(Received for publication, July 28, 1995; and in revised form, September 20, 1995)
From the
Protein-protein interactions can be guided by contacts between
surface loops within proteins. We therefore investigated the hypothesis
that novel protein-protein interactions could be created using a
strategy of ``loop grafting'' in which the amino acid
sequence of a biologically active, flexible loop on one protein is used
to replace a surface loop present on an unrelated protein. To test this
hypothesis we replaced a surface loop within an epidermal growth factor
module with the complementarity-determining region of a monoclonal
antibody. Specifically, the HCDR3 from Fab-9, an antibody selected to
bind the -integrins with nanomolar affinity (Smith, J.
W., Hu, D., Satterthwait, A., Pinz-Sweeney, S., and Barbas, C. F.,
III(1994) J. Biol. Chem. 269, 32788-32795), was grafted
into the epidermal growth factor-like module of human tissue-type
plasminogen activator (t-PA). The resulting variant of t-PA bound to
the platelet integrin
with
nanomolar affinity, retained full enzymatic activity, and was
stimulated normally by the physiological co-factor fibrin. Binding of
the novel variant of t-PA to integrin
was dependent on the presence of divalent cations and was
inhibited by an RGD-containing peptide, demonstrating that, like the
donor antibody, the novel t-PA binds specifically to the ligand-binding
site of the integrin. These findings suggest that surface loops within
protein modules can, at least in some cases, be interchangeable and
that phage display can be combined with loop grafting to direct
proteins, at high affinity, to selected targets. In principle, these
targets could include not only other proteins but also peptides,
nucleic acids, carbohydrates, lipids, or even uncharacterized markers
of specific cell types, tissues, or viruses.
Development of the ability to create novel protein-protein interactions promises to provide important new therapeutic agents as well as unique tools and reagents for the study of key biological processes. Such advances in protein engineering may also provide seminal information about how proteins interact.
One strategy for
manipulating protein-protein interactions is to employ random or nearly
random (e.g. alanine scanning) (1) site-directed
mutagenesis to identify amino acid residues critical for binding
affinity and specificity. Cumbersome, large scale mutagenesis efforts
followed by laborious, time-consuming assays of individual, mutated
proteins can sometimes extend this approach to create new molecular
interactions. For example, every residue in human growth hormone was
scanned for activity prior to re-engineering the molecule to bind the
prolactin receptor(2) , and a similar strategy facilitated the
construction of a variant of interleukin-3 that binds the monomeric
receptor with higher affinity than it binds the interleukin-3
receptor(3) . An important current challenge,
therefore, is to create more rapid and efficient strategies to engineer
proteins with new binding properties. A recent approach that can
obviate the need to perform large numbers of binding assays with
individual, mutated proteins is biopanning using phage display systems.
Although originally conceived as a means of screening vast numbers of
peptide motifs(4) , it is now apparent that whole proteins and
domains within proteins can be manipulated using the phage selection
strategy(5) .
Another strategy that has been used to modify
protein-protein interactions is the addition, deletion, or substitution
of entire domains within
proteins(6, 7, 8, 9, 10, 11, 12) .
Although it is often successful, this strategy provides an extremely
low resolution picture of protein-protein interactions; consequently,
manipulation of entire domains is often followed by the construction
and analysis of numerous point mutants as described above. Severe
limitations also arise if a protein domain of interest carries more
than one important biological activity; maintaining one activity (e.g. functionally significant domain-domain interactions)
while altering another (e.g. high affinity binding to a
co-factor or receptor) can be problematic. An approach that might
overcome both of these limitations would be to transfer between
proteins not entire domains but rather defined structural elements
within protein domains. We therefore focused in this study on grafting
flexible protein loop structures, which often guide protein binding
phenomena(13) . Flexible loops are found on the surface of most
protein modules and exist as short stretches of amino acids that
connect regions of defined secondary structure. Although
crystallographic and NMR studies show that these loops are usually less
well defined than helices and -sheets, their conformational
freedom is normally restricted substantially compared with free
peptides. Consequently, the binding activities of surface loops in
proteins usually differ significantly from those of the corresponding
linear amino acid sequence(14) .
To substantially increase
the speed and efficiency by which protein engineering can be used to
confer novel binding activities to a selected protein, we adopted a
strategy that combined phage display and loop grafting. To test this
strategy, we replaced amino acids in a surface loop within the
epidermal growth factor (EGF) ()domain of tissue-type
plasminogen activator with residues forming one CDR of a monoclonal
antibody that was directed against the adhesive integrin receptor
and had been subjected to
mutagenesis and ``affinity maturation'' using a phage display
system. The resulting variant of t-PA, LG-t-PA, bound
with nanomolar affinity, possessed
full activity toward both synthetic and natural substrates, and was
stimulated normally by the co-factor fibrin. Because of its novel
integrin binding properties, this new variant of t-PA may display
enhanced thrombolytic potency toward the platelet-rich thrombi that
precipitate acute myocardial infarction.
Following mutagenesis, single-stranded DNA corresponding to the entire 437-bp HindIII-KpnI fragment was fully sequenced to assure the presence of the desired mutation and the absence of any additional mutations. Replicative form DNA was prepared for appropriate phage, and the mutated 437-bp fragment was recovered after digestion of replicative form DNA with HindIII and KpnI and electrophoresis of the digestion products on an agarose gel. The isolated HindIII-KpnI fragment was used to reconstruct a full-length cDNA encoding LG-t-PA.
Binding of the
novel t-PA to each integrin was measured with an assay adapted from the
ligand receptor binding assays previously reported (25, 26) . Purified integrin was immobilized in
Titertek 96-well plates in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl containing 1 mM CaCl and 1 mM
MgCl
. For coating, the integrin was diluted to a
concentration of 5 µg/ml. Integrin was allowed to coat plastic
plates for 18 h at 4 °C. Following coating, the nonspecific
protein-binding sites on the plate were blocked by incubation with 50
mM Tris-HCl, pH 7.4, 100 mM NaCl containing 20 mg/ml
bovine serum albumin. For binding measurements, conditioned medium
containing wild type or modified t-PA was diluted to the appropriate
enzyme concentrations in binding buffer (50 mM Tris-HCl, pH
7.4, 100 mM NaCl, 0.5 mM CaCl
containing
1 mg/ml bovine serum albumin). The t-PA was applied to integrin
immobilized in microtiter wells and allowed to bind for 2 h at 37
°C. After binding, free t-PA was removed from the plate by three
rapid washes with binding buffer, and bound t-PA was detected using an
activity assay for t-PA as described previously(21) .
Nonspecific binding of t-PA was determined by the addition of 5 mM EDTA to chelate divalent cations that are required for integrin
ligand binding function(23) . In some cases the peptide GRGDSP
(synthesized by Coast Scientific) was used as a competitor. This
indirect assay was used for measuring t-PA binding to integrin because
of technical limitations encountered using purified t-PA. Following
purification, t-PA is maintained in solution with 200 mM arginine. We found that this concentration of arginine interfered
with all of the integrin binding assays and precluded attempts to use
direct methods of measuring binding affinity. Thus, the indirect
binding assay was chosen. This type of measurement is expected to give
an underestimate of the binding of loop-grafted t-PA to integrins
because COS cells secrete other integrin-binding proteins that compete
with LG-t-PA for binding to integrin (data not shown).
Figure 1:
A representation of the NMR structure
of murine epidermal growth factor adapted from Kohda et al. (28). Residue 23 of the murine protein corresponds to residue 65
of human t-PA. The -turn formed by residues 23-28 of the
murine protein may be extended in t-PA due to the occurrence of a
three-amino acid insertion,
YFS
, at the
location indicated by the large arrow. The primary sequence of
the substituted region of LG-t-PA and the corresponding region of wild
type t-PA are indicated at the bottom of the figure. Dashes in the LG-t-PA sequence indicate identity to the wild
type enzyme.
Table 2presents the results of a kinetic assay of plasminogen
activation in the presence of the co-factor fibrin by t-PA and LG-t-PA.
The kinetic constants of LG-t-PA for plasminogen activation are very
similar to those of wild type t-PA; k/K
values for the two
enzymes vary by approximately 10% in this assay. LG-t-PA, therefore,
maintained full enzymatic activity not only toward small synthetic
substrates but also toward the natural protein substrate plasminogen.
The activity of wild type t-PA is stimulated by fibrin, fibrinogen, and cyanogen bromide fragments of fibrinogen, and we and others have reported that mutations mapping to at least five distinct regions of the enzyme can differentially affect stimulation of t-PA by these distinct co-factors(29, 30) . To examine whether mutations present in LG-t-PA affected stimulation of the enzyme by any of these three co-factors, we performed standard indirect chromogenic assays of the two enzymes in the presence of each of the co-factors. The results of these assays are depicted in Fig. 2and indicate that the mutations present in LG-t-PA have not significantly compromised interaction of the enzyme with any of the co-factors.
Figure 2:
Standard indirect chromogenic assay of
plasminogen activation by wild type (WT) and LG-t-PA in the
presence of buffer (), fibrin monomers (
), fibrinogen
(
), or cyanogen bromide fragments of fibrinogen
(
).
The role, if any, of the EGF domain of t-PA in mediating stimulation
of the enzyme by fibrin has generated controversy and conflicting
reports (9, 12, 31) . Although this study
will not resolve the issue, our results do argue strongly that residues
63-71, which form part of an antiparallel -sheet and a
-turn on surface of the EGF domain, do not play a role in
stimulation of the enzyme by fibrin or fibrinogen.
Figure 3:
The
binding of LG-t-PA to platelet integrin was measured using purified integrin as described under
``Materials and Methods.'' Nonspecific binding was determined
by parallel incubation with 10 mM EDTA. The absolute amount of
LG-t-PA bound was calculated based upon the specific activity of
LG-t-PA.
Figure 4:
RGD-containing peptide blocks the binding
of LG-t-PA to platelet integrin . A
binding assay was performed as described under ``Materials and
Methods.'' The binding of LG-t-PA to integrin
was challenged with synthetic
peptides of sequence GRGDSP (
) or SPGDRG
(
).
The major finding of this study was that amino acids forming a biologically active, flexible surface loop on one protein could be grafted into another, unrelated protein and still maintain their initial binding activity. The protein sequence of the grafted loop was originally optimized by mutagenesis and affinity maturation of the donor protein, monoclonal antibody Fab-9, using phage display. To our knowledge these results provide the first example in which a flexible protein loop, optimized by phage display, has been inserted into another protein backbone to successfully endow the recipient protein with novel, high affinity binding properties.
This study differs
significantly both in approach, and in end result, from previously
reported studies where RGD sequences have been inserted into
non-adhesive proteins(33, 34, 35) . Although
these efforts have successfully targeted recipient proteins to
integrins, substantial quantitation of binding affinities has not been
reported for these proteins nor has the integrin target been
identified. In these studies an RGD sequence was inserted into either
lysozyme or calpastatin, and the apparent K of the
resulting, mutated protein for integrin appeared, based on cell
adhesion or cell spreading assays, to be approximately 400 or 50
nM, respectively. In a previous report we developed an
antibody against integrin that possesses 50-400-fold higher
affinity, with a K
approaching 1
nM(14, 27) . In this report we show that
grafting the amino acid sequence of HCDR3 of the optimized antibody
into the extended loop of an EGF module can transfer nanomolar affinity
for integrin to the EGF domain. The high affinity of LG-t-PA for
integrin almost certainly depends on maintenance of important
biophysical properties of the loop; both linear and cyclic synthetic
peptides containing the amino acid sequence of the Fab-9 HCDR3 exhibit
approximately 100-fold lower affinity for integrins than LG-t-PA or
Fab-9.
The development of a general method to accomplish the ab initio design of a three-dimensional structure and underlying amino acid sequence of a peptide motif that can be placed into a chosen recipient protein and will bind, at high affinity, to a selected target molecule will require substantially greater understanding of protein folding and molecular recognition than is presently available. Consequently, current efforts to use protein engineering to endow proteins with new binding properties often resort to very large scale mutagenesis protocols followed by laborious and time-consuming screening of individual variants of the protein of interest. It is now evident that phage display systems can simplify these types of mutational strategies by identifying active motifs from libraries containing vast numbers of distinct amino acid sequences. Phage display itself, however, is also often subject to severe limitations that prevent it from being a completely general approach; many proteins, for example, cannot be displayed on the surface of phage in a biologically active form, and significant problems with proteolysis of proteins displayed on phage are also frequently experienced. Many of these technical difficulties could be overcome, however, if biologically active structures could be identified in one or more protein contexts that are well behaved in phage display studies and then grafted into other proteins of interest. Such a system would also eliminate the need to create new libraries for each protein studied. A set of libraries using modules that can be easily manipulated by phage display could serve as the source of biologically active protein motifs of different size, composition, and structure. These biologically active motifs could then be grafted into several different positions of the recipient protein to create a novel variant with altered binding properties. Such a combinatorial approach has the potential to be substantially more rapid and less laborious than current approaches. Although it remains completely unclear how generally applicable this strategy will prove to be, as shown above, our initial attempt at ``loop grafting'' was successful.
Although the primary goal of this study was to test the feasibility of a protein loop-grafting strategy, it is possible that the resulting variant of t-PA merits consideration as an improved thrombolytic agent for the treatment of acute myocardial infarction and other thromboembolic disorders. Efforts to target plasminogen activators to blood clots by conjugating the enzyme to antibodies or Fab fragments of antibodies directed against either fibrin (36, 37) or platelet integrins (38) have been previously reported and have often demonstrated that this strategy can enhance the thrombolytic potency of the plasminogen activator both in vitro and in vivo. For example, chemical conjugation of monoclonal antibody 7E3 (anti-IIb-IIIa) to urokinase yielded a molecule that was 25-fold more potent in lysing platelet-rich clots than wild type urokinase(38) . LG-t-PA, therefore, due to its novel binding properties, may also exhibit enhanced potency toward the platelet-rich arterial thrombi that precipitate acute myocardial infarction.