(Received for publication, May 15, 1995)
From the
The thirteenth type III domain of fibronectin binds heparin
almost as well as fibronectin itself and contains a so-called
heparin-binding consensus sequence,
Arg
The interaction of heparin or heparan-sulfate glycosaminoglycans
(GAGs) ( The present study concerns fibronectin, a large modular glycoprotein
that interacts with a variety of macromolecules in the extracellular
matrix and with cell-surface molecules such as integrins and
heparan-sulfate proteoglycans. Such interactions are important in
regulating cell behavior including growth, adhesion, spreading,
migration, and differentiation(15) . Recent studies have shown
that certain cell types require both the GAG binding and cell binding
regions of fibronectin for efficient spreading and formation of focal
contacts(16, 17, 18) . The interaction of
fibronectin with heparin is dominated by the COOH-terminal hep-2 region
which can be isolated as a 30-kDa proteolytic fragment consisting of
fibronectin type III domains 12 through 14. Previous work has shown
that domain III-13, when isolated by further proteolysis or expressed
in Escherichia coli as an independent domain, binds heparin in
solution with almost the same affinity as the parent
fragment(19) . A cationic cluster,
Arg Fibronectin type III
domains are among the most ubiquitous of protein modules, occurring in
about 2% of animal proteins(20) . The three-dimensional
structures of several type III domains have been elucidated, and they
all show a similar
With the exception of the R54S mutant, all fusion proteins were
soluble and monomeric. All cleaved and purified III-13 domains were
homogeneous by analytical size-exclusion chromatography on Superdex-75
(Pharmacia) and/or SDS-polyacrylamide gel electrophoresis in
8-25% gradient polyacrylamide gels (Pharmacia Phast system). All
mutations were confirmed by sequencing at both the DNA and the protein
level. Protein sequencing was done with a Hewlett Packard G1000S
protein sequencing system. With the Arg
where [titrant] is the free concentration of fragment
(or peptide),
Figure 1:
Three-dimensional
structure of the heparin-binding domain III-13 modeled after the known
structure of a homologous type III domain from tenascin. Negatively
charged side chains are red, and positively charged Arg and
Lys side chains are green; there are no His
residues.
Figure 2:
Melting of recombinant fibronectin domain
III-13 and its mutants. Samples were equilibrated in TBS at
Figure 3:
Titration of fluorescein-labeled heparin
(0.1 µM) with recombinant fibronectin domain III-13 and
its mutants at room temperature in 0.02 M Tris-HCl, pH 7.4,
0.02% NaN
The results presented here indicate that binding of
fibronectin to heparin at physiological ionic strength is more complex
than previously appreciated. It involves at least 6 basic residues
within type III domain 13. These include Arg All of the single site mutants exhibited cooperative reversible
unfolding transitions as detected by sigmoidal changes in fluorescence
upon heating. This proves that the recombinant domains were folded into
a compact structure similar to that of the wild type and decreases the
likelihood that the observed effects of the mutations on binding are
due to improper folding. The choice of Ser as the substitute for Arg or
Lys in the mutants has the effect of replacing the cationic side chain
with a hydroxylated methlyene group which preserves a measure of
hydrophillic character. Furthermore, the new group is similar to those
which are abundant in the GAG thus minimizing the possibility that the
mutation would actually interfere with heparin binding. The variation
of T A three-dimensional model of
domain III-13 suggests that these 6 cationic residues, all of which are
conserved in human, bovine, rat, chick, and xenopus
fibronectin(15, 34, 35) , are clustered on
one side of the domain in a region devoid of negative charge. This
situation resembles that recently described for lactoferrin, which
contains two BBXB sequences that are separated by 24 residues
and form a cationic groove or ``cradle'' into which the
anionic polysaccharide was proposed to fit(8) . In fibronectin
III-13 only a single BBXB sequence is involved, perhaps
accounting for its It is
worth mentioning that domain III-14 of fibronectin has little affinity
for heparin in spite of the fact that it has 13 basic and only 7 acidic
residues for a net positive charge of +6, compared to +2 for
domain III-13. Yet, the affinity of III-14 for heparin is at least
10-fold lower in comparison to III-13(19) . A model of domain
III-14 (not shown) suggests that the cationic residues are dispersed
more or less randomly over its surface with no obvious clusters that
resemble anything like the cradle in III-13. The 3 contiguous basic
residues Arg-Lys-Lys (residues 87-89 in the model) fall in close
proximity to Glu The RRAR sequence, when present in a synthetic
peptide, has weak affinity for heparin in comparison to larger
fragments containing this sequence(19, 40) .
Furthermore, a proteolytic fragment of fibronectin that contains type
III domain 12 plus a 17-residue COOH-terminal extension that includes
the RRAR sequence of III-13 also has negligible affinity (fragment
10K12 in (19) ). It was argued that the low affinity might be
due to the failure of the peptide, when isolated or appended to domain
12, to assume a tertiary structure consistent with its configuration in
the folded domain. The present results show, however, that this
sequence by itself does not promote high affinity for heparin, even
when presented in the context of a properly folded domain, as in
the R23S and K25S mutants, both of which bind substantially more weakly
than the wild type domain. This same RRAR sequence also exists in the
carboxyl-terminal lobe of lactoferrin where it is not functional as a
GAG-binding site(8) . These observations help to explain why
the list of actual heparin-binding proteins is much shorter than the
list of proteins containing a putative heparin-binding ``consensus
sequence''(7) . Many of the latter probably lack the
additional strategically positioned cationic residues that are
necessary for tight binding to heparin.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-Arg
-Ala
-Arg
(residues 1697-1700 in plasma fibronectin). Barkalow and
Schwarzbauer (Barkalow, F. J., and Schwarzbauer, J. E. (1991) J.
Biol. Chem. 266, 7812-7818) showed that mutation of
Arg
-Arg
in domain III-13 of recombinant
truncated fibronectins abolished their ability to bind
heparin-Sepharose. However, synthetic peptides containing this sequence
have negligible affinity for heparin (Ingham, K. C., Brew, S.
A., Migliorini, M. M., and Busby, T. F.(1993) Biochemistry 32,
12548-12553). We generated a three-dimensional model of
fibronectin type III-13 based on the structure of a homologous domain
from tenascin. The model places Arg
, Lys
, and
Arg
parallel to and in close proximity to the
Arg
-Arg
-Ala
-Arg
motif,
suggesting that these residues may also contribute to the
heparin-binding site. Domain III-13 and six single-site mutants
containing Ser in place of each of the above-mentioned basic residues
were expressed in Escherichia coli. All of the purified mutant
domains melted reversibly with a Tm near that of the
wild type indicating that they were correctly folded. When
fluorescein-labeled heparin was titrated at physiological ionic
strength, the wild type domain increased the anisotropy in a hyperbolic
fashion with a K
of 5-7
µM, close to that of the natural domain obtained by
proteolysis of fibronectin. The R54S mutant bound 3-fold weaker and the
remaining mutants bound at least 10-fold weaker than wild type. The
results point out that the
Arg
-Arg
-Ala
-Arg
consensus sequence by itself has little affinity for heparin
under physiological conditions, even when presented in the context
of a folded domain. Thus, the heparin-binding site in fibronectin
is more complex than previously realized. It is formed by a cluster of
6 positively charged residues that are remote in the sequence but
brought together on one side of domain III-13 to form a ``cationic
cradle'' into which the anionic heparin molecule could fit.
)with proteins occurs in a variety of physiological
processes including blood coagulation, lipoprotein metabolism, cell
adhesion and migration, and regulation of growth factor
activity(1, 2, 3, 4) . Efforts to
understand the nature of such interactions are hampered by the fact
that GAGs are generally heterogeneous in their size and charge
density(5) . Furthermore, there is no example of a detailed
structure of a GAG
protein complex determined by x-ray or NMR
methods, although some attempts at molecular modeling have been
made(6, 7, 8, 9) . That
electrostatic forces are involved is evident from the fact that the
complexes can be disrupted by increasing ionic strength. It is clear
that positively charged Arg and/or Lys residues on the protein play an
important role because their modification by chemical (10, 11) or recombinant (12, 13, 14) means usually leads to a
reduction in the affinity for heparin, the most widely studied GAG.
This is supported by numerous studies with synthetic heparin-binding
peptides. However, in cases where the affinities have been measured,
the peptides rarely bind as tightly as the parent protein on which
their sequence is based. This probably means that the tertiary
structure of the protein holds the peptide in a conformation that is
more complementary to the GAG or that other residues not contained in
the peptide contribute to the binding site in the protein, or both.
-Arg
-Ala
-Arg
,
located near the NH
terminus of III-13, matches one of two
patterns that are commonly found in heparin-binding proteins, namely
BBXB, where B represents a basic residue, Arg, Lys, or His,
and X represents any residue(7) . Arg
and
Arg
were shown to be critical in that their simultaneous
mutation abolished the ability of truncated recombinant fibronectins
(``deminectins'') to bind heparin-Sepharose under
physiological conditions(12) . However, synthetic peptides
containing this cluster have negligible affinity for heparin under
physiological conditions, indicating that tertiary structure and/or
other residues are important (19) .
-sandwich fold which resembles that of the
immunoglobulin C
domain(21, 22, 23, 24) . To gain
insight into the three-dimensional arrangement of cationic residues in
the folded structure of III-13, a molecular model was generated based
on the known three-dimensional structure of a homologous type III
domain in tenascin(21) . The model predicted that in addition
to the above-mentioned arginines 6, 7, and 9, which would be expected
to fall close to each other, 3 additional cationic residues should be
located nearby in which case they might also contribute to the
heparin-binding site. Based on the model, all 6 residues were
individually mutated, and the resulting expression products were tested
for the presence of compact structure and their ability to bind heparin
in the fluid and solid phases. The results are embodied in the title of
this paper.
Molecular Modeling
The initial model of
fibronectin domain III-13 was generated using as a template the
three-dimensional structure of a homologous domain III-3 from
tenascin(21) . The amino acid sequences of III-13 that differed
from tenascin III-3 were replaced and adjusted manually to avoid
stearic overlap using the program FRODO(25) . This initial
model was then subjected to energy minimization and molecular dynamics
using the X-PLOR 3.1 program package(26) . In the first step of
this process, the model was subjected to 150 cycles of conjugate
gradient energy minimization to further reduce stearic contacts. During
this initial minimization, coordinates of side chain atoms of
conservative residues and all C atoms were held near
their initial positions by applying additional harmonic potential
restraints. The coordinates were modified by subjecting the model to a
two-stage molecular dynamics simulation. In the first stage the model
constrained as above was heated from 0 to 300 K in 5 ps and then
equilibrated for 50 ps. To increase the mobility of the side chains in
this simulation, the partial atomic charges were reduced to 0.5 of
their initial values, and hydrogen bond energy terms were turned off.
In the second stage of the simulation, the model was equilibrated for
another 50 ps with the harmonic restraints of the model removed, the
partial atomic charges restored to their original values, and the
hydrogen bond energy terms turned on. The resulting model was then
subjected to a final 200 cycles of conjugate gradient energy
minimization to optimize the geometry and stereochemistry of the final
structure.
Expression of Recombinant Domains
Expression of
recombinant III-13 and its mutants as fusion proteins with
maltose-binding protein was accomplished in E. coli using a
modified pMAL-p2 expression vector (New England Biolabs) in a manner
similar to that previously described(27) . cDNA fragments
encoding residues 1-89 of domain III-13, i.e. Asn-Thr
of plasma
fibronectin(28) , were prepared by PCR using 21-base synthetic
primers flanking the desired regions and cDNA encoding human
fibronectin kindly provided by Dufour et al.(29) . The
primers also contained the sequence for the BamHI and HindIII restriction sites for ligation into the pMAL-p2
expression vector. Approximately 20 ng of the ligated DNA was used to
transform TB1 E. coli cells by electroporation. The
electroporated cell/DNA mixture was grown in LB media at 37 °C for
1 h. Serial dilutions were spread onto plates covered with LB media
containing ampicillin and incubated at 37 °C overnight. After
screening for the presence of insert by restriction analysis and/or PCR
analysis, individual colonies were grown overnight at 37 °C with
shaking at 225 revolutions/min, and diluted 100-fold with fresh
ampicillin-containing media. After growing at 37 °C for about 2.5 h
or until the absorbance at 600 nm reached about 0.6, the cells were
induced with isopropyl-1-thio-
-D-galactopyranoside at a
final concentration of 0.3 mM and grown for an additional 3 h.
Although the fusion proteins were preceded by a signal peptide
sequence, the bulk of the product was found in the cytoplasm. The cells
were harvested by centrifugation, lysed by sonication in 0.005% Triton
X-100, and the fusion protein was purified immediately by affinity
chromatography on immobilized amylose and/or heparin. The yield of
fusion protein varied between 5 and 15 mg/liter. For unknown reasons,
storage of the lysate at 4 °C resulted in progressively lower yield
of material binding to the amylose resin. Similarly, the efficiency of
rebinding of maltose-binding protein and fusion protein to the amylose
resin, even after exhaustive dialysis, was too low to be useful in
further steps of purification. Therefore, after digestion of the fusion
proteins with factor Xa(30) , the liberated intact III-13
domains were purified to homogeneity by rechromatography on
heparin-Sepharose and/or QMA anion-exchange media (Waters) and
size-exclusion chromatography as needed. The amino terminus of the
final products contained 2 additional residues, Ile and Leu, that are
not part of the III-13 domain.
Mutagenesis
Arg Ser and Lys
Ser
mutations were prepared by the method of Ho et
al.(31) . Synthetic complementary 21-base primers
containing the desired mutation and spanning the region to be mutated,
as well as two primers exterior to the type III-13 insert, were
annealed to the wild type III-13 cDNA template. PCR extension of these
primers resulted in two cDNAs that were then annealed to each other
through their 21-base overlapping region (containing the mutation) and,
using the primers exterior to the III-13 insert, extended again to
produce the complete mutated inserts. The 6-fold degeneracy in the
codon for serine was exploited, when possible, to introduce unique
restriction sites in the mutated insert. In those cases, digestion with
the unique restriction enzyme followed by electrophoresis could be used
as an initial test for the presence of the mutation before the insert
was ligated into the plasmid. The R6S mutation (TCG for AGA) introduced
a second TaqI site (T-CGA), the R7S mutation (TCG for AGG)
added a third MboI site (-GATC), the R9S mutation (TCT for
CGT) inserted a unique XmnI site (GAANN-NNTTC), the R23S
mutation (TCG for AGA) produced a unique SalI site (G-TCGAC),
the K25S mutation (TCG for AAG) introduced a second TaqI site
(T-CGA), the R47S mutation added a unique SalI site (G-TCGAC),
and the R54S mutation inserted a unique XhoI site (C-TCGAG).
After initial confirmation of the mutation by digestion of the PCR
products at these unique sites, the mutated inserts were ligated into
the pMAL-p2 plasmid, transformed, expressed, and purified as outlined
above. Repeated attempts to generate the K25S mutation using an AGT
codon for the serine (unique ScaI site) were unsuccessful;
only about 25% of the plasmid was cleavable, and induced cells produced
no fusion protein. This was not a problem when the TCG codon was used.
, Arg
,
Arg
, Arg
, and Lys
mutants, this
was done on the intact domains because the mutations were close enough
to the NH
terminus of the domain. With the Arg
and Arg
mutants, it was necessary to first cleave
chemically or enzymatically, sequence the resulting mixture of
peptides, and compare the results with the known sequence of domain
III-13 using custom software developed by G. Argraves (Shelton, CT).
Thermal Stability
The structural integrity of the
recombinant III-13 domains was assessed by heating a solution of the
domain at a concentration of 0.1 mg/ml at
1 °C/min in the
SLM 8000C spectrofluorometer while monitoring the ratio of fluorescence
at 350 nm to that at 320 nm with excitation at 280 nm(32) . The
fluorescence ratio provides a convenient and sensitive means of
detecting the spectral shift that accompanies denaturation. Changes in
the fluorescence ratio permitted detection of melting transitions and
assessment of relative stability and the degree of reversibility of the
denaturation upon cooling.
Fluorescence Anisotropy
Measurements were made
with the SLM-8000C spectrofluorometer in the T format with excitation
and emission wavelengths of 493 and 524 nm, respectively. All
experiments described here were performed with Sephadex G-100 fraction
no. 4 of fluoresceinamine-labeled heparin (FA-heparin) having an
average molecular mass of 15,000 daltons(33) . Titrations
of 0.1 µM FA-heparin with recombinant III-13 domains were
performed in 0.02 M Tris buffer, pH 7.4, containing 0.02%
NaN
and no NaCl (TB) or 0.15 M NaCl (TBS). Small
amounts of a stock solution of the recombinant peptides were added
continuously with a motorized syringe controlled by the same computer
that controls the fluorometer. The change in anisotropy,
A, as a function of titrant concentration was fitted to a
single class of equivalent binding sites on the FA-heparin by using the
following equation:
A
is the maximum anisotropy
change that would be produced at saturating concentrations of titrant,
and K
is the apparent dissociation
constant of the heparin-fragment complex(19) . Since in all
cases the concentration of FA-heparin was low compared to the range of
concentration of fragment, the free fragment concentration was taken as
the total. The concentrations of the fragments were determined from the
absorbance at 280 nm, using a molar extinction coefficient,
= 10,800 M
cm
.
Analytical Affinity
Chromatography
Heparin-Sepharose was prepared as
described(33) . The recombinant peptides or fusion proteins
were applied at 1 ml/min to a 1.7-ml column of heparin-Sepharose in
either TB or TBS using a Pharmacia fast protein liquid chromatography
system. A linear gradient to 0.6 M NaCl was used for elution,
which was monitored by fluorescence at 340 nm with excitation at 280
nm.
Molecular Modeling of Domain III-13
The
fibronectin type III domains form a sandwich with a folding
topology similar to that of the immunoglobulin C
domain(21, 22, 23, 24) . Domain
III-13 of fibronectin was modeled after domain III-3 from
tenascin(21) . The results are illustrated in Fig. 1where the charged side chains are highlighted. Arginines
6, 7, and 9 conform to one of the sequence patterns identified in a
number of heparin-binding proteins(1, 7) . Arginines 6
and 7 are the ones whose mutation by Barkalow and Schwarzbauer (12) abolished binding of truncated fibronectins to
heparin-Sepharose at physiological ionic strength. The model suggests
that additional positively charged residues Arg
,
Lys
, and Arg
might also contribute to heparin
binding since they lie in close proximity to arginines 6, 7, and 9.
Although the positions of the side chains are not precisely determined
by the model, it is clear that 6 of the 10 positive charges in this
domain lie within a few nanometers of each other on one side of the
structure, in a region devoid of negative charge. A similar structure
has been identified as the GAG-binding site in human lactoferrin and
has been termed a ``cationic cradle''(8) . In an
effort to evaluate the relative importance of all 6 of these residues,
each of them was mutated separately to a serine residue. The resulting
constructs were expressed in E. coli and evaluated for folding
integrity and heparin binding. Arginine 47, which in this model lies on
the opposite side of the domain and is not predicted to be part of the
binding site, was also mutated as a control for the possibility that
simply reducing the net positive charge might affect heparin binding.
Reversible Unfolding of Recombinant III-13 and Its
Mutants
The thermal stability of the recombinant III-13 domains
was assessed as a measure of their structural integrity. The various
recombinant peptides were heated at 1 °C/min in the fluorometer
while monitoring the ratio of fluorescence at 350 nm to that at 320 nm
as a measure of the spectral shift that accompanies
unfolding(32) . As shown in Fig. 2, all of the products
underwent a cooperative sigmoidal unfolding transition similar to that
observed previously with natural fragments derived from the parent
protein by proteolysis(32) . The transitions were highly
reversible in that the fluorescence ratio returned to a value close to
the original upon cooling. The T values
varied between 60 and 71 °C (Table 1). These results show
that all of the recombinant III-13 domains, as isolated, were folded
into compact structures with stabilities similar to one another and to
that of the natural domain.
0.1
mg/ml and heated at 1 °C/min while monitoring the ratio of
fluorescence intensity at 350 nm to that at 320 nm with excitation at
280 nm. The dashed curves indicate reversibility on
cooling.
Analytical Affinity Chromatography on
Heparin-Sepharose
Affinity chromatography was used as a
qualitative test of the ability of the recombinant III-13 domains to
bind heparin(19) . All of the products bound in high yield to
the heparin-Sepharose column at room temperature, whether applied in
the presence (TBS) or absence (TB) of 0.15 M NaCl. Natural
III-13, derived from plasma fibronectin by proteolysis(19) ,
and wild type rIII-13 eluted similarly between 0.43 and 0.45 M in a gradient of NaCl (Table 1) indicating that the
recombinant and natural domains are functionally similar. The remaining
mutants all eluted earlier, between 0.25 and 0.38 M NaCl. The
final concentration of NaCl required for elution was similar whether
starting from 0.0 M (TB, Table 1) or 0.15 M
(TBS, not shown). The R47S control mutant also bound and was eluted at
the same salt concentration as the natural and recombinant wild type
fragments.Titration of Fluorescent-labeled Heparin
A
fluorescence polarization anisotropy assay was used to obtain a more
quantitative estimate of the effect of the various mutations on the
affinity for heparin in the fluid phase. The results are presented in Fig. 3where solid curves represent best fits to . In
TB, all of the products caused a dose-dependent increase in
fluorescence anisotropy of FA-heparin. Dissociation constants ranged
from 0.5 to 1.4 µM for the natural, wild type and R47S
control domains and all of the mutants fell within this same range (Table 1). At physiological ionic strength, in TBS, the K values of the wild type and R47S
control domains were close to each other and to that of the natural
domain although the values for all three were approximately 10-fold
higher than at low ionic strength. In contrast, the binding of the R6S,
R7S, R9S, and R23S mutants was too weak to cause a significant increase
in the anisotropy at the concentrations of the domains that were
achieved (Fig. 3). The K
for these
mutants is conservatively estimated at
100 µM, based
on the reasonable assumption that the change in anisotropy caused by
saturation with these mutants would be similar to that caused by the
wild type. The same assumption was used in fitting the data for the
K25S mutant, which caused a slight increase in anisotropy but still
failed to achieve a substantial fractional saturation of the response
at the highest concentration employed. The R54S mutant had the highest
affinity of all the mutants with a K
of
19 µM, still significantly weaker than the wild type or
R47S control. The results indicate that the affinity of domain III-13
for heparin, as measured in the fluid phase at physiological ionic
strength, is reduced at least 10-fold by elimination of a positive
charge at positions 6, 7, 9, or 23, slightly less at position 25, and
approximately 3-fold at position 54. By contrast, the affinity is
essentially unaffected by neutralizing the positive charge at position
47 and, when measured at low ionic strength, is not sensitive to any of
these mutations.
, in the absence (TB) and presence (TBS) of 0.15 M NaCl. Solid lines represent best fits of the data
to . The corresponding values of K
are given in Table 1.
,
Arg
, Arg
, Arg
, Lys
,
and Arg
. Mutation of any 1 of these residues to a serine
decreased the affinity of the isolated domain for fluorescent heparin
in the fluid phase in TBS by at least an order of magnitude in four
cases and to a lesser but still significant extent in the other cases.
The mutant domains also exhibited decreases in the concentration of
NaCl required for their elution from heparin-Sepharose. By contrast, in
0.02 M Tris without added salt, where the fluid-phase
interaction between domain III-13 and FA-heparin is about 10-fold
tighter than in TBS, the mutant domains bind as well as the wild type
domain. This latter observation should serve as a cautionary note to
those who would attempt to interpret the physiological significance of
GAG binding measurements conducted at subphysiological ionic strength.
between the different mutants was
outside the range of experimental error suggesting some subtle changes
in stability. This could arise from the loss of attractive or repulsive
electrostatic interactions involving the mutated residues or of
hydrophobic interactions involving the methylene groups in their side
chains. Note also the 9 °C lower T
of
wild type III-13 relative to its natural counterpart which was derived
from fibronectin by proteolysis. The natural fragment is significantly
longer, containing additional residues at the COOH-terminal end that
are derived from domain III-14 and might participate in a stabilizing
interaction with III-13(32) .
50-fold lower affinity for FA-heparin relative
to that of lactoferrin. The other 3 basic residues that contribute to
the GAG-binding site in III-13 are recruited from positions that are
14, 16, and 45 residues distant from Arg
in the primary
structure. Mutation of Arg
, which according to the model
is located on the opposite side of the domain, had no effect on
binding, indicating that mere reduction of the net positive charge from
+2 to +1 cannot account for the observed effects of the other
mutations. Other examples of proteins in which positively charged
groups that are remote in sequence are brought together in the folded
protein to form a GAG-binding site include antithrombin
III(36) , lipoprotein lipase(37) , fibroblast growth
factors(38) , and platelet factor 4(9, 39) .
In the latter case, the clustering of positive charge is enhanced
through self-association of the protein to form dimers and tetramers
with progressively higher affinity for heparin(39) .
which would diminish their cationic
potential. Similarly, although basic residues 26, 28, and 76 are
predicted to be in close proximity to each other, their cationic
potential would be diminished by proximity to Asp
. Thus,
the relative affinities of these two homologous domains for heparin are
consistent with the distribution of charge predicted by their
respective models.
We are indebted to Dr. David Mann for helpful
suggestions throughout the course of this work and for valuable
discussions during the preparation of the manuscript. Thanks also to
Mary Migliorini for confirming the presence of mutations in all of the
expressed recombinant domains by amino acid sequencing and to Gary
Argraves for development of the sequence analysis and titration
software.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.