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*
Department of Clinical Chemistry, Lund University, University Hospital Malmö, Malmö, Sweden; and
Department of Laboratory Medicine, Lund University, Lund, Sweden
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain is crucial for the C4b-C4BP
interaction. To extend this observation, and to investigate the
interaction with M proteins, we constructed and characterized a total
of nine mutants of C4BP. We identified a key recognition surface for M
proteins that overlaps with the C4b binding site because substitution
of R64 and H67 by Gln dramatically reduces binding to both ligands.
However, the analysis of all mutants indicates that the binding sites
for C4b and M proteins are only overlapping, but not identical.
Furthermore, M proteins were able to displace C4BP from immobilized
C4b, whereas C4b only weakly affected binding of C4BP to immobilized M
proteins. We found that the molecular mechanisms involved in these two
interactions differ because the binding between M proteins and C4BP is
relatively insensitive to salt in contrast to the C4BP-C4b binding. In
addition, six mAbs directed against the -chain interfered with
C4b-C4BP interaction, whereas only two of them efficiently inhibited
binding of C4BP to M proteins. Collectively, our results suggest that
binding between C4b and C4BP is governed mostly by electrostatic
interactions, while additional noncovalent forces cause tight binding
of C4BP to streptococcal M proteins. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chains and one ß-chain (4, 5). As shown by electron
microscopy and biochemical studies, C4BP consists of a 
160-kDa
central core that can be released by chymotrypsin treatment and several
extended tentacles formed by the -chains (6, 7). The
- and ß-chains are connected by disulfide bridges and consist of
eight and three CCP (complement control protein) units, respectively
(8). CCP units are often encountered in complement factors
and consist of 60 aa forming a compact hydrophobic core surrounded
by five or more ß-strands organized into ß-sheets (9).
The mechanism by which C4BP functions as a factor I cofactor in the
degradation of C4b is still unclear. However, we have recently shown
that a cluster of positively charged amino acids present on the
interface between CCP1 and CCP2 of the C4BP -chain is crucial for
binding of C4b and for factor I cofactor function
(10). An interesting property of C4BP is its ability to bind to Streptococcus pyogenes (group A streptococcus) and Bordetella pertussis, two bacterial pathogens that are major causes of disease in humans (11, 12). The binding of C4BP to S. pyogenes is mediated by surface M proteins, which are important virulence factors (11, 13). The M proteins form fibrillar coiled-coil dimers on the streptococcal surface and have been studied extensively, due to their important ability to inhibit phagocytosis (14, 15).
Many strains of S. pyogenes bind C4BP (11), and the available evidence indicates that binding of C4BP to M proteins plays an important role in pathogenesis. In particular, studies of several different M proteins showed that the binding site for C4BP is localized to the hypervariable N-terminal region of the M proteins (16). This finding implies that the interaction with C4BP is of physiological importance, because the ability to bind C4BP has been retained in spite of extensive sequence variation in the hypervariable region. The role of bacteria-bound C4BP may be to down-regulate complement activation in the bacterial microenvironment, thereby protecting the bacterium against opsonization and phagocytosis (11).
In a study employing whole S. pyogenes bacteria, the binding
site in C4BP was mapped to an area of C4BP encompassing CCP1-CCP3, and
it was suggested that the binding site of surface M proteins mimics
that of C4b (17). In this study, we report a comparison
between the binding sites for C4b and M proteins in the C4BP -chain,
and we point out important differences in the molecular mechanisms
governing these two interactions. Using a set of C4BP mutants, we first
considerably extended our previous study on the binding site for C4b
(10) and confirmed the conclusion that a cluster of
positively charged amino acid residues at the CCP1-CCP2 interface is
essential for the binding of C4b. All nine C4BP mutants were then
analyzed for ability to bind two purified streptococcal M proteins,
Arp4 and Sir22 (11, 18), which have different N-terminal
regions responsible for binding of C4BP (16). We concluded
that a key binding site on C4BP for Arp4 and Sir22 lies on the
interface between CPP1 and CCP2. However, the recognition sites in C4BP
for C4b and M proteins are only overlapping, but are not identical.
Furthermore, the intermolecular forces involved in these interactions
are different. Collectively, these results extend our knowledge about
the binding properties of C4BP and provide new insights into the
molecular mechanisms by which S. pyogenes interacts with the
immune system of the infected host. The present characterization of
binding mechanisms may apply to other regulatory proteins of the
complement system and to other pathogens that have evolved similar
strategies to avoid complement attack.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Human plasma C4BP, C4, and the streptococcal Arp4 and Sir22 proteins were purified, as described previously (18, 19, 20). Arp4 and Sir22 are equal to Emm4 and Emm22 in an alternative nomenclature (13). Protein concentrations were determined by measurement of absorbance at 280 nm, and the extinction coefficients (1%, 1 cm) used were 14.1, 8.3, and 19.4 for C4BP, C4, and Arp4, respectively. Due to a very low content of aromatic amino acids in Sir22, it is not possible to use absorbance measurement. Instead, concentration of Sir22 was determined from amino acid composition analysis after 24-h hydrolysis in 6 M HCl.
C4b-like molecules (C4 met) were prepared by incubation of purified C4 with 100 mM methylamine, pH 7.6, for 1 h at 37°C and subsequent dialysis against 100 mM Tris-HCl, 150 mM NaCl, pH 7.5. It has been demonstrated previously that C4 molecules treated this way are functionally equivalent to the C4b that results from cleavage by C1s component of the complement pathway (21). Throughout the study, the C4 met derivative was used, but will be referred to as C4b for reasons of clarity.
cDNA clones for the C4BP mutants R39Q, R64Q/R66Q, and R39Q/R64Q/R66Q and purification of the recombinant proteins were described previously (10). Briefly, the proteins were expressed from pcDNA3 vector (Invitrogen, San Diego, CA) in a human kidney cell line (293; American Type Culture Collection (Manassas, VA) catalog number 1573-CRL) and purified by affinity chromatography on a mAb 104 column. Mutations K63Q, R64Q, R66Q, H67Q, and K79Q were introduced according to QuikChange kit (Stratagene, La Jolla, CA); primers used were: K63Q (5'-ACC TTC TGT ATC TAC CAA CGA TGC AGA CAC-3'); R64Q (5'-TTC TGT ATC TAC AAA CAA TGC AGA CAC CCA-3'); R66Q (5'-ATC TAC AAA CGA TGC CAA CAC CCA GGA GAG-3'); H67Q (5'-AAA CGA TGC AGA CAA CCA GGA GAG TTA CGT-3'); K79Q (5'-GGG CAA GTA GAG ATT CAA ACA GAT TTA CTA TTT-3'); and their antisense counterparts. Nucleotides corresponding to mutated amino acids are underlined. All of the mutations were confirmed by DNA sequencing (Perkin-Elmer, Norwalk, CT). For construction of the R39Q/R64Q/R66Q/K79Q mutant, we used as a template cDNA coding for R39Q/R64Q/R66Q C4BP. Concentrations of rC4BP mutants were determined from amino acid composition analysis after 24-h hydrolysis in 6 M HCl. Proteins were labeled with 125I using the chloramine T method. The sp. act. was 0.4–0.5 MBq/µg of protein.
Abs used in this study were: mAb 67 recognizing CCP4 of the -chain;
mAb 70, CCP1 of the -chain; mAb 92, mAb 96, mAb 102, and mAb 104
binding to CCP1-CCP2 of the -chain (22). In the
previous study, it was shown that only mAb 70 could displace 70% of
the binding of C4BP to mAb 96; all other Abs bound to different
epitopes (22).
C4b/Arp4/Sir22 ligand-binding assay
Microtiter plates (Maxisorp; Nunc, Naperville, IL) were
incubated overnight at 4°C with 50 µl of solution containing 10
µg/ml C4b, Arp4, or Sir22 in 75 mM sodium carbonate, pH 9.6. The
wells were washed three times with 50 mM Tris-HCl, 0.15 M NaCl, and
0.1% Tween, pH 7.5 (washing buffer), and then incubated at room
temperature with 200 µl of quench solution (washing buffer
supplemented with 3% fish gelatin). After another three washes,
increasing concentrations of plasma-purified C4BP or recombinant
proteins diluted in 50 mM Tris-HCl, 150 mM NaCl, pH 8 (TBS),
supplemented with 0.1% BSA and 0.1% Tween 20 were added, and the
plates were incubated for 4 h at room temperature. The plates were
then washed three times and incubated with biotinylated mAb mAb 67
diluted in quench solution. The Ab recognizes the middle part (CCP4) of
the -chain (22). After 1 h of incubation, the
plates were washed and incubated for 1 h with
streptavidin-conjugated HRP (Dakopatts, Glostrup, Denmark), washed
again, and developed according to the manufacturer’s instructions. The
results are shown as a percentage of the maximal binding of wild-type
rC4BP obtained in each set of experiments. Experiments were repeated
three times, and means ± SD were calculated.
Competition assay
Microtiter plates were incubated overnight at 4°C with 50 µl
of solution containing 10 µg/ml C4b, Arp4, or Sir22 in 75 mM sodium
carbonate, pH 9.6. The wells were washed three times with washing
buffer and then incubated at room temperature with 200 µl of quench
solution. After another three washes,
125I-labeled C4BP (125I-C4BP) was
added (20 kcpm/well) together with enough plasma-purified C4BP in 50 mM
Tris-HCl, 150 mM NaCl, 0.1% Tween 20, and 0.1% BSA, pH 7.5, to
achieve 75% of the binding obtained with the tracer alone. Thus,
0.7 nM or 4.7 nM C4BP was added when C4b or Arp4/Sir22 was immobilized,
respectively. Increasing concentrations of various mAbs were also added
to the samples (mAb 67, 70, 92, 96, 102, and 104) or C4b, Arp4, and
Sir22. In the experiments testing the influence of salt concentration
on the binding, 125I-C4BP was added to plates
covered with C4b, Arp4, or Sir22 in buffer with NaCl content ranging
from 25 mM NaCl to 1.5 M NaCl in 20 mM Tris-HCl, pH 7.4. The samples
were incubated for 4 h at room temperature and washed five times,
and the amount of radioactivity associated with each well was measured
in a gamma counter. In experiments with varying salt concentrations,
the plates were washed with buffer consisting of 50 mM Tris-HCl, pH
7.5, and 0.1% Tween 20. The results are shown as percentage of the
maximal binding of 125I-C4BP obtained in each
experiment. Approximately 70–75% or 18–24% of applied
125I-C4BP bound when Arp4/Sir22 or C4b was
immobilized. Experiments were repeated three times, and means ±
SD were calculated.
Binding of C4BP to S. pyogenes
S. pyogenes strain AL168, expressing Sir22 (18), was used for binding experiments, and a nonbinding mutant was used as a negative control. S. pyogenes strains were grown overnight in Todd-Hewitt broth (Difco, Detroit, MI) at 37°C. The streptococci were diluted in a suspension of the non-C4BP-binding Escherichia coli strain LE392, which was cultivated overnight at 37°C in LB medium. Binding assays were performed essentially as described (11). Briefly, the bacteria were washed twice in PBSAT (120 mM NaCl, 30 mM sodium phosphate, 0.02% NaN3, 0.05% Tween 20, pH 7.2) and resuspended to 109 organisms/ml. Duplicate samples of bacteria (200 µl) were mixed with 20 µl of 125I-C4BP variants (15 kcpm) and incubated at room temperature for 2 h. After addition of PBSAT (2 ml), the samples were centrifuged at 4200 x g for 12 min and the supernatants were discarded. This washing step was repeated once. Radioactivity in the pellets was determined, and binding was expressed as the percentage of radioactivity added that remained in the bacterial pellet. The mean of the duplicate samples was calculated, and nonspecific binding (<7%), recorded with strain LE392, was subtracted. The experiment was repeated three times, and means ± SD were calculated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We have previously shown that a cluster of positively charged
amino acids, present at the interface between CCP1 and CCP2 of the C4BP
-chain, is crucial for C4b binding and factor I cofactor function
(10). In that study, we used three mutants of C4BP, in
which either one, two, or three Arg residues were replaced by polar
Gln, generating mutants R39Q, R64Q/R66Q, and R39Q/R64Q/R66Q. We found
that all three variants of C4BP showed decreased binding ability for
C4b and were poor cofactors for factor I. We decided to generate
additional C4BP mutants to investigate in detail the interaction with
C4b and other ligands that might bind to the same site. Fig. 1
shows the 3D model of the structure for
CCP1 to CCP3 in human C4BP (8, 10) that we used to design
these mutations. Five new single amino acid mutants and one quadruple
mutant were constructed. Thus, a total of nine different C4BP mutants
were used, including the three mutants described previously (see Figs. 1 and 2 for the designations of these
nine mutants). In all nine cases, we exchanged positively charged,
surface-exposed amino acids to Gln residues. These changes were
designed taking the 3D model structure into account and were thus
expected to be structurally well tolerated. The rC4BP molecules were
expressed in the human kidney cell line 293 and purified from culture
media by affinity chromatography. Fig. 2A shows the
recombinant proteins stained with Coomassie brilliant blue after
separation on a 10% polyacrylamide gel under reducing conditions.
Unreduced samples were applied on a 5% gel and stained with Coomassie
brilliant blue (Fig. 2B) or transferred to a polyvinylidene
difluoride membrane and detected with mAb 67, recognizing CCP4 of C4BP
(Fig. 2C). All mutant proteins were expressed by the cells
at similar levels and had similar mobilities upon SDS-PAGE. They were
then probed with a panel of mAbs directed against -chain of C4BP
(mAb 67, 70, 92, 96, 102, and 104). Approximately 50% binding to
immobilized wild-type C4BP was observed at 0.4–1 nM of each Ab
(results not shown). Similar results were obtained for all variants of
C4BP. This shows that the mutations did not affect the recognition of
the C4BP by the mAbs, suggesting that the conformation of the C4BP
variants was correct.
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The interaction between C4b and rC4BP mutants was probed using a
direct binding assay. Purified C4BP was added to immobilized C4b, and
the amount of bound protein was detected using biotinylated mAb 67.
Fig. 3A (left
panel) shows binding curves for the six available single amino
acid mutants of C4BP. Due to the multimeric nature of C4BP, this
analysis only allowed a qualitative analysis of the effect of the
mutations on interactions, but did not permit calculation of affinity
constants. Maximal binding of wild-type rC4BP was set as 100%, and the
apparent affinities for each mutant were estimated from the midpoint of
the corresponding binding curve. The different apparent affinities are
shown relative to that obtained for wild-type rC4BP (Fig. 3B, left panel). Plasma-purified and wild-type
rC4BP bound to C4b with similar apparent affinities, confirming that
293 kidney cells are able to assemble correct C4BP molecules.
Approximately 3 nM of each protein was required to reach half-maximal
binding. All introduced mutations resulted in decreased binding ability
of C4BP, in accordance with our hypothesis that positively charged
residues at the CCP1-CCP2 interface are crucial for binding of C4b
(10). In particular, substitution of
Arg64, His67,
Lys63, and Arg39 caused a
strong decrease in apparent affinity between C4b and C4BP.
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To investigate whether the electropositive cluster of amino acids
at the interface between CCP1 and CCP2 was also important for the
interaction with streptococcal M proteins, we analyzed the ability of
the different C4BP mutants to bind the two M proteins Arp4 and Sir22.
This was of particular interest because it has been suggested that
S. pyogenes binds to a site in C4BP indistinguishable from
the C4b binding site (17). The interaction between C4BP
mutants and streptococcal M proteins was evaluated in a direct binding
assay (as described for the analysis of C4b binding), using purified
preparations of M proteins (Fig. 3
A). The apparent
affinities of the different C4BP mutants for the M proteins were
estimated from the midpoint of the binding curves and are shown in Fig. 3B as relative change when compared with wild-type rC4BP.
Approximately 0.3 nM of wild-type rC4BP was needed to obtain
half-maximal binding to both M proteins. Several of the C4BP mutants
were strongly affected in their ability to bind the two M proteins.
We found that two of the tested single amino acid mutants, R64Q and H67Q, showed dramatically decreased apparent affinity to Arp4, by 180- and 40-fold, respectively. This result clearly indicates that these two amino acids constitute a vital part of the Arp4 recognition site on C4BP. R39Q and K63Q showed only a slight decrease in binding ability, whereas R66Q and K79Q bound to Arp4 better than to a wild-type rC4BP. Analysis of the double mutant R64Q/R66Q and the triple mutant R39Q/R64Q/R66Q was of particular interest, because the R66Q mutation alone increased binding. Both mutants showed strongly decreased binding, once again emphasizing the importance of R64 for efficient binding. However, the quadruple mutant R39Q/R64Q/R66Q/K79Q gave a surprising result, in that it bound to Arp4 almost as well as wild-type rC4BP.
The effects of mutations obtained for the two different M proteins were in most cases alike, but they were less pronounced for Sir22. Using direct binding assay, we found that H67Q, R64Q, and K63Q bound with a lower apparent affinity to Sir22, similar to what we observed for Arp4. R39Q, R66Q, and K79Q bound with increased affinity to Sir22. Results obtained for double, triple, and quadruple mutants were comparable with those described for Arp4. The results acquired from the direct binding assay were confirmed by competition experiments, in which radiolabeled wild-type rC4BP was allowed to compete with various C4BP mutants for binding to C4b and Arp4 (results not shown).
Observations made for C4b and the two M proteins (Fig. 3) were similar
for several of the C4BP mutants. For example, the H67Q mutant showed
strongly decreased binding to all three ligands. However, some
differences are noteworthy. In particular, none of the C4BP mutants
showed increased binding to C4b, while R66Q and K79Q showed
considerably increased binding to the two M proteins. Moreover, the
quadruple mutant, which was almost unaffected in its ability to bind M
proteins, showed strongly reduced binding to C4b (but also in the case
of C4b the quadruple mutant bound better than the triple mutant). Taken
together, these data suggest that C4BP has overlapping, but not
identical, binding sites for C4b and M proteins.
We also tested whether the point mutations introduced in C4BP had
similar effect on the binding to whole S. pyogenes bacteria
as to purified M proteins. In this experiment, bacteria in suspension
were analyzed for ability to bind wild-type
125I-rC4BP or various mutants. Binding was
analyzed for the Sir22-expressing strain AL168, and the results were in
agreement with those obtained with purified Sir22. As shown in Fig. 4, up to 60% of wild-type rC4BP bound to
this strain. The binding of R64Q, H67Q, and R64Q/R66Q was decreased as
compared with recombinant wild type. Results obtained for remaining
mutants were also similar to those obtained with purified Sir22 (not
shown). The fact that comparable results were obtained with purified
streptococcal proteins and proteins expressed on the bacterial surface
shows that physiologically relevant conclusions can be drawn from our
experiments.
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Results obtained with mutants of C4BP prompted us to perform
competition assays to assess whether or not C4b and M proteins utilize
the same binding site on C4BP. C4b, Arp4, or Sir22 was immobilized in
wells of microtiter plates, and 125I-C4BP was
added together with various fluid-phase competitors: C4BP, C4b, Arp4,
or Sir22. Both Arp4 and Sir22 were able to displace
125I-C4BP from the immobilized C4b (Fig. 5, upper panel). In a
reciprocal experiment, C4b had only a weak effect on binding of
125I-C4BP to immobilized Arp4 or Sir22 (Fig. 5, middle and lower panels). These results suggested
that either the affinities between the C4b-C4BP and M protein-C4BP
interactions are very different and/or that these proteins bind to
nonequivalent sites on C4BP. Similar results have been
obtained previously in a competition assay in which C4b was allowed to
compete with Arp4 for binding to immobilized C4BP (11). In
such a set up, Arp4 displaced 125I -labeled C4b
from C4BP, whereas the addition of C4b did not prohibit binding between
125I-labeled Arp4 and immobilized C4BP.
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To further characterize the binding sites for C4b and S.
pyogenes, we tested whether these interactions were inhibited in
the presence of increasing salt concentration. To this end,
plasma-purified 125I-C4BP was incubated with C4b,
Arp4, or Sir22 immobilized in wells of a microtiter plate. The buffer
(20 mM Tris-HCl, pH 7.4) was supplemented with 0.1% BSA, 0.1% Tween
20, and NaCl concentrations ranging from 25 mM to 1.5 M. After 4-h
incubation and washing with 50 mM Tris-HCl, pH 7.5, supplemented with
0.1% Tween 20, bound C4BP was measured in a gamma counter. Fig. 6 shows that binding between C4BP and C4b
(black bars) wassensitive to salt concentration and that
binding was abolished already at 0.3 M NaCl. In contrast, binding
between Arp4 and C4BP (gray bars) was relatively insensitive to salt
concentration and only decreased by 30% at 1.5 M NaCl. The interaction
between Sir22 and C4BP (white bars) was slightly more sensitive to NaCl
concentration as compared with Arp4 and decreased by 50% at 1 M NaCl.
These data support the conclusion that C4b and M proteins use different
mechanisms to interact with C4BP.
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A competition assay was used to study the effects of several mAbs
against C4BP on the binding of C4b, Arp4, and Sir22. Trace amounts of
plasma-purified 125I-C4BP were added together
with increasing amounts of various mAbs: mAb 67, 70, 92, 96, 102, and
104. In agreement with results reported previously, we found that all
of these Abs, which have similar affinities for the -chain of C4BP,
inhibited the interaction with C4b (22). As shown in Fig. 7 (upper panel), mAb 104 was
most efficient in inhibiting interaction with C4b: about 0.8 nM Ab was
required to reach 50% inhibition of the binding, while other Abs were
5- to 10-fold less efficient. The same experiment was performed with
immobilized Arp4 (Fig. 7, middle panel). In this system, mAb
104 and also mAb 102 had a strong inhibitory effect on the interaction.
However, higher concentration had to be used to obtain similar
inhibition as for the C4b-C4BP interaction. Several Abs influenced the
interaction with Arp4 weakly or had no effect. In particular, mAbs 67
and 92 had little or no influence on the binding between C4BP and Arp4,
in contrast to their effect on binding between C4BP and C4b. Results
obtained for Sir22 were similar to Arp4 (Fig. 7, lower
panel). However, when Sir22 was immobilized, only mAb 102 and mAb
104 significantly inhibited the interaction with C4BP at the
concentrations tested.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain
involved in the binding of these ligands. To address this question, we
used nine C4BP mutants and compared the ability of these molecules to
interact with C4b and with the M proteins Arp4 and Sir22. Effects of
NaCl and mAbs were also tested, and the experimental results were then
evaluated in conjunction with structural analysis of a recently
reported 3D model structure of human (8) and mouse C4BP.
Taken together, our data show that the key binding region for C4b
overlaps with the surface interacting with Arp4/Sir22 and is located on
CCP1 and CCP2. However, the recognition areas are not identical and the
molecular mechanisms involved in these two processes differ. Previously published data from our group (10) together with the present results show that Arg39, Lys63, Arg64, and His67 are crucial for C4b binding. This observation indicates that an essential interaction site is located at the interface between the CCP1-CCP2 modules and on CCP2. These results are in good agreement with other published observations showing that the key binding site for C4b lies within the CCP1-CCP3 region (6, 17, 22, 23). We suggest that C4b displays a negative surface that could complement the electropositive region in C4BP. This hypothesis is supported by a recent report showing that negatively charged residues in C3b (homologous to C4b) are essential for the binding to factor H and to complement receptor 1 (CR1; similar in structure and function to C4BP) (24). Thus, it is possible that C4BP, factor H, and CR1 all display a positively charged binding region, while the binding area at the surface of C3b or C4b is electronegative. Indeed, Krych and colleagues observed that altered binding of C3b or C4b to CR1 always correlated with mutations that removed a positive charge or added a negative charge in CR1 (25). Accordingly, it has been observed that binding affinity of cofactors to C3b and C4b increases concomitantly with decrease in ionic strength of the buffer (26). Long-range electrostatic forces together with favorable charge-charge interactions could be crucial for the formation of many short-lived macromolecular complexes within the complement cascade.
Previously, Accardo and co-workers have shown that the entire binding
site for S. pyogenes lies within CCP1–3 of the C4BP
-chain and suggested that this site is indistinguishable from the
C4b binding site (17). These authors have also shown that
replacing Arg64 and His67
by uncharged amino acids decreased the ability of C4BP to bind whole
S. pyogenes bacteria and C4b-Sepharose. Analysis of our
results clearly shows that the cluster of positively charged amino
acids between CCP1 and CCP2, which is crucial for binding of C4b, is
also part of the site of interaction for the streptococcal M proteins
Arp4 and Sir22. However, the mechanism of the latter binding appears to
be different, and other forces than electrostatic most likely play an
important role. This conclusion is based on the differential effects of
increasing NaCl concentration on the binding of C4b and of M proteins
and on analysis of the C4BP mutants.
Interactions between proteins involve complex mechanisms and are
predominantly dictated by electrostatic forces, hydrogen bonds, and van
der Waals contacts (27). Furthermore, binding surfaces are
more hydrophilic than the protein core, but they also tend to be more
hydrophobic than the average nonbinding surface areas (28, 29). Characterization of binding mechanisms by interpretation of
mutagenesis results can be complicated because the side chains of some
amino acids, like these of Arg or Lys, have dual, hydrophobic, and
charged/polar characteristics. In this study, we attempted to
differentiate between two binding mechanisms, one which seems to be
highly dependent on electrostatic forces and the other being favored by
hydrogen bonding and hydrophobic contacts. It is generally accepted
that NaCl in the 0.1–1 M range reduces long-range electrostatic
interactions and limits the formation of salt bridges
(30), without significantly affecting their stability once
they are formed at the interface of a specific protein-protein
recognition site. In contrast, hydrophobic interactions between
hydrophobic side chains present at protein interfaces together with
hydrogen bonds are short ranged, and NaCl concentrations within the
range used in this study should not significantly disturb such contact.
The relatively minor effect of salt on the C4BP-Arp4/Sir22 interactions
stands in sharp contrast to the C4b-C4BP binding that was entirely
abolished already at 300 mM NaCl (Fig. 6). The most likely hypothesis
that could account for these effects is that long-range electrostatic
forces and ion pairings are essential for the attraction and binding of
C4b to C4BP, while the binding of M proteins to C4BP also involves
numerous hydrophobic interactions and hydrogen bonds. Possibly, the
C4b-C4BP complex requires only short
t1/2 for physiological reasons (i.e.,
C4b interacts with C4BP, is cleaved by factor I, and then released),
while the S. pyogenes-C4BP interaction probably has evolved
to escape complement attack requiring different types of forces to
further stabilize the binding with C4BP. These two different mechanisms
could then result in the observed 10-fold difference in apparent
affinity constants. It seems that although the electropositive region
on C4BP CCP1 and CCP2 lies within the binding site for Arp4, the
interaction is essentially depending on hydrogen bonds and hydrophobic
contact. Interestingly, similar hydrophobic interaction seems to be
used by measles virus, as it interacts with a hydrophobic loop present
on the surface of CD46 (31).
Residues Lys63, Arg64, and
His67 in C4BP are directly involved in the
binding of Arp4 and Sir22 because changes of these residues resulted in
greatly decreased binding of both streptococcal proteins. It seems that
the binding involves the hydrophobic character of
Lys63 and Arg64, but not
their ability to form salt bridges, in contrast to what is expected for
the C4b-C4BP interaction, because the Arp4/Sir22-C4BP binding is rather
insensitive to salt. There are slight differences between Arp4 and
Sir22 with respect to their interaction with C4BP mutants, which is
consistent with the fact that, although these two proteins share an
overall sequence identity of 78%, there is only 25% identity
within the hypervariable region binding C4BP (estimated with Lasergene;
DNAstar, Madison, WI). The hypervariable region in Arp4 and Sir22
contains many negatively charged amino acids that could attract (in a
nonspecific manner) the electropositive region on C4BP. However,
negatively charged amino acids are also present in the hypervariable
region of protein M5 that does not interact with C4BP
(16). These findings agree with our results and imply that
other forces than electrostatic play an important role in the
interaction between C4BP and M proteins.
The conclusion that C4b and Arp4/Sir22 have overlapping but not
identical binding sites on the surface of C4BP was confirmed by using a
set of mAbs, directed against the -chain of C4BP. Inhibition of the
C4b-C4BP binding by all six Abs was reported previously, and our
present results are in agreement with these data (22).
Only two of these mAbs (no 104 and 102) could completely inhibit the
binding of both Arp4 and Sir22 to C4BP. In particular, mAb 104 was the
most efficient inhibitor both for C4b and for Arp4/Sir22. However, it
should be noted that mAbs 67 and 92 had little or no effect on the
binding of Arp4/Sir22, but could completely block binding of C4b,
confirming that the two different ligands do not have identical binding
sites. There are three additional lines of evidence for the conclusion
that C4b and M proteins bind in different ways to C4BP. First, we have
found that the pH dependencies of the C4b-C4BP and Arp4/Sir22-C4BP
interaction differ. Binding between C4b and C4BP showed two maxima
around pH 6 and 8, whereas Sir22-C4BP interaction did not change in the
interval of pH 6–9 (results not shown). Second, the positively charged
cluster on C4BP binds the electronegative polysaccharide heparin, which
therefore competes with C4b for binding to C4BP (8), but
influences the binding between C4BP and Arp4 to a much smaller extent
(8). Third, both Arp4 and Sir22 block binding of C4b to
C4BP, while C4b is not able to efficiently compete with M proteins for
binding to C4BP even at a concentration of 10 µM (Fig. 5)
(11). This observation suggests that the interaction
between C4BP and M proteins on the surface on the bacteria is
physiologically relevant even during a massive activation of the
complement system when high concentrations of C4b are generated
locally.
Because human and mouse C4b interact with C4BP CCP1 to CCP3
(23), but only human and higher primate C4BP molecules
interact with S. pyogenes (17), we decided to
compare the molecular surface of 3D models of human and murine C4BP
aiming at finding regions that present important amino acid differences
(Fig. 8). Positively charged residues at
the interface between CCP1-CCP2 are conserved in the two species, which
is consistent with the hypothesis that they are crucial for the
attraction and transient binding of C4b (via electrostatic forces).
During evolution, the molecular surfaces of M proteins may have evolved
in such a way that they not only occupy the area overlapping with the
C4b binding site on the surface of C4BP, but also allow for M proteins
to remain tightly bound. The surfaces of CCP3 in the two species are
relatively similar, and the fact that S. pyogenes binds only
to human C4BP but not to murine C4BP molecule suggests that CCP3 is not
a key binding site for M proteins. There are striking differences
within the linker region between CCP1 and CCP2, caused by the fact that
it is one residue longer in mouse C4BP as compared with human C4BP
(Fig. 8). This region is therefore a good candidate for constituting an
epitope recognized by some of the tested mAbs. The differences in
length and amino acid distribution in this region could also account
for the lack of interaction between mouse C4BP and streptococcal M
proteins. Some differences are also seen at the surface of CCP2, which
in human C4BP displays more polar surfaces (hydrogen bonds forming)
that together with hydrophobic-aromatic regions at the surface of CCP1
could be crucial for M proteins binding. The latter forces may be
individually weak, but since many such contacts can be established when
C4BP binds to Arp4 or Sir22, the overall result is the formation of a
tight, stable, and specific complex. It is possible that the molecular
surface required for binding of C4b involves at least CCP1–3, and that
it is larger than the binding area for M proteins, which appears to
mainly involve CCP1–2. This could in part be explained by the six
times larger molecular size of the C4b molecule as compared with the M
proteins.
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Björn Dahlbäck, Department of Clinical Chemistry, University Hospital Malmö, S-205 02 Malmö, Sweden.
3 Abbreviations used in this paper: C4BP, C4b-binding protein; 3D, three-dimensional; CCP, complement control protein domain; CR1, complement receptor 1; 125I-C4BP, 125I-labeled C4BP.
Received for publication October 6, 1999. Accepted for publication March 8, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-chain is crucial for C4b binding and factor I cofactor function. J. Biol. Chem. 274:19237.-chain is crucial for C4b binding and factor I-cofactor function. Biochem. J. 323:469.
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