(Received for publication, November 20, 1995)
From the
Phage display technology has been exploited to study in detail
the interaction between plasminogen activator inhibitor 1 (PAI-1) and
either thrombin or an essential positively charged ``loop''
of tissue-type plasminogen activator (t-PA), denoted variable region 1
(VR1). For this purpose, a PAI-1 mutant phage library was used that
served as a reservoir of PAI-1 proteins potentially deficient in the
interaction with either VR1 or thrombin. A stringent two-step selection
procedure was developed. (i) A negative selection was performed by
incubating the pComb3/PAI-1 mutant library with an excess of a thrombin
mutant with its VR1 domain substituted with that of t-PA
(thrombin-VR1). (ii) The remaining phages were complexed with t-PA
(positive selection) and selected by panning with an immobilized
anti-t-PA monoclonal antibody. Four consecutive panning rounds yielded
an enrichment of pComb3/PAI-1 mutant phages of 50-fold. Sequence
analysis of 16 different cDNAs, encoding PAI-1 mutants that are
hampered in the binding to thrombin-VR1, revealed the following
mutations. Four independent variants share a mutation of the P4`
residue (Glu
Lys). Nine independent PAI-1 variants
share a substitution of P1` (Met
Lys), whereas
three others share a P2 substitution (Ala
Asp).
Kinetic analysis of representative PAI-1 mutants provides evidence that
the P4` residue is essential for the interaction with the VR1 domain,
consistent with the data of Madison et al. (Madison, E. L.,
Goldsmith, E. J., Gething, M. J., Sambrook, J. F., and Gerard, R.
D.(1990) J. Biol. Chem. 265, 21423-21426), whereas the
P1` and P2 residues confer thrombin specificity. Concordant with the
design of the selection procedure, mutants were obtained that inhibit
thrombin-VR1 at least 100-fold slower than wild-type PAI-1, identifying
residues that are central to the interaction with either thrombin or
VR1. This study demonstrates that phage technology can be used to
analyze large numbers of mutants defective in their interaction with
other (domains of) proteins, provided an adequate selection scheme is
devised.
Plasminogen activator inhibitor 1 (PAI-1) ()is the
main inhibitor of the serine proteases tissue-type plasminogen
activator (t-PA) and urokinase-type plasminogen activator and therefore
a major physiological regulator of the fibrinolytic system (reviewed in (1) ). A number of studies have delineated regions or amino
acids of PAI-1 that are involved in the interaction with t-PA: first,
the reactive-site P1 residue (Arg
) of PAI-1 that
interacts with the catalytic center of t-PA; second, the region between
amino acids 110 and 145 of PAI-1 that binds to an unknown domain on
t-PA(2) ; and third, presumably negatively charged residues on
PAI-1 that are involved in an interaction with the positively charged
variable region 1 (VR1) on the protease domain of
t-PA(3, 4, 5) . Initially, evidence for the
importance of the VR1 ``loop'' of t-PA for the interaction
with PAI-1 was provided by mutagenesis experiments: deleting the VR1
loop or replacement of the positively charged amino acids of VR1 by
negatively charged residues reduced the second-order rate of inhibition (k
) for PAI-1 by at least 3 orders of magnitude
(from 2
10
to
10
M
s
)(4) .
Consistent with these data, we demonstrated that replacement of the VR1
domain of thrombin by the corresponding region of t-PA created a
protein (denoted thrombin-VR1) that acts as an efficient
``target'' serine protease for PAI-1, in contrast to native
thrombin (k
= 2
10
versus
10
M
s
, respectively)(6) . Although native
thrombin is virtually not inhibited by PAI-1, we and others recently
reported that, in the presence of either high molecular weight heparin
or vitronectin, the rate of thrombin inhibition by PAI-1 is increased
2-3 orders of magnitude(7, 8, 9) . The
binding of PAI-1 to these cofactors has been extensively studied, and
the interaction sites on the PAI-1 protein have been precisely
localized(10, 11, 12, 13) .
In this paper, we employ a large library of PAI-1 mutants that are displayed on the surface of phages(14) . This library serves as a reservoir of tools to further delineate structure-function relationships of this multifunctional protein. We devised a stringent two-step selection procedure to isolate mutants of PAI-1 that are hampered in the binding to either the VR1 domain or thrombin. For this purpose, the PAI-1 mutant phage library was incubated with an excess of thrombin-VR1, and subsequently, nonbinding phages were captured by complexing with t-PA and an immobilized anti-t-PA monoclonal antibody. The selection procedure was specifically devised to obtain PAI-1 mutants that inhibit thrombin-VR1 at a rate that is at least 100-fold slower than that of wild-type PAI-1. Accordingly, amino acid residues were identified that are central to the interaction with either the VR1 domain or thrombin.
Figure 1:
Scheme for the selection
of PAI-1 mutants, expressed on the surface of phages, defective in the
interaction with thrombin-VR1. The library of freshly grown
pComb3/PAI-1 phages (PAI-1 ), which had been previously
selected for active PAI-1 mutants, was incubated for 1 h at 37 °C
with an excess of thrombin-VR1 (IIa-VR-1) to form complexes
with PAI-1 mutants that have retained the ability to bind thrombin-VR1
(``negative selection'' (A)). Subsequently, t-PA was
added to the mixture to form t-PA
PAI-1 phage complexes with PAI-1
mutants that do not bind to thrombin-VR1 (``positive
selection'' (B)). Finally, the mixture was transferred to
a well that was coated with an anti-t-PA mAb (CLB-16) to capture the
t-PA
PAI-1 phage complexes (C). Binding of phages to a
well coated with an irrelevant mAb (CLB-CAg 69) served as a negative
control, whereas incubation of phages with t-PA only served as a
positive control. This selection and panning procedure was repeated
three times.
Figure 2:
Inhibition of t-PA or thrombin-VR1 by the
preselected PAI-1 mutant library or by the library obtained after four
consecutive panning rounds. Increasing numbers of phages, derived from
the preselected pComb3/PAI-1 library (selected only for active PAI-1
variants) or from the library obtained after four panning rounds, were
incubated with t-PA or thrombin-VR1 for 90 min at 37 °C in a volume
of 25 µl as described under ``Experimental Procedures.''
Residual amidolytic activity of t-PA or thrombin-VR1 (indicated as
percentage) was assayed by the addition of 150 µl of HBST buffer
and 25 µl of 4 mM Pefachrome tPA or S2238, respectively,
followed by continuous recording of the absorbance at 405 nm in a
Titertek Twinreader. , t-PA inhibition by preselected pComb3/PAI-1
phage library;
, thrombin-VR1 inhibition by preselected
pComb3/PAI-1 phage library;
, t-PA inhibition by pComb3/PAI-1
phage library obtained after four consecutive panning rounds;
,
thrombin-VR1 inhibition by pComb3/PAI-1 phage library obtained after
four consecutive panning rounds.
Figure 3:
Inhibition of t-PA or thrombin-VR1 by
wild-type PAI-1 or PAI-1 Met
Lys. Increasing
amounts of purified wild-type PAI-1 (A) or purified PAI-1
Met
Lys (B) were incubated in microtiter
wells for 1 h at 37 °C in HO buffer, employing a total volume of 25
µl containing 3.3 nM t-PA or thrombin-VR1. The residual
activity of t-PA or thrombin-VR1 was measured as described under
``Experimental Procedures.''
, t-PA inhibition by
wild-type PAI-1;
, thrombin-VR1 inhibition by wild-type PAI-1;
, t-PA inhibition by PAI-1 Met
Lys;
,
thrombin-VR1 inhibition by PAI-1 Met
Lys.
Kinetic analysis of wild-type
PAI-1 used in this study demonstrated a second-order rate constant for
t-PA inhibition of 8.3 10
M
s
, comparable with published
data(18, 19) . Rate constants for inhibition of
thrombin-VR1 or thrombin in the presence of one of the cofactors by
wild-type PAI-1 were somewhat lower than described
previously(6, 20) due to the use of a different
storage buffer of PAI-1. All PAI-1 mutants reveal a strongly decreased
potency for the inhibition of thrombin-VR1 relative to t-PA (at least
4.7-fold). Interestingly, the P4` mutant (Glu
Lys)
displays similar rate constants for thrombin in the presence of either
one of the cofactors. In addition to the P4` mutant, variants were
selected that are deficient in the interaction with both thrombin-VR1
and native thrombin. The P1` mutant (Met
Lys)
exhibits markedly lower rate constants for inhibition of thrombin-VR1
or thrombin in the presence of either heparin or vitronectin, whereas
t-PA inhibition is only slightly reduced. Results obtained with the P1`
mutants are in good agreement with published data: replacement of P1`
Met
by a Lys residue results in a slight reduction of the
second-order rate constant for t-PA inhibition(21) . Similar to
the P1` mutant (Met
Lys), t-PA inhibition by the
P2 mutant (Ala
Asp) is slightly reduced,
consistent with the observation of York et al.(22) .
By contrast, inhibition of thrombin-VR1 or thrombin in the presence of
one of the cofactors by the P2 mutant (Ala
Asp) is
virtually abolished (k
6
10
M
s
).
This study illustrates that phage display of a mutant
library, combined with an appropriate selection procedure and DNA
sequencing, may rapidly provide the location of amino acids involved in
a particular interaction. The PAI-1 mutant library employed in this
study has been constructed by error-prone polymerase chain reaction and
contains an average of two mutations/unit length of PAI-1 cDNA (1137
base pairs), corresponding to 46% single and 34% double mutants and 20%
wild-type proteins(14) . Due to the moderate extent of
mutations, amino acids are altered solely by single base pair
substitutions. Consequently, only a limited set of amino acid
substitutions will be encountered as exemplified by, for example, the
PAI-1 P1` (Met
Lys) and P2 (Ala
Asp) mutants, resulting from a single base pair change.
Obviously, our restrained mutagenesis procedure restricts the range of
amino acid alterations, whereas a more rigorous random mutagenesis
procedure would result in a library containing a relatively large
portion of inactive proteins. It should be emphasized that the design
of the selection protocol will dictate the properties of the selected
mutants. The PAI-1 mutant phage library was incubated with a 100-fold
excess of thrombin-VR1, corresponding to pseudo first-order reaction
conditions that allow the following calculations. To capture
97%
of a specific PAI-1 mutant, the selected incubation period (1 h) should
be equal to five times the half-time of the reaction (t
= 12 min). From the equation t
= ln 2/(k
E
), where E
is the concentration of
thrombin-VR1 (150 nM), it is deduced that PAI-1 mutants with a
second-order rate constant for thrombin-VR1 inhibition exceeding 6.4
10
M
s
will be complexed. Consequently, our selection procedure was
devised to specifically select mutants that reacted at least 100-fold
slower with thrombin-VR1 than wild-type PAI-1 (k
= 2
10
M
s
)(6) . Clearly, this expectation is
borne out by the experimental data: the second-order rate constants for
inhibition of thrombin-VR1 by the selected PAI-1 P1` (Met
Lys), P2 (Ala
Asp), and P4`
(Glu
Lys) mutants are 5.1
10
,
6
10
, and 2.3
10
M
s
, respectively (Table 2).
The dominant contribution of the P1` Met, P2 Ala,
and P4` Glu residues of PAI-1 to the interaction with thrombin-VR1 can
be assigned either to binding to the VR1 loop of t-PA or to binding to
the thrombin moiety. This distinction could be made upon measuring the
inhibition of native thrombin in the presence of a cofactor (heparin or
vitronectin). In this respect, the rate of inhibition of thrombin is
decreased 50-100-fold for the P1` (Met
Lys)
and P2 (Ala
Asp) mutants as compared with
wild-type PAI-1, whereas the inhibition rate for the PAI-1 P4` mutant
(Glu
Lys) is unaltered. Similarly, the rate of
inhibition of t-PA by the PAI-1 P1` (Met
Lys) and
P2 (Ala
Asp) mutants is decreased 14- and 8-fold,
respectively, whereas the rate of t-PA inhibition by the PAI-1 P4`
mutant (Glu
Lys) is only slightly reduced
(2.6-fold). Nevertheless, altering the P1` or P2 residue preferentially
compromises the interaction of PAI-1 with thrombin rather than the
inhibition of t-PA. This conclusion agrees with a recent report stating
that positively charged residues at the P1` position are not tolerated
for thrombin inhibition (23) due to ionic repulsion of the S1`
subsite of thrombin(23, 24) . Taken together, it is
concluded that efficient thrombin inhibition requires uncharged amino
acid residues at both the P1` and P2 positions.
Substitution of P4`
Glu with Lys does not affect thrombin inhibition, but
severely diminishes binding to the VR1 domain of t-PA. The selection of
the PAI-1 P4` mutant (Glu
Lys), created by a
single base pair substitution, was anticipated on the basis of our
selection strategy combined with the data reported by Madison et
al.(5) . Initially, these investigators replaced the
positively charged residues of the VR1 loop of t-PA by Glu residues,
rendering the protein fairly resistant to PAI-1 (k
10
M
s
)(4) . Subsequently, they showed that
substitution of the negatively charged P4` Glu
of PAI-1
with the positively charged Arg residue virtually restored the
inhibition of this t-PA variant(5) . These observations have
now received substantial support by the preferential selection of the
P4` mutant (Glu
Lys), demonstrating the dominant
contribution of Glu
in the interaction with the VR1
domain of t-PA. However, it is thought that several positively charged
residues of the VR1 loop contribute to the interaction with PAI-1 and,
consequently, that several negatively charged residues on PAI-1 are
involved in this interaction. Further assessment of PAI-1 residues
involved in the interaction with the VR1 loop probably requires an
adjustment of the selection protocol to isolate mutants with a
second-order rate of inhibition for thrombin-VR1 between 6.4
10
M
s
and,
as argued before, that of wild-type PAI-1. Clearly, two options are
available to reduce the stringency of the selection. First, the time
allowed for complex formation between thrombin-VR1 and phage-displayed
PAI-1 mutants can be decreased, ultimately resulting in mutants with
higher inhibition constants. Second, assuming that the interaction
between the VR1 domain and PAI-1 is predominantly of an ionic nature,
the ionic strength during complex formation can be raised. Currently,
these options are being assessed to optimally explore the phage display
technology for analyzing the structure and function of PAI-1. The
approach described in this report is applicable to many other proteins,
provided these proteins can be functionally displayed on the surface of
phages and if an adequate selection procedure is designed.