serpin consensus and replaced them with the corresponding residues from the representative inhibitory serpins a1PI and ATIII As there are no discrepancies between the PAI-1 sequence and the serpin consensus in
hF, we chose alanine-scanning mutagenesis as a means of identifying hF-residues with importance for RCL insertion Likewise, residues from b-sheet A with putative contacts to
hF or the hF/s3A-loop were individually replaced by alanine In total, 38 PAI-1 variants were characterized in terms of latency transition and functional behaviour upon interaction with uPA and several side chains involved in the retardation of latency transition, and hence of importance for the functional stability of stressed PAI-1, were identified Finally, we describe PAI-1 variants that can adapt remarkably stable conformations and predom-inantly behave as substrates for uPA As will be discussed, the data presented can probably be extrapolated to other serpins and thus provide further insight into the molecular details of the stressed-to-relaxed transition of these proteins [27]
Fig 1 Ribbon diagrams of relaxed, latent PAI-1 (right) and stressed, active PAI-1 stab (left) [18] Insertion of the reactive centre loop (red) as strand 4
in b-sheet A (pink) requires mobility of hF and the hF/s3A-loop (orange).
Trang 3Materials and methods
Cloning, mutagenensis and purification of PAI-1
The cDNA for human PAI-1 was modified to include a
N-terminal His6-tag plus a recognition motif for heart
muscle kinase and cloned into the Escherichia coli
expres-sion vector pT7-PL [28] Mutations in PAI-1 were
intro-duced with the QuickChange Site-Directed Mutagenesis Kit
(Stratagene) as described by the manufacturer, except that
the final product was electroporated into E coli DH5a cells,
and confirmed by sequencing using the Thermo Sequenase
II Dye Terminator Cycle Sequencing Kit (Amersham
Pharmacia Biotech) and a 373A ABI sequencer (Applied
Biosystems) Numbering of residues in PAI-1 was S1-A2-V3
-H4-H5… [29]
For gene expression, individual colonies of transformed
E coli cells BL21(DE3)pLysS (Novagen) were inoculated
into 2· TY broth (16 gÆL)1 tryptone, 10 gÆL)1 yeast
extract, 5 gÆL)1 NaCl) supplemented with 100 lgÆmL)1
ampicillin and 34 lgÆmL)1chloramphenicol and incubated
overnight at room temperature The cultures were diluted
1 : 20 and incubated at 37C until a D600between 0.7 and
0.9 A final concentration of 0.5 mM isopropyl thio-b-D
-galactoside was added to induce gene expression and the
incubation was continued for 2 h From this point, protein
purification was performed at 4C The cells were harvested
by centrifugation (7000 g, 20 min), resuspended in 35 mL
phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl,
1.4 mM KH2PO4, 4.3 mM Na2HPO4) and opened with
sonication The bacterial lysates were cleared by
centrifu-gation (15 000 g, 30 min) and filtration (0.22 lm),
supple-mented with 2MNaCl, 10 mMimidazole and 5% glycerol,
and applied to a 5-mL Ni-nitrilotriacetic acid column
(Qiagen) equilibrated in the same buffer After extensive
washing with the equilibration-buffer, PAI-1 was eluted by
increasing the concentration of imidazole to 200 mM The
eluted protein was subjected to gel filtration on a Superdex
75 column (1.6· 60 cm, Amersham Pharmacia Biotech)
that had been equilibrated in Hepes-buffered saline (10 mM
Hepes, 0.14MNaCl, pH 7.4 at 37C) supplemented with
5% glycerol and a final concentration of 1M NaCl
Fractions containing PAI-1 were pooled, the concentration
determined from A280 using the calculated extinction
coefficient 0.77 mLÆmg)1Æcm)1[30], and stored at )80 C
until used This procedure routinely gave 2–15 mg PAI-1
per litre of culture and N-terminal sequencing revealed that
the recombinant protein had the expected N terminus, i.e
(M)GSMGSHHHHHHGSRRASV3…, where the
initi-ating M in parentheses is missing The phosphorylation site
for heart muscle kinase (HMK, underlined) allows
radio-active labelling of the molecule, a feature not used in
the present study The reactivity of His- and HMK-tagged
PAI-1 in terms of uPA inhibition and vitronectin binding
was not affected by this modification [31]
SDS/PAGE analysis of functional behaviour
Reactions between recombinant PAI-1 (100 lgÆmL)1) and
uPA (200 lgÆmL)1, Wakamoto Pharmaceutical Company,
Tokyo, Japan) were performed in HBS at 37C for 30 min
and quenched by boiling in SDS sample buffer The reaction
products were subjected to nonreducing SDS/PAGE in 11% acrylamide gels followed by staining with Coomassie brilliant blue For time-course experiments, PAI-1 (200 lgÆmL)1) was incubated in HBS at 37C for up to
24 h before reaction with uPA, followed by SDS/PAGE Band intensities were determined by scanning densitometry Determination of functional half-lives
The general buffer for the assay described was HBS (pH 7.4
at 37C) supplemented with 0.25% gelatine and unless stated otherwise, all incubations were at 37C PAI-1 was incubated at a concentration of 20 lgÆmL)1and at various time-points, aliquots were taken for preparation of a twofold dilution series in a 96-well plate with 100 lL PAI-1 per well in concentrations ranging from 20 to 0.0098 lgÆmL)1 Immediately thereafter, 100 lL 0.5 lgÆmL)1uPA (0.25 or 0.125 lgÆmL)1uPA for PAI-1 variants with activity below 20%) was added to each well followed by incubation for at least 5 min to allow complex formation between uPA and PAI-1 The remaining uPA activity in each well was determined as the absorbance
at 405 nm after addition of 25 lL 0.3 mgÆmL)1 S-2444 (Chromogenix, Sweden) and further incubation for 40 min The specific inhibitory activity of PAI-1 at the various time-points, i.e the fraction of the total amount of PAI-1 forming
a stable complexwith uPA, was calculated from the amount
of PAI-1 required to inhibit half the uPA The half-life of PAI-1 was finally calculated from an exponential decay plot
of the data obtained Generally, only one preparation of each PAI-1 variant was investigated, but the following were investigated with two independent preparations, giving indistinguishable results: wild-type, 1(T96A), PAI-1(F100A), PAI-1(V126A), PAI-1(F128A), PAI-1(I137A), PAI-1(I138A), PAI-1(N139A), PAI-1(W141A), PAI-1(T146A) and PAI-1(M149K)
Structural analysis Structural analysis was based on the following depositions
in the Protein Data Bank ([32]; PDB-ID is given in parenthesis): active (1DVM) and latent (1DVN) PAI-1 [18], active a1-PI (1QLP) [33], cleaved a1-PI (7API) [34], active and latent antithrombin (1E05) [35].SWISS PDB-VIEWER
v3.51 (http://www.expasy.ch/spdbv/) was used for visu-alization and structural alignments
Statistical analysis Rates of latency transition were compared using an unpaired t-test
Results
Reversions to the serpin consensus in the hF/s3A-loop
of PAI-1
To determine if the hF/s3A-loop governs latency transition
of PAI-1 by more readily allowing insertion of the intact RCL than the corresponding loop from other inhibitory serpins, we prepared PAI-1 variants with hF/s3A-loops that mimic those found in the inhibitory serpins aPI and ATIII,
Trang 4respectively Table 1 shows a structure-based sequence
alignment of hF/s3A-loops from the relaxed serpin
structures cleaved a1PI [34], latent PAI-1 [18], and latent
ATIII [35] Also, the consensus serpin sequence of the
hF/s3A-loop is included in Table 1 (adapted from [2])
Alignment of the three structures was performed
accord-ing to the Ca atoms in the rigid serpin fragment 2c [36],
encompassing residues 129–155 in PAI-1 The
hF/s3A-loop of PAI-1 deviates from the consensus serpin
sequence at position 149 (M instead of a basic residue,
e.g K168 in a1PI) and position 152 (Asn instead of an
acidic residue, e.g D171 in a1PI) Also, the stretch
GKGA(155–158) appears more flexible in PAI-1 than the
corresponding stretch in most other serpins, either because
of its length (e.g compared to a1PI) or the lack of Pro
residues (e.g compared to ATIII) (Table 1 and [2]) We
prepared the PAI-1 variants PAI-1(M149K),
1(N152D), 1(G155K, D(156–157), A158E) and
PAI-1(G155P, K156S, G157E) where the latter two have the
stretch between position 155 and 158 replaced by the
corresponding stretch from a1PI and ATIII, respectively
(Table 1) These variants were all found to be not
significantly different from the wild-type in terms of
specific inhibitory activity (Table 2) Thus, the introduced
mutations did not compromise the correct folding of PAI-1
in its active conformation
Compared with the wild-type protein, PAI-1(M149K),
and to a lesser extent PAI-1(G155K, D(156–157), A158E),
had a decreased rate of latency transition; PAI-1(N152D)
had a similar rate; and a slightly increased rate for PAI-1(G155P, K156S, G157E) was counteracted by introducing the N152D mutation (Table 2)
The two mutations M149K and N152D were introduced individually or together in the PAI-1(G155K, D(156–157), A158E) background and the resulting variants were found
to behave as wild-type PAI-1 towards uPA in terms of inhibitory activity (Table 2) Combining the M149K and [G155K, D(156–157), A158E] mutations did not decrease the rate of latency transition compared to M149K alone (T½¼ 156 ± 13 min vs 136 ± 24 min, P¼ 0.16) (Table 2)
Alanine scanning mutagenesis The s5A residues K325 and K327 have been suggested
to coordinate a chloride ion between b-sheet A and the hF/s3A-loop [18] The s6A residues E283 and E285 are potential partners for electrostatic interactions with K325 and K327, and E285 makes contact with the hF/s3A-loop in PAI-1stab[18] Finally, T96 (s2A) forms a hydrogen bond
to the hF-residues H145 in latent PAI-1 and W141 in PAI-1stab[18] and F100 (s2A), V126 (s1A) and F128 (s1A/ hF-loop) form part of the hydrophobic interface between
hF and b-sheet A These eight side chains (E283, E285, K325, K327, T96, F100, V126 and F128), each of the residues in hF (i.e S129 to K147), and the hF/s3A-loop residues M149 and N152 (see above) were substituted with
A and the resulting variants were characterized in terms of
Table 2 Reversions to the serpin-consensus in the hF/s3A-loop For each PAI-1 variant, the specific inhibitory activity towards uPA was determined
in a peptidolytic assay and expressed as percentage of the theoretical maximum The activity was monitored over time and the rate of latency transition expressed as the functional half-life, t ½ The averages and standard deviations for at least three independent experiments are given.
* Significantly different from the corresponding value for wild-type (P < 0.005).
Table 1 Structure-based sequence alignment of the hF/s3A-loops from the three inhibitory serpins PAI-1, a 1 PI and ATIII Residue numbering is according to PAI-1 [29] The alignment is based on the three-dimensional structures of the relaxed conformations of the serpins (see text for details) Also shown is the serpin consensus sequence for this region, adapted from [2].
E
V
L I
Trang 5specific inhibitory activity, functional behaviour and the
rate of latency transition
Among the variants tested, the following had a more than
threefold reduced specific inhibitory activity: F100A,
V126A, F128A, I137A, N139A, W141A, T146A, M149A,
N152A, and K327A (Table 3) Their functional behaviour
was analysed by treatment with uPA followed by SDS/
PAGE and scanning densitometry (Fig 2) In that analysis,
inhibitory active PAI-1 will migrate as a complexwith uPA
while PAI-1 exhibiting substrate behaviour will migrate
slightly faster than native PAI-1 due to cleavage of the
C-terminal 33 amino acids Latent PAI-1 or PAI-1
other-wise inert to uPA will comigrate with native PAI-1 This
analysis showed that the substitutions F100A, V126A,
F128A, I137A, N139A, W141A, T146A and N152A
increased the fraction of PAI-1 molecules behaving as a
substrate for uPA to between 40 and 60%, compared to
15% for the wild-type protein (Fig 2) They thus showed
a readily distinguishable substrate behaviour To assay the stability of this substrate behaviour, PAI-1 variants were incubated for up to 24 h at 37C prior to reaction with uPA and SDS/PAGE analysis After 24 h, the fraction of molecules behaving as a substrate for uPA decreased approximately twofold for PAI-1(V126A), PAI-1(F100A), PAI-1(F128A) and PAI-1(W141A) with a concomitant increase in the fraction being inert to uPA Substrate behaviour remained almost constant for PAI-1(I137A), PAI-1(N139A), PAI-1(T146A) and PAI-1(N152A) for 24 h (Fig 3 and not shown) The lower specific inhibitory activity of M149A and K327A was associated solely with
an increased fraction in a form comigrating with native PAI-1, and thus in an inert, probably latent conformation (Fig 2)
The rate of latency transition was determined for all variants Typical assays are depicted in Fig 4, and the data for all the variants are summarized in Table 3 Replacing any of the residues E132, R135, D140, K147, M149, E283 and K327, respectively, with an A increased the rate of latency transition more than twofold Three variants, I137A, V142A, and N152A, had a biphasic loss of activity, one component with a significantly faster latency transition rate and another component with a significantly slower latency transition rate The activity of the three variants PAI-1 (N139A), PAI-1(W141A) and PAI-1(T146A) remained almost invariant for several hours at 37C (Table 3) The K325A substitution slightly delays latency transition
in PAI-1, which is in agreement with our previous obser-vations [37,38] Less pronounced, but still significantly slower latency transition were observed with the substitu-tions T96A and I138A The E285A substitution also slightly delays latency transition of wild-type PAI-1 whereas in the PAI-1stabbackground, it accelerates latency transition
Discussion
The only noteworthy stabilizing effect resulting from reversions to the serpin consensus in the hF/s3A-loop was seen for PAI-1(M149K) (Table 2) In the relaxed serpin conformation, M149 in PAI-1 and the corresponding K168
Table 3 Alanine-scanning For each PAI-1 variant, the specific
inhibitory activity towards uPA was determined in a peptidolytic assay
and expressed as percentage of the theoretical maximum The activity
was monitored over time and the rate of latency transition expressed as
the functional half-life, t ½ The averages and standard deviations for at
least three independent measurements are given for each variant.
PAI-1 variant Activity (%) t ½ (min)
PAI-1 stab (E285A) 76 ± 3 680 ± 35* , **
a
A biphasic loss of activity was observed, suggesting a
hetero-geneity in the active fraction Note that the wild-type residue at
position 134 is an A * Significantly different from the
corres-ponding value for wild-type (P < 0.005); ** Significantly different
from the corresponding value for PAI-1 stab (P < 0.005).
Fig 2 Reaction products following reaction of PAI-1 variants with uPA PAI-1 variants (100 lgÆmL)1), indicated by their amino acid substitution, were reacted with uPA (200 lgÆmL)1) at 37 C in HBS,
pH 7.4 for 30 min and the products were separated by nonreducing SDS/PAGE (11% acrylamide) followed by staining with Coomassie brilliant blue The migration of the uPA–PAI-1 complex, uPA, intact (inert) PAI-1 and cleaved PAI-1 is indicated on the right.
Trang 6in a1PI are located at a critical position at the top of the hF/
s3A-loop right above the inserted s4A while in the stressed
conformation of PAI-1staband a1PI, they stack against the
aromatic moiety of the s3A residues Y172 and F189,
respectively [18,33,34] (Fig 5) Accordingly, we assume that
the aliphatic moiety of the introduced K in PAI-1(M149K)
allows the side chain to adapt the same orientation as the
original M and therefore the stabilizing effect of the M149K
substitution is likely to be governed by the introduced
positive charge In stressed ATIII, the RCL is partially
inserted as the top of s4A and the equivalent of M149 from PAI-1, i.e R197, is located right above the bifurcation between s3A and s5A [35] In light of this, we suggest that a positively charged side chain close to the point of initial insertion of the RCL [36] represents an obstacle for the local structural rearrangements required for the movements of the RCL during latency transition N152 is often replaced
by D in PAI-1 variants carrying several mutations that lead
to a decreased rate of latency transition [20,39] However, the N152D mutation does not per se delay latency transition
in PAI-1 (Table 2) Substitution of the stretch GKGA(155– 158) in PAI-1 with the corresponding stretch from a1PI or ATIII had only modest effects on the rate of latency transition (Table 2) Therefore, besides the M at position
149, deviations from the serpin consensus in the hF/s3A-loop of PAI-1 does not contribute to the rate of latency transition
Alanine-scanning mutagenesis identified side chains that contribute to the functional stability of PAI-1 as their removal increased the rate of latency transition more than twofold (Table 3) In principle, this observation can imply two things: (a) the side chain in question contributes to the thermodynamic stability of the stressed serpin conforma-tion, which is why its removal makes the latency transition energetically more favourable; (b) alternatively, the side chain in question is instrumental in obstructing the conformational changes occurring during the latency transition Accordingly, we propose that the hF side chains
of D140, K147 (forming a salt-bridge to s2A), M149 (packing against Y172 in s3A, Fig 5), and the salt-bridge between E132 and R135 [18] contribute to the thermo-dynamic stability of the stressed conformation and/or are important for the positioning of hF in a way delaying the proper movements of the intact RCL during latency transition PAI-1(I137A), PAI-1(V142A) and
Fig 3 Time-course experiment showing the substrate behaviour of selected variants PAI-1 (200 lgÆmL)1) was incubated at 37 C in HBS pH 7.4, and at the indicated time points, aliquots were reacted with a twofold molar excess of uPA for 30 min Reaction products were analysed by nonreducing SDS/PAGE followed by staining with Coomassie brilliant blue The migration of the uPA–PAI-1 complex, uPA, intact (inert) PAI-1 and cleaved PAI-1 is indicated on the right.
Fig 4 Time-course experiment showing the inhibitory activity towards
uPA of representative PAI-1 variants as measured in a peptidolytic
assay PAI-1 (20 lgÆmL)1) was incubated in HBS supplemented with
0.25% gelatine at 37 C and at the indicated time-points, samples were
taken and the inhibitory activity determined Activity was plotted
semilogarithmically against time The experiment shown is a typical
one out of a total of at least three.
Trang 7PAI-1 (N152A) all showed a biphasic loss of activity
suggesting a heterogeneity in the active fraction of these
variants That substituting either of the juxtaposed residues
E283 (s6A) or K327 (s5A) with an A increases the rate of
latency transition may be related to the proposed role for
K327 in the coordination of a stabilizing chloride ion [18] or
suggest the existence of a stabilizing salt-bridge between the
two side chains (Fig 5)
In contrast, substituting T96 in s2A, I138 in hF, K325 in
s5A or E285 in s6A with A increased the half-life of latency
transition by 24–68% (Table 3) The T96A substitution is a
reversion to the serpin consensus A/G (G115 in a1PI) [2],
suggesting that the absence of a side chain beyond the C
atom at this position increases the functional stability of the stressed serpin I138 is highly conserved among serpins (I157
in a1PI) [2], buried between hF and b-sheet A (Fig 5) and may be instrumental in promoting the translocation of hF during RCL insertion K325 is also conserved among serpins (K335 in a1PI) [2] and its substitution for A in a1PI,
a1ACT and ATIII has been suggested to stabilize the stressed conformation of these serpins by relieving the strain
of side chain overpacking between the K325 side chain and residues in the hF/s3A-loop, i.e the conserved I150 and L153 in PAI-1 ([40,41], see Table 1) This is in good agreement with our observation of a decreased rate of latency transition for PAI-1(K325A) (Table 3 and [37,38]) The side chains of K325 and E285 are juxtaposed, which is why the E285A substitution may provide a spatial relief mimicking the effect of the K325A substitution (Fig 5) Of note is that the side chain of E285 forms a hydrogen bond to the backbone of the hF/s3A-loop in PAI-1stab[18] (Fig 5) and in contrast with the wild-type protein, the functional stability of this variant is decreased by the E285A substi-tution (Table 3) This advocates that the contact between the E285 side chain and the hF/s3A-loop contributes to the functional stability of PAI-1staband that a similar contact is not present in the stressed conformation of the wild-type protein
Inhibitory activity and substrate behaviour of PAI-1(N139A) and PAI-1(T146A) were found to be invariant for several hours (Fig 3 and Table 3) Both events require
an exposed RCL, and it therefore seems that insertion of the intact RCL during latency transition as well as insertion of the cleaved RCL during complexformation with uPA is retarded in these variants [42] Both N139 and T146 are highly conserved among serpins (N158 and T165, respectively, in a1PI) [2] and form hydrogen bonds to the hF/s3A-loop [18] (Fig 5) Considering the almost identical phenotypes of PAI-1(N139A), PAI-1(T146A), and the close spatial proximity and similar structural role of N139 and T146, we find it likely that the structures of these variants, with the RCL exposed, are similar and contains a distorted
hF that delays insertion of the RCL
Substituting W141 with an A leads to substrate behaviour and a low, stable inhibitory activity (Fig 2 and Table 3) This W is located in the cleft between hF and s2A (Fig 5), and the presence of an aromatic side chain at this position is common in serpins [2] Mutation
of the equivalent Y160 in a1PI to A or W resulted in decreased or increased thermodynamic stability, respect-ively, and in line with our observation for PAI-1(W141A),
a1PI(Y160A) displayed a marked increase in substrate behaviour [43]
Alanine substitution of the residues F100, V126, F128 and I137, respectively, led to increased substrate beha-viour and a low unstable inhibitory activity, and for I137A a biphasic loss of activity (Fig 2 and Table 3) The phenotype of these substitutions is therefore different from that of N139A, T146A and W141A The F residues at positions 100 and 128 are highly conserved among serpins (F119 and F147, respectively, in a1PI) [2] and buried in the hydrophobic interface between hF and b-sheet A (Fig 5) The substrate behaviour and low activity, which could not be increased by refolding in vitro (not shown),
of these variants suggest that these residues are pivotal for
Fig 5 Selected residues important for the interactions between hF, the
hF/s3A-loop and b-sheet A, as seen in the structure of PAI-1 stab [18] (for
an overview, see Fig 1) H-bonds are indicated in green, a-helixF in
orange and parts of b-sheet A in pink Backbone atoms of the
s1A/hF-loop (residues D127 to E130) and of the top of hF and the
hF/s3A-loop (residues T146 to L154) are shown in CPK colours The following
side chains are shown in CPK colours and numbered (the secondary
structure element is given in parenthesis): 1, T96 (s2A); 2, F100 (s2A);
3, V126 (s1A); 4, F128 (s1A/hF-loop); 5, I137 (hF); 6, I138 (hF); 7,
N139 (hF); 8, W141 (hF); 9, V142 (hF); 10, T146 (hF); 11, M149 (hF/
s3A-loop); 12, Y172 (s3A); 13, E283 (s6A); 14, E285 (s6A); 15, K325
(s5A); 16, K327 (s5A) Note that in the structure of latent PAI-1, T96
forms a hydrogen bond with H145 from hF [18] (data not shown).
Trang 8the correct folding of the stressed serpin conformation.
V126 (conserved among serpins; V145 in a1PI [2]) and
I137 (not conserved) are partially exposed in the cleft
between hF and s1A (Fig 5) The substrate behaviour of
the corresponding A-substituted variants suggests that
these residues are instrumental for the movements of hF
during complexformation
As detailed above, several of the residues investigated in
this study are conserved among serpins and substituting any
of these with A changed the characteristics of PAI-1 In
addition, our observations for the M149A, M149K, N152A
and N152D variants of PAI-1 implicate the conserved side
chains K168 and D171 in a1PI (corresponding to M149 and
N152, respectively, in PAI-1) as contributors to the stability
of the stressed serpin conformation The K335A
substitu-tion in a1PI, corresponding to the K325A substitution in
PAI-1, stabilizes a1PI by 6.5 kcalÆmol)1[40], suggesting that
the modest decrease in the rate of latency transition resulting
from the K325A substitution (Table 3) could reflect a
substantial stabilization of the stressed PAI-1 conformation
We cannot, however, exclude the possibility that the
observed delay of latency transition resulting from amino
acid substitutions reflects features of PAI-1 not shared by
other serpins
In contrast, none of the residues in hF that were
replaceable by A without functional consequences (i.e
S129, E130, V131, R133, F136, K143, T144 and H145) are
conserved among serpins [2] This supports the general
notion that conservation of a residue indicates its
import-ance for protein function Furthermore, with the exception
of H145, these residues are pointing away from the
hF/s3A-loop and b-sheet A, suggesting that functionally important
residues should be sought in the interfaces between
secon-dary structural elements
Conclusively, through mutagenesis we have now
provi-ded further evidence that the positioning of hF and its
movements relative to b-sheet A helps regulate the
stressed-to-relaxed transition of serpins in general and latency
transition of PAI-1 in particular The data presented
provide novel insights into the determinants of serpin
stability located in and around hF and support the presence
of a novel serpin conformation that, due to rearrangements
in the top of hF, has a considerably delayed rate of RCL
insertion, both during latency transition and during
com-plexformation with uPA
Acknowledgements
The excellent technical assistance of A Christensen is gratefully
acknowledged The work was supported by grants from the Danish
Cancer Society, The Danish Research Agency, and the Novo-Nordisk
Foundation.
References
1 Huntington, J.A., Read, R.J & Carrell, R.W (2000) Structure of a
serpin-protease complexshows inhibition by deformation Nature
407, 923–926.
2 Irving, J.A., Pike, R.N., Lesk, A.M & Whisstock, J.C (2000)
Phylogeny of the serpin superfamily: implications of patterns of
amino acid conservation for structure and function Genome Res.
10, 1845–1864.
3 Gils, A & Declerck, P.J (1998) Structure-function relationships in serpins: current concepts and controversies Thromb Haemost 80, 531–541.
4 Ye, S & Goldsmith, E.J (2001) Serpins and other covalent pro-tease inhibitors Curr Opin Struct Biol 11, 740–745.
5 Wind, T., Hansen, M., Jensen, J.K & Andreasen, P.A (2002) The molecular basis for anti-proteolytic and non-proteolytic functions
of plasminogen activator inhibitor type-1 Roles of the reactive centre loop, the shutter region, the flexible joint-region and the small serpin fragment Biol Chem 383, 21–36.
6 Mottonen, J., Strand, A., Symersky, J., Sweet, R.M., Danley, D.E., Geoghegan, K.F., Gerard, R.D & Goldsmith, E.J (1992) Structural basis of latency in plasminogen activator inhibitor-1 Nature 355, 270–273.
7 Carrell, R.W., Stein, P.E., Fermi, G & Wardell, M.R (1994) Biological implications of a 3 A˚ structure of dimeric antithrombin Structure 2, 257–270.
8 Lomas, D.A., Elliott, P.R., Chang, W.S., Wardell, M.R & Car-rell, R.W (1995) Preparation and characterization of latent
a 1 -antitrypsin J Biol Chem 270, 5282–5288.
9 Chang, W.S & Lomas, D.A (1998) Latent a 1 -antichymotrypsin.
A molecular explanation for the inactivation of a 1 -antichymo-trypsin in chronic bronchitis and emphysema J Biol Chem 273, 3695–3701.
10 Lawrence, D.A., Olson, S.T., Palaniappan, S & Ginsburg, D (1994) Engineering plasminogen activator inhibitor 1 mutants with increased functional stability Biochemistry 33, 3643–3648.
11 Tucker, H.M., Mottonen, J., Goldsmith, E.J & Gerard, R.D (1995) Engineering of plasminogen activator inhibitor-1 to reduce the rate of latency transition Nat Struct Biol 2, 442–445.
12 Sui, G.C & Wiman, B (1998) Stability of plasminogen activator inhibitor-1: role of tyrosine221 FEBS Lett 423, 319–323.
13 Sui, G.C & Wiman, B (2000) The B b-sheet in the PAI-1 molecule plays an important role for its stability Thromb Haemost 83, 896–901.
14 Ma˚ngs, H., Sui, G.C & Wiman, B (2000) PAI-1 stability: the role
of histidine residues FEBS Lett 475, 192–196.
15 Ngo, T.H., Hoylaerts, M.F., Knockaert, I., Brouwers, E & Declerck, P.J (2001) Identification of a target site in plasminogen activator inhibitor-1 that allows neutralization of its inhibitory properties concomitant with an allosteric up-regulation of its antiadhesive properties J Biol Chem 276, 26243–26248.
16 Berkenpas, M.B., Lawrence, D.A & Ginsburg, D (1995) Mole-cular evolution of plasminogen activator inhibitor-1 functional stability EMBO J 14, 2969–2977.
17 Sharp, A.M., Stein, P.E., Pannu, N.S., Carrell, R.W., Berkenpas, M.B., Ginsburg, D., Lawrence, D.A & Read, R.J (1999) The active conformation of plasminogen activator inhibitor 1, a target for drugs to control fibrinolysis and cell adhesion Struct Fold Des 7, 111–118.
18 Stout, T.J., Graham, H., Buckley, D.I & Matthews, D.J (2000) Structures of active and latent PAI-1: a possible stabilizing role for chloride ions Biochemistry 39, 8460–8469.
19 Nar, H., Bauer, M., Stassen, J.M., Lang, D., Gils, A & Declerck, P.J (2000) Plasminogen activator inhibitor 1 Structure of the native serpin, comparison to its other conformers and implications for serpin inactivation J Mol Biol 297, 683–695.
20 Stoop, A.A., Eldering, E., Dafforn, T.R., Read, R.J & Panne-koek, H (2001) Different structural requirements for plasminogen activator inhibitor 1 (PAI-1) during latency transition and pro-teinase inhibition as evidenced by phage-displayed hypermutated PAI-1 libraries J Mol Biol 305, 773–783.
21 Komissarov, A.A., Declerck, P.J & Shore, J.D (2002) Mechan-isms of conversion of plasminogen activator inhibitor 1 from a suicide inhibitor to a substrate by monoclonal antibodies J Biol Chem 277, 43858–43865.
Trang 922 Bijnens, A.P., Gils, A., Knockaert, I., Stassen, J.M & Declerck, P.J.
(2000) Importance of the hinge region between a-helixF and the
main part of serpins, based upon identification of the epitope of
plasminogen activator inhibitor type 1 neutralizing antibodies.
J Biol Chem 275, 6375–6380.
23 Schousboe, S.L., Egelund, R., Kirkegaard, T., Preissner, K.T.,
Rodenburg, K.W & Andreasen, P.A (2000) Vitronectin and
substitution of a b-strand 5A lysine residue potentiate
activity-neutralization of PA inhibitor-1 by monoclonal antibodies against
a-helixF Thromb Haemost 83, 742–751.
24 Wind, T., Jensen, M.A & Andreasen, P.A (2001) Epitope
map-ping for four monoclonal antibodies against human plasminogen
activator inhibitor type-1 Implications for antibody-mediated
PAI-1-neutralization and vitronectin-binding Eur J Biochem.
268, 1095–1106.
25 Vleugels, N., Gils, A., Bijnens, A.P., Knockaert, I & Declerck,
P.J (2000) The importance of helixF in plasminogen activator
inhibitor-1 Biochim Biophys Acta 1476, 20–26.
26 Gettins, P.G (2002) The F-helixof serpins plays an essential,
active role in the proteinase inhibition mechanism FEBS Lett.
523, 2–6.
27 Carrell, R.W & Stein, P.E (1996) The biostructural pathology of
the serpins: critical function of sheet opening mechanism Biol.
Chem Hoppe Seyler 377, 1–17.
28 Christensen, J.H., Hansen, P.K., Lillelund, O & Thøgersen, H.C.
(1991) Sequence-specific binding of the N-terminal three-finger
fragment of Xenopus transcription factor IIIA to the internal
control region of a 5S RNA gene FEBS Lett 281, 181–184.
29 Andreasen, P.A., Riccio, A., Welinder, K.G., Douglas, R.,
Sar-torio, R., Nielsen, L.S., Oppenheimer, C., Blasi, F & Danø, K.
(1986) Plasminogen activator inhibitor type-1: reactive center and
amino-terminal heterogeneity determined by protein and cDNA
sequencing FEBS Lett 209, 213–218.
30 Gill, S.C & von Hippel, P.H (1989) Calculation of protein
extinction coefficients from amino acid sequence data Anal.
Biochem 182, 319–326.
31 Jensen, J.K., Wind, T & Andreasen, P.A (2002) The vitronectin
binding area of plasminogen activator inhibitor-1, mapped by
mutagenesis and protection against an inactivating
organochem-ical ligand FEBS Lett 521, 91–94.
32 Berman, H.M., Westbrook, J., Feng, Z., Gilliland, G., Bhat, T.N.,
Weissig, H., Shindyalov, I.N & Bourne, P.E (2000) The Protein
Data Bank Nucleic Acids Res 28, 235–242.
33 Elliott, P.R., Pei, X.Y., Dafforn, T.R & Lomas, D.A (2000) Topography of a 2.0 A˚ structure of a 1 -antitrypsin reveals targets for rational drug design to prevent conformational disease Protein Sci 9, 1274–1281.
34 Engh, R., Lobermann, H., Schneider, M., Wiegand, G., Huber, R.
& Laurell, C.B (1989) The S variant of human a 1 -antitrypsin, structure and implications for function and metabolism Protein Eng 2, 407–415.
35 Skinner, R., Abrahams, J.P., Whisstock, J.C., Lesk, A.M., Carrell, R.W & Wardell, M.R (1997) The 2.6 A˚ structure of antithrombin indicates a conformational change at the heparin binding site.
J Mol Biol 266, 601–609.
36 Whisstock, J.C., Skinner, R., Carrell, R.W & Lesk, A.M (2000) Conformational changes in serpins I The native and cleaved conformations of a 1 -antitrypsin J Mol Biol 296, 685–699.
37 Hansen, M., Busse, M.N & Andreasen, P.A (2001) Importance
of the amino-acid composition of the shutter region of plasmi-nogen activator inhibitor-1 for its transitions to latent and sub-strate forms Eur J Biochem 268, 6274–6283.
38 Jensen, S., Kirkegaard, T., Pedersen, K.E., Busse, M., Preissner, K.T., Rodenburg, K.W & Andreasen, P.A (2002) The role of b-strand 5A of plasminogen activator inhibitor-1 in regulation of its latency transition and inhibitory activity by vitronectin Biochim Biophys Acta 1597, 301–310.
39 Stoop, A.A., Jespers, L., Lasters, I., Eldering, E & Pannekoek, H (2000) High-density mutagenesis by combined DNA shuffling and phage display to assign essential amino acid residues in protein– protein interactions: application to study structure–function of plasminogen activation inhibitor 1 (PAI-I) J Mol Biol 301, 1135–1147.
40 Im, H., Seo, E.J & Yu, M.H (1999) Metastability in the in-hibitory mechanism of human a 1 -antitrypsin J Biol Chem 274, 11072–11077.
41 Im, H & Yu, M.H (2000) Role of Lys335 in the metastability and function of inhibitory serpins Protein Sci 9, 934–941.
42 Lawrence, D.A., Olson, S.T., Muhammad, S., Day, D.E., Kvassman, J.O., Ginsburg, D & Shore, J.D (2000) Partitioning
of serpin-proteinase reactions between stable inhibition and sub-strate cleavage is regulated by the rate of serpin reactive center loop insertion into b-sheet A J Biol Chem 275, 5839–5844.
43 Cabrita, L.D., Whisstock, J.C & Bottomley, S.P (2002) Probing the role of the F-helixin serpin stability through a single trypto-phan substitution Biochemistry 41, 4575–4581.