(Received for publication, November 21, 1995)
From the
Tissue-type plasminogen activator (t-PA) has evolved to optimize
cleavage of plasminogen (Plg) while minimizing cleavage of other
potential protein and peptide substrates. We find that the S2 and S2`
subsites of t-PA are important determinants of specificity, and
occupancy of the S3 subsite is essential for catalysis. t-PA
efficiently hydrolyzes a protein substrate which incorporates an
optimized substrate sequence, revealing the ability of the protease to
participate in the highly selective cleavage of protein fusions.
Surprisingly, t-PA cleaves this engineered protein substrate with a K that is reduced 950-fold relative to
the K
for hydrolysis of the same target
sequence within a peptide. This reduction of K
suggests that binding is facilitated by interactions between
protein substrate and protease that are distant from the P4-P2`
residues. We use this kinetic data to derive a model in which several
distinct mechanisms contribute to the remarkable specificity of t-PA.
Highly specific proteases efficiently hydrolyze target proteins while leaving other proteins intact. To achieve selectivity, these enzymes must possess mechanisms to solve a challenging problem of molecular recognition-the same chemical reaction, peptide bond hydrolysis, must be favored within one protein context while being disfavored in all others. During the evolution of chymotrypsin-like serine proteases such stringent substrate specificity has been achieved, and this evolutionary feat has provided the basis for both the blood-clotting cascade and the fibrinolytic system. Understanding the molecular basis for specificity would facilitate rational design of proteases for the selective hydrolysis of peptide bonds, affording valuable reagents for peptide mapping and the isolation of protein domains. In addition, the ability to direct proteolytic activity to desired targets, and thereby selectively activate or inactivate extracellular proteins, would be likely to have wide ranging therapeutic applications.
Tissue-type plasminogen activator (t-PA) ()is an attractive model for the study of the evolution of
highly specific proteolysis(1) . t-PA is a chymotrypsin-like
serine protease that initiates the fibrinolytic cascade by cleaving a
single bond (Arg
-Val
) in the
circulating zymogen plasminogen (Plg). This bond is the only known
substrate for t-PA in vivo, a remarkably stringent specificity
given that t-PA shares 40% sequence identity with trypsin, a
nonspecific protease(2) . Part of this specificity is due to
the formation of a ternary complex involving t-PA, Plg, and fibrin,
which serves to reduce the K
of t-PA for
plasminogen by more than 400-fold(2, 3) . However,
even in the absence of fibrin, t-PA retains stringent specificity for
Plg, and this specificity is an inherent property of the protease
domain of t-PA(2) . A detailed understanding of the mechanisms
employed by t-PA to ensure selectivity would provide new insight into
the evolution of the endogenous fibrinolytic system, and might suggest
effective, knowledge-based strategies for design of novel proteases
with unique substrate specificities. In this study we characterize the
restriction of t-PA's substrate specificity by enhanced
discrimination against suboptimal occupancy of the P4-P2` subsites. We
also show that interactions between t-PA and protein substrates that
are distant from the active site play an important role in the binding
of substrate. t-PA utilizes these distinct mechanisms concomitantly to
efficiently recognize Plg while remaining inert to other potential
substrates.
as described previously by Wells(6) .
G
represents the difference
between two substrates in the free energy required to reach the
transition state complex during hydrolysis. The degree of subsite
interdependence was investigated by plotting the sums of free energy
changes for single substitutions against free energy changes for
multiple substitutions at the same sites.
To begin to gain additional insights into the determinants of specificity of t-PA, we altered the highly conserved P2 and P2` residues of this peptide and examined the effects on catalysis of the amino acid substitutions. We synthesized two sets of peptides containing sequences GGSGPFXRSALVPEE or GGSGPFGRSXLVPEE, where X is Gly, Ala, Ile, Phe, Lys, or Asp. Each peptide was kinetically characterized as a substrate for both trypsin and t-PA (Table 1) to determine whether the conservation of residue identity observed in the phage display screen is predictive of optimal subsite occupancy, to quantitate contributions of subsite preferences to selectivity, and to identify properties of substrate residues which account for enhanced discrimination.
For occupancy of the S2 subsite in t-PA, the order
of preferences is Gly > Ala > Lys > Ile > Phe > Asp. The
preferences of trypsin (Ala > Gly > Lys > Phe > Ile >
Asp) are qualitatively similar to those of t-PA (Table 1); both
enzymes prefer alanine or glycine, and cleave peptides with negatively
charged side chains poorly. Quantitatively, t-PA is more discriminating
than trypsin toward nonpreferred residues at this subsite, displaying
an 11-fold greater difference between its most efficiently cleaved and
least efficiently cleaved substrates. At the S2` subsite, the order of
preferences in subsite occupancy is Ala > Gly > Phe > Ile >
Asp > Lys for t-PA, and Ile > Ala > Phe > Lys > Gly
Asp for trypsin. Again, t-PA exhibits greater discrimination (by
7-fold) against suboptimal interactions at this subsite with the
exception of occupation of the subsite by aspartic acid, which reduces
the catalytic efficiency of trypsin by 1700-fold while reducing the
catalytic efficiency of t-PA only 10-fold. Similar preferences for
occupancy of the S` subsites of trypsin have been mapped previously by
monitoring acyl transfer rates of varied peptide
nucleophiles(11, 12) .
The greater preference of t-PA for optimal occupancy at the S2 and S2` subsites relative to trypsin strongly suggests that one mechanism for narrowing the specificity of t-PA is enhanced discrimination against suboptimal subsite occupancy. Kinetic differences among substrates with variations at individual residues are not dramatic but cumulatively become a major barrier to nonselective hydrolysis. The preferences for subsite occupancy correlate well with the frequency of occurrence of residues during the phage display screen(10) . For example, glycine was found at P2 three times as often as alanine during the screen, and the peptide containing P2 glycine was cleaved approximately twice as efficiently as the analogous peptide containing alanine.
A substrate containing aspartic acid at P2 is
inefficiently cleaved by both trypsin and t-PA (Table 1). Based
on the position of the P2 residue in a complex of trypsin and bovine
pancreatic trypsin inhibitor (BPTI)(13) , a P2 aspartic acid
may be within 2.6-3 Å from His of the
catalytic triad and may interfere with its functions during catalysis.
For t-PA, whose lowered catalytic efficiency is expressed entirely as a
reduction in k
, decreased catalysis may result
from a direct negative influence on the catalytic machinery. An
alternate possibility is that the presence of aspartic acid induces
nonproductive binding which subtly alters the positioning of the
scissile bond.
Aspartic acid at P2` also reduces catalytic
efficiency of both t-PA and trypsin, but the negative effect is
170-fold greater for trypsin (Table 1). Modeling of an aspartic
acid at the P2` residue in the trypsin-BPTI complex suggests a possible
candidate for interaction with its side chain carboxyl may be the
backbone amide of Gly. Since the backbone amide of
Gly
forms part of the oxyanion hole, interaction of P2`
with this amide is likely to reduce transition state stabilization. An
aspartic acid at the P2` position of substrate may also produce a
buried negative charge in the enzyme/substrate complex which trypsin
possesses no apparent means of neutralizing. t-PA, by contrast, may be
able to at least partially neutralize this charge; modeling based on
the BPTI-trypsin complex suggests that a P2` aspartic acid would
approach within
4Å of arginine 304 of t-PA, a residue which
is analogous to tyrosine 39 of trypsin. Neutralization of unfavorable
electrostatic interactions that disturb catalytic function or substrate
binding may be one way to modulate the specificity of serine proteases.
A likely example of a protease employing this mechanism is
enteropeptidase(14) , which possesses the basic sequence, RRRK,
at residues 886-889 (96-99 in chymotrypsin) lining the
binding cleft. These positive charges have been proposed to neutralize
negatively charged substrate residues and account for the strong
selectivity of enteropeptidase for aspartic acid at P4, P3, and P2.
To further probe the subsite occupancy requirements of t-PA and trypsin we assayed the hydrolysis of peptides that were acylated at the amino terminus and amidated at the carboxyl terminus. These modifications remove potential electrostatic effects on subsite affinity that might be produced by the charged termini of peptides XII-XIX and allow effects of steric occupancy of subsites to be probed more directly. Peptides based on the parent sequence FGRSAL spanning P3 to P3`, XIX, and XX, which contained charged and uncharged termini, respectively, were assayed for hydrolysis by trypsin and t-PA and were observed to yield similar kinetic constants (Table 2). Shorter uncharged peptides XXI and XXII which lack P2` or P3` residues continued to be cleaved with relatively high efficiency, results that are in sharp contrast to the greatly impaired hydrolysis observed for peptides XIII and XIV. This observation suggests that unfavorable electrostatic interactions within the S2` or S3` pockets may prevent efficient catalysis of peptides that contain charged termini. Occupation of these subsites, however, does not appear to be necessary for efficient catalysis, implying that binding energy derived from the S2` and S3` pockets does not contribute to lowering the activation barrier for the rate-limiting step for peptide bond hydrolysis, nor is subsite occupation necessary to properly position a substrate.
By contrast to the lack of effect
observed from removal of P2` and P3` residues, uncharged peptides XXIII
and XXIV lacking P3 and P2 residues are hydrolyzed significantly less
efficiently by trypsin and t-PA, although the effect is smaller than
that observed for charged peptides. Removal of P2 affects trypsin and
t-PA to a similar extent, with both enzymes exhibiting approximately
150 fold reduction in catalytic efficiency. The P1 terminal amine of
substrate XXIII is within 3.5 Å of Ser and
Ser
. Acylation of this amine removes a charge which may
interfere with catalysis or proper substrate geometry. Analysis of the
hydrolysis of peptide XXIV shows that the removal of the P3 residue has
a much greater effect on the K
for t-PA than for
trypsin, again suggesting that t-PA is more dependent on occupancy of
P3 than is trypsin.
Figure 1:
Values of k/K
for peptides
which vary by 1, 2, or 4 amino acids at P4, P3, P1`, and P2` from the
parent sequences YKKSPGRVVGGSKY (containing the P6-P4` sequence from
plasminogen) and GGSGPFGRSALVPEE (containing P4-P2` residues selected
for efficient t-PA-mediated catalysis by substrate phage display) were
used to calculate
G
values
reported in Table 3. Values obtained for multiple substitutions
are plotted against the sum of the values for the matching combination
of individual substitutions. The linear equations describing these
plots are y = 0.40 + 0.98x; R = 0.98 for t-PA, and y = 0.20 +
0.76x; R = 0.93 for
trypsin.
The observed additivity is
consistent with the possibility that substrate residue side chains
contact separate surfaces of the enzyme. This is also consistent with
the -sheet like conformation of subsite residues evaluated here
within the three dimensional structures of many serine
protease-inhibitor complexes, where side chains alternately extend from
the backbone in opposite directions(17) . Mutagenesis of
residues forming the protein surface and further kinetic analysis will
be necessary to fully establish the predictability and independence of
changes within non-S1 subsites. True independence of subsite
interactions may allow the engineering of unique specificities through
sequential mutation of subsites. Manipulation of non-S1 subsites would
maintain the S1 site and the catalytic triad intact, thus retaining
structural features necessary for efficient catalysis. The retention of
efficient catalysis might be combined with the potential for
significant diversity of substrate specificity, as alterations of the
four sites examined here could theoretically allow as many as 1.3
10
distinct specificities.
We found that ODC-PFGRSA was
efficiently hydrolyzed by t-PA (Fig. 2). Surprisingly, this
activity was characterized by a greatly reduced K relative to hydrolysis of an analogous peptide substrate (Table 4). Kinetic analysis of cleavage of ODC-PFGRSA yielded k
and K
values of 0.075
± 0.048 s
and 3.7 ± 2.7
µM, respectively. The enhanced catalytic efficiency of
hydrolysis of ODC-PFGRSA compared with the analogous peptide is
therefore due to a 950-fold reduction in K
, which
compensates for a 56-fold reduction in k
.
Cleavage of ODC-PFGRSA by the isolated protease domain of t-PA occurred
with a catalytic efficiency very similar to that observed with the
full-length enzyme (k
= 0.075 ±
0.015 s
, K
= 4.8
± 3.2 µM, and k
/K
= 1.6
10
M
s
),
demonstrating that the reduction in K
is an
inherent property of the protease domain. By contrast to the relatively
efficient catalysis of ODC-PFGRSA, analysis of cleavage of ODC-SPGRVV
by t-PA yielded a similar K
(1.0 ± 0.44
µM), but revealed a k
(1.0 ±
0.16
10
s
) which was three
orders of magnitude smaller. The sequence PFGRSA, therefore, continues
to exert a favorable influence on k
when
introduced into the context of a protein. The recognition of PFGRSA as
a labile site and a reduction of K
mediated by
enzyme-substrate interactions which are independent of the binding
cleft surrounding P1 combined to yield efficient hydrolysis of
ODC-PFGRSA. These data demonstrate the importance of distal
interactions in the recognition of substrates by t-PA, a feature that
may characterize other chymotrypsin-family proteases. Cleavage of
proteins also demonstrates the ability to adapt t-PA as a highly
selective protease, a property which may prove valuable for peptide
mapping and the hydrolysis of protein fusions.
Figure 2:
Specific cleavage of ODC-PFGRSA by t-PA
between mature ODC and an amino-terminal extension composed of a
His purification tag and a TEV protease cleavage site.
Incubations of each sample loaded were carried out for 1 h at 37
°C. Lane 1 contains 25 nM t-PA only; lanes 2 and 3 contain 1.57 µM ODC-PFGRSA with or
without 25 nM t-PA; lanes 4 and 5 contain
1.57 µM wild type ODC with or without 25 nM t-PA.
The kinetics of
hydrolysis of ODC-PFGRSA were remarkably similar to those reported for
activation cleavage of Lys-plasminogen by t-PA (2, 3) (Table 4). ODC-SPGRVV, however, was
cleaved with a k 2000-fold lower than that for
Plg, in spite of the identity of primary sequence with Plg at P4 to
P2`. This observation suggests that t-PA utilizes productive secondary
interactions with Plg which are distinct from those at P4-P2` to
overcome unfavorable subsite contacts in the vicinity of the scissile
bond and to promote efficient cleavage of Plg. ODC-SPGRVV does not
possess the potential for such interactions, so that insertion of
SPGRVV into ODC is not sufficient to overcome the extremely low k
observed for catalysis of peptide substrates
containing this P4-P2` sequence. Interactions with fibrin, which lower
the apparent K
of t-PA for Plg by 400-fold also
appear to be unique to Plg. No significant effect on catalysis for any
ODC variant was observed when fibrin was present, suggesting that the
mechanism of fibrin activation does not make a general contribution to
the binding and cleavage of proteins, including substrates like
ODC-PFGRSA which are as susceptible as Plg to cleavage by t-PA in the
absence of fibrin.
At least two explanations could account for the
reduction in K for the hydrolysis of sequences
within proteins relative to the same sequences within peptides.
Insertion of these sequences into a protein context may reduce the
number of conformations available to the amino acid sequence and thus
reduce the entropic cost of binding to the protease. Because a large
reduction in K
is observed for hydrolysis of
substrate sequences within a flexible amino-terminal extension which
would not be expected to be significantly constrained, however, this
argument does not seem compelling. Alternatively, the protein may
possess additional sites for interaction with t-PA that are distant
from the active site. The ability of t-PA to cleave two entirely
unrelated proteins, Plg and ODC, with nearly identical kinetic
parameters strongly suggests that at least one determinant of these
interactions with t-PA is nonspecific. Trypsin has also been observed
to exhibit a significant decrease in K
for
cleavage of protein relative to peptide substrates(2) , and the
favorable influence of tertiary structure on binding of proteinaceous
substrates may be a general characteristic of serine proteases and an
essential part of the physiological mechanism of protein cleavage.
The sequence PFGRSA was placed into L. donovani ODC at an internal site at which the wild-type ODC was known to be readily cleaved by a catalytically impaired variant of trypsin, H57A trypsin (8) . H57A trypsin hydrolyzes peptide substrates 100-1000-fold less efficiently than does trypsin, which is similar to the 1000-fold difference between t-PA and trypsin. It was surprising, therefore, that the ODC variant containing PFGRSA at the trypsin-accessible internal site was not detectably cleaved by 100 nM t-PA within 32 h of incubation time. Recent studies of sites of trypsin-mediated proteolysis in proteins suggest that, to productively bind substrate, trypsin must be able to disrupt the local structure of 10-12 residues surrounding the cleavage site(18) , and comparison of B factors of labile sequences with their solvent accessible surface area suggests that a target sequence's accessibility is more important than its flexibility(19) . The inability of t-PA to cleave this substrate suggests that, relative to trypsin, t-PA may require that substrate sequences within proteins be more flexible and achieve greater localized distortions of protein structure to participate in productive binding. The more stringent dependence of t-PA on occupancy of the S3 subsite may be one reason for this requirement, as precise binding of the P3 residue of the substrate protein may be necessary for catalysis. The ability of a protein surface to form favorable interactions with diverse protein partners is reminiscent of the serine protease inhibitor ecotin (20) which inhibits a broad spectrum of proteases by deemphasizing the importance of interactions at the S1 site in favor of extensive complementarity with more distant surface residues. We speculate that surfaces interacting with ecotin from widely variant serine proteases may be conserved to facilitate binding and catalysis of protein substrates.
The existence of general mechanisms to reduce and restrict catalysis
by t-PA suggests that mechanisms must also be present to enhance the
specific hydrolysis of Plg. Such positive contributions to peptide bond
cleavage seem particularly likely since the P4 to P2` residues
surrounding the labile bond in Plg are apparently not optimized to
yield efficient catalysis. t-PA and Plg are therefore likely to
interact productively at secondary sites distant from the S4 to S2`
subsites. At least one of these secondary sites may be able to interact
with many protein substrates, as they reduce the K for cleavage of a protein, ODC, that is unrelated to Plg. These
interactions, however, are not sufficient to permit efficient cleavage
of the ODC variant containing the native cleavage site from
plasminogen. Overcoming the negative effect on catalysis by the P4 to
P2` residues of Plg apparently requires secondary interactions that are
specific to t-PA and Plg. The exact nature of these specific
interactions is unclear but may involve; (i) conformational changes in
t-PA upon binding Plg which enhance its activity, (ii) adoption by the
cleavage site in Plg of a conformation that has particularly favorable
subsite occupancy and that lowers the energy of the resultant
transition state, or (iii) utilization of interactions other than those
involving Plg's P4-P2` residues to achieve a productive
interaction in spite of weak or inefficient subsite interactions.
Finally, the efficiency of Plg activation by t-PA is specifically
enhanced by formation of a ternary complex which includes fibrin and
lowers K
by a factor of
440(2, 3) . Cumulatively, these effects greatly reduce
the potential of the trypsin-like protease domain of t-PA for
proteolysis of virtually all proteins and then effectively restore
efficient proteolysis of one selected substrate. As the exact molecular
details of this impressive natural engineering become more clear, the
ability to rationally design proteases combining novel specificities
with high activity will be greatly enhanced.