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*
Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama, Birmingham, AL 35294;
School of Life Sciences, Keele University, Keele, Staffordshire, United Kingdom; and
Biomedical Sciences Research Center "A. Fleming," Vari, Greece
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-helix 169–176, particularly Tyr175.
Mutational analysis of residues participating in the formation of this
pocket demonstrates that Asp112 and Tyr175 are
important contact residues for C1q binding, that Glu88
influences the conformational change in C1q necessary for complement
activation, and that Asn158 and His38 probably
contribute to the correct geometry of the binding site. Thus, it
appears that the pocket at the open end of the cleft is the C1q-binding
site of CRP. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RI and FcRIIa receptors (10, 11). Thus, CRP represents an important first line of innate host
defense.
Our previous mutational studies have shown that residue
Asp112 of CRP plays a critical role in the
formation of the C1q-binding site, while Lys114
appears to hinder binding of C1q to CRP (12). Subsequent
determination of the crystal structure of human CRP (2, 3)
revealed a cyclic 5-fold symmetric aggregation of protomers, each
having a flattened jellyroll appearance, consisting of
anti-parallel 
-strands arranged into two -sheets. Two calcium
ions are ligated in close proximity to each other to side chains and
main chain carbonyls of each protomer. They are integral structural
elements of the PCh binding site of the protomer (13) and
all are located on the same recognition face of the CRP pentamer.
Because complement activation and opsonization by CRP require its
binding to an appropriate multivalent ligand through the PCh binding
site, it seems that the structural elements involved in CRP-C1q and
CRP-FcR interactions must be located on the face of the pentamer
opposite the ligand-binding site. This effector face is characterized
by an unusual extended deep cleft (2), which starts at
about the center of each protomer and extends to the central pore of
the pentamer. Residue Asp112 is located within
the relatively open end of the cleft toward the center of the pentamer,
suggesting that this region of the cleft may be involved in C1q
binding.
In the present study we have used site-directed mutagenesis to define the topology and structure of the C1q binding site of CRP. The results indicate that the site is located at the shallow end of the cleft toward the pentamer’s central pore and that in addition to Asp112, Tyr175 is critical for C1q binding and complement activation.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The CRP cDNA clone HLCRP-23 in the expression vector p91023 (14) was used as template for construction of mutant CRP cDNA. Mutagenesis was conducted using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. Mutagenic oligonucleotides were synthesized on a model 394 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA). All mutations were verified by nucleotide sequencing using the chain termination method (15). Two independent clones for each mutant were purified by two successive CsCl gradient ultracentrifugations, sequenced, and used for protein expression.
COS cells used for transfection were maintained in culture as reported previously (14). Forty micrograms of each recombinant plasmid containing wild-type (wt) or mutant CRP cDNA was transfected into 4 x 106 COS cells by electroporation at 350 V/500 µF using a Gene Pulsar apparatus (Bio-Rad, Richmond, CA). Transfected cells were replated in tissue culture dishes containing 10 ml of medium. The culture media were changed after 16 h and harvested 96 h after electroporation.
Purification of CRP and C1q
Native human CRP was purified from ascitic fluid by affinity chromatography on a PCh-conjugated agarose column (Pierce, Rockford, IL) as described previously (16). After affinity chromatography, CRP was further purified by fast protein liquid chromatography anion exchange chromatography on a MonoQ column (Pharmacia, Piscataway, NJ) eluted with a 0.15- to 1.0-M NaCl gradient in 20 mM Tris buffer, pH 7.8. Recombinant wt and mutant CRP were purified from COS cell culture supernatants using a single PCh affinity chromatography step as reported previously (13). C1q was purified from human plasma according to the method of Tenner et al. (17). The purity of all protein preparations was assessed by 5–20% polyacrylamide gradient SDS-gel electrophoresis.
CRP ELISA and ligand binding assays
The concentration of CRP was measured by a solid phase sandwich-type ELISA, using a sheep anti-human CRP serum (Cappel, Durham, NC) as capture Ab and the anti-CRP mAb HD2.4 (18) as reporter. Standard curves were constructed using purified native CRP (6.25–200 ng/ml). Wells were developed with affinity-purified alkaline phosphatase-conjugated goat anti-mouse IgG (Pierce) followed by Sigma 104 phosphatase substrate. Color development was measured at 405 nM using an ELISA plate reader (Molecular Devices, Menlo Park, CA).
The binding avidity of CRP for PCh ligands was measured by two assays using PCh-substituted BSA (PCh-BSA) or PnC in the solid phase. PCh-BSA (9 mol of PCh/mol BSA) was synthesized according to the method of Chesebro and Metzger (19). PnC was isolated from S. pneumoniae, strain R36A, as described by Liu and Gotschlich (20), except that the final enzymatic step was omitted. Both assays used protein A-purified HD2.4 mAb as reporter. Tris-buffered saline, pH 7.2, containing 0.1% BSA, 0.01% Nonidet P-40, 5 mM CaCl2, and 0.02% NaN3 (PCh binding buffer) was used throughout the assays. Standard curves were constructed by using purified native CRP (6.25–200 ng/ml). Purified wt and mutant CRP were diluted to a concentration of 50 ng/ml and tested in duplicate. Wells were developed as in the ELISA, using affinity-purified alkaline phosphatase-conjugated goat anti-mouse IgG and Sigma 104 phosphatase substrate.
C1q binding and C3 deposition assays
C1q binding assays used either PCh-BSA or PnC in the solid phase. Microtiter wells were coated with 100 µl of either PCh-BSA or PnC at a concentration of 10 µg/ml in Tris-buffered saline, pH 7.2. Standard curves were constructed using serial dilutions of purified native CRP, covering the concentration range from 50–1600 ng/ml in PCh binding buffer. Purified wt and mutant CRP were diluted to appropriate concentrations in the PCh binding buffer and tested in duplicate. Wells accounting for nonspecific binding received only buffer without CRP. After overnight incubation at 4°C, the wells were aspirated, washed twice with PCh binding buffer, and rinsed once with C1q binding buffer (10 mM Tris-HCl (pH 7.2), 100 mM NaCl, 5 mM CaCl2, 0.1% BSA, 0.01% Nonidet P-40, and 0.02% NaN3). Then 100 µl of 0.5 or 50 µg/ml of purified C1q in C1q binding buffer was added to wells coated with PCh-BSA or PnC, respectively. After 3-h incubation at room temperature, the wells were washed once with C1q binding buffer and twice with PCh binding buffer. Then, 100 µl of 5 µg/ml caprylic acid-purified rabbit anti-C1q IgG in PCh binding buffer were added to each well and incubated for 1 h at room temperature. Rabbit anti-human C1q sera were raised in this laboratory. After washing, the plates were developed using alkaline phosphatase-conjugated goat anti-rabbit IgG (Pierce) and Sigma 104 phosphatase substrate.
For C3 deposition assays, microtiter wells were coated with 100 µl of PnC (40 µg/ml) in PBS, pH 7.2. After 2 h at room temperature the wells were aspirated, washed once with PBS, blocked for 45 min at room temperature with 400 µl of PBS containing 1% BSA, and rinsed with buffer A (PBS containing 0.1% BSA and 1 mM CaC12). Purified dialyzed wt and mutant CRP were diluted to appropriate concentrations in buffer B (buffer A containing 0.01% Nonidet P-40) and added to duplicate PnC-coated wells. After 1-h incubation at 37°C the wells were washed twice with buffer B and rinsed once with buffer C (PBS containing 1% BSA, 0.15 mM CaC12, 0.5 mM MgC12, and 0.02% NaN3). Then 100 µl of normal human serum (NHS) diluted 1/10 in buffer C was added to each well and incubated for 30 min at 37°C. The NHS used was obtained from laboratory volunteers, and it was absorbed with PnC-conjugated agarose for 2 h at 0°C to remove any CRP and anti-PnC Abs. PnC was linked to tresyl-activated agarose (Schleicher & Schuell, Keene, NH) according to the manufacturer’s instructions. The wells were then washed twice with buffer C, and 100 µl of 1 µg/ml of anti-C3d mAb (Quidel, San Diego, CA) diluted in buffer C was added to each well. After 1 h at room temperature, the wells were washed and developed with alkaline phosphatase-conjugated goat anti-mouse IgG as in the ELISA. Standard curves were constructed using serial dilutions of purified native CRP in buffer B, covering the concentration range from 0.1–3.2 µg/ml. Wells accounting for nonspecific binding received buffer B without CRP.
Statistics
Differences among the binding activities of the various forms of CRP were assessed using unpaired Student’s t test. Unless otherwise mentioned, all comparisons were to wt CRP.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). Asp112,
Lys114, Tyr175,
Thr90, and Glu88 describe
the shallow end of the cleft, while His38 and
Asn158 are located deeper within the cleft. The
previously described mutants D112N, D112A, and K114A (12)
were included in the present study for completeness. All mutant CRP
cDNAs were expressed successfully and purified by affinity
chromatography on PCh-conjugated agarose. SDS-PAGE demonstrated that
the apparent m.w. of the subunits of recombinant wt and mutant CRP was
identical with that of native CRP. Similarly, the elution volume of all
CRP species from gel filtration columns was identical with that of
native CRP, indicating a pentameric structure. The functional integrity
of the mutants was evaluated by ligand binding assays, using PCh-BSA or
PnC in the solid phase. Known differences between the two ligands
suggested the use of both. Compared with BSA-PCh, PnC has a higher
density of PCh residues arranged in a symmetrical array, thus allowing
multipoint high affinity attachment of CRP. However, PnC also could
bind CRP through sugar residues and, therefore, BSA-PCh provides a
better model for ligands binding CRP only through PCh residues. The
results are summarized in Fig. 2. Values
shown are binding activities relative to native CRP and reflect
specific avidity for PCh-BSA or PnC. In this and all subsequent assays
two independent clones for each mutant were tested. The data shown are
for one clone; the second clone gave similar results. Each mutant CRP
was purified from two different transfections and was assayed three to
eight times. As shown, the PCh and PnC binding activities of wt and all
mutant CRPs, except E88R, did not differ from each other, and all were
close to unity. The small observed differences among these CRP species
were not significant. E88R bound to both ligands poorly, which we
interpret to indicate a gross conformational change apparently
affecting the PCh-binding site. For this reason E88R was not tested
further.
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, mutants D112N, D112A, and K114A gave
results similar to those published previously (12), i.e.,
mutation of Asp112 or
Lys114 resulted in a significant decrease or
increase, respectively, in C1q binding. Substitution of Ala for
Tyr175 resulted in a decrease in C1q binding
similar to that observed for the Asp112 mutants.
Substituting Ala for His38 did not affect the
C1q-binding activity of either PCh-BSA- or PnC-complexed CRP. However,
substituting Gln for His38 resulted in a
significant, although <50%, decrease in C1q-binding activity. The
most dramatic change in C1q-binding activity occurred when
His38 was replaced by Arg. H38R CRP bound 5- to
10-fold less C1q than wt CRP. Mutations of residues
Glu88, Thr90, and
Asn158 did not cause significant changes in C1q
binding. To further evaluate the C1q-binding activity of selected
mutants, CRP dose-response experiments were performed. Fig. 4 shows the results of a representative
experiment. As shown, no significant C1q binding to D112N, Y175A, or
H38R mutants bound to PCh-BSA was detected even at a concentration of
>1.5 µg/ml. Low C1q binding was detected at the highest
concentrations of the three mutants when PnC was used in the solid
phase. No substantial differences in C1q binding activity were observed
among native, recombinant wt, and E88A CRP bound to either PCh-BSA
or PnC.
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are consistent with those of the
C1q-binding assay (Fig. 3
), although the effects of mutations were more
pronounced in the C3 deposition assay. Thus, the D112N, D112A, Y175A,
and H38R mutants, which express 
5- to 10-fold lower C1q-binding
activity than wt, are at least 100-fold less efficient at complement
activation. Similarly, E88Q, E88A, N158A, and H38Q, which display only
20–40% reduction in C1q binding, have less than half the complement
activating ability of wt CRP. Conversely, K114A, which binds 4.5
times more C1q than wt, has >15-fold increased complement-activating
efficiency. To ensure that the results of the C3 deposition assay truly
reflected the complement-activating potential of the CRP mutants,
dose-response experiments were performed. As shown in Fig. 6, Y175A CRP did not activate complement
at any of the concentrations used, while D112N and H38R activated
complement negligibly and only at concentrations of >2 µg/ml. The
dose-response curves of H38Q, E88A, E88Q, and N158A are parallel to
each other and to native and wt CRP, indicating that differences among
them were maintained throughout the dose-response
range.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-helix 169–176,
particularly Tyr175 (Fig. 1). Residues
His38 and His95 line the
floor of the pocket, while Asn158 is located
upstream toward the center of the cleft. The topology and structure of
the shallow end of the cleft led to the suggestion that it may be the
C1q-binding site of the protomer (2). The fact that
Asp112 is both an integral part of this
structural feature and is critical for C1q binding is certainly
consistent with this view. The present data further support this
proposal.
Our mutational analysis demonstrates that in addition to
Asp112, Tyr175 is a major
determinant for C1q binding to complexed CRP. Substitution of Ala for
this residue led to almost complete loss of C1q-binding activity when
PCh-BSA was the CRP ligand and greatly reduced activity when PnC was
the ligand (Figs. 3 and 4). A similar difference between the two
ligands was observed for the D112N mutant and can probably be
attributed to the regularly arrayed and more closely spaced PCh groups
of PnC compared with PCh-BSA, a feature that allows a higher number of
the CRP pentamer’s PCh-binding sites to engage. The implication, then,
is that multipoint attachment leads to a conformational change or a
tilting of the protomers, exposing the C1q-binding site to the surface.
His38 presents a special case. The H38R mutant
expresses greatly reduced C1q binding, while the H38Q mutant only
moderately reduces binding, and H38A has essentially wt binding
activity. A likely explanation of these results is that the Arg
substitution at this position places the long, positively charged, side
chain in a position that favors the formation of a 3-Å salt bridge
with the negatively charged side chain of Asp112.
Neutralization of the negative charge of Asp112
obviously has the same effect on C1q binding as the D112N mutation.
Alternatively, the Arg side chain of the H38R mutant could adopt the
same configuration as the corresponding residue of serum amyloid P
(22), forming a hydrogen bond to the main chain of
Tyr175, again interfering with C1q binding.
Because residue H38 contributes to the topology of the floor of the
putative C1q-binding site, the reduction of binding observed for the
Gln substitution can perhaps be attributed to a less favorable
stereochemistry than that offered by the side chain of His, while the
small side chain of Ala apparently does not interfere with the geometry
of the C1q-binding site. Finally, as noted before, substitution of Ala
for Lys at position 114, results in a >4-fold increase in C1q binding.
We interpret this result to indicate that the positive charge of the
Lys side chain interferes with C1q binding. Observation of the
structure of the proposed binding site (Fig. 1) leads us to propose
further that the effect is exercised by the side chain of
Lys114' from the neighboring protomer.
Investigation of the ability of the PnC-complexed CRP mutants to form
an effective C3 convertase gave an interesting picture in which all the
effects observed for C1q binding were greatly amplified (Figs. 5 and 6). Thus, D112N, D112A, Y175A, and H38R essentially failed to form a C3
convertase even when very large amounts of CRP were used, while
cleavage of C3 by the convertase formed by K114A was increased by
>15-fold over that effected by the wt control. More instructive were
the results for mutants that did not significantly affect C1q binding.
E88Q, E88A, and N185A had significantly reduced ability to form the C3
convertase, while their C1q-binding ability was only modestly decreased
(compare Figs. 3 and 5). In contrast, T90A, N158S, and H38A, all of
which bound C1q with an affinity similar to that of wt CRP, also
expressed wt C3 cleaving activity. Two nonmutually exclusive
explanations can be offered for the observed amplification effect of C3
cleavage compared with the C1q binding. Firstly, several enzymatic
steps separate the results of the two assays, accounting at least in
part for the observed amplification. Secondly, formation of a C3
convertase requires binding of C1q in a way that induces a
conformational change that, in turn, causes the reorientation and
mutual activation of the two C1r zymogens, which then activate the two
C1s zymogens to active serine proteases. It seems possible that certain
mutations inhibit C1q binding to complexed CRP only minimally, but
prevent or attenuate the induction of the fully active conformation of
the bound C1q, affecting the kinetics of C1r and C1s activation and
resulting in the formation of smaller amounts of C3 convertase.
Taken together, the results of C1q binding and C3 activation assays
strongly indicate that the pocket at the shallow end of the CRP cleft
is the C1q-binding site of the molecule. The site is formed by residues
Tyr175, Asp112,
Glu88, Lys114' from the
neighboring protomer, His38, and
Asn158. Asp112,
Tyr175, and Glu88 appear to
be contact residues, with Glu88 influencing the
conformational change in C1q, while Asn158 and
His38 probably contribute to the geometry of the
site. It is of interest that substitution of Glu for
Leu176, a residue participating in the formation
of the wall of the deeper and narrower part of the cleft upstream of
the proposed C1q-binding site, results in significantly reduced binding
of CRP to FcRI (10). Thus, it appears that the C1q- and
FcRI-binding sites partially overlap each other and that they are
both located within the vicinity of the CRP cleft.
C1q is a large (460-kDa) molecule composed of six identical subunits, each consisting of three structurally similar, but distinct, polypeptide chains, A, B, and C (reviewed in Ref. 23). Each subunit has a triple-helical collagen-like region and a globular head region. The C1r2C1s2 proenzyme complex is associated with the collagenous part of the molecule, while the heterotrimeric globular heads have been shown to contain binding sites for IgG and IgM (24). The exact topology and structure of the Ig-binding site(s) is not known, but ionic bonds are probably involved, and Arg residues of C1q have been implicated (25). For evolutionary and structural reasons we propose that the CRP-binding site is also located on the globular heads of C1q, although evidence for its location on the collagen part of the molecule has been presented (26, 27). C1q is a member of a large family of proteins, termed collectins (28), which share a similar structure consisting of globular heads and collagen stems. In all collectins the globular heads mediate recognition functions through ligand-binding sites, while the collagen parts carry out effector functions. Evidence for the presence of the CRP-binding site on the neck or tail collagen region of C1q (26, 27) was presented before determination of the structure of CRP (2, 3) and did not benefit from the knowledge that the PCh-binding sites are on one face of the pentamer rather than on the perimeter of the ring. The only physically reasonable access to a CRP pentamer positioned with its recognition face against a cell wall is then provided by the opposite, effector face, and this would be effectively inaccessible to the neck or tail region of C1q.
Assuming there is a CRP-binding site on each C1q globular head, the
number of functional C1q-binding sites per CRP pentamer is determined
by the relative size of the reacting partners. The diameter of the C1q
globular head has been estimated from electron microphotographs to be
about 50 Å (29), while the overall dimensions of the CRP
pentamer are 102 Å outside diameter with a 30 Å diameter central
pore and a protomer diameter of 36 Å. Therefore, it would appear that
only a single globular C1q head from a C1q molecule can bind to a CRP
pentamer bound flat on the surface of a ligand. Consequently, more than
one CRP molecule in close proximity to each other would be necessary
for complement activation, which is also a condition for complement
activation by complexed IgG. The question then arises as to whether
there is one or more C1q-binding site per CRP pentamer. The C of the
five Tyr175 of the pentamer lie on a circle of 38
Å diameter, and the C of the five Asp112 are
at a similar distance. Although it seems possible that a single binding
site is formed by these residues, which are perhaps brought closer
together or exposed more fully to the solvent following binding of CRP
to an effector ligand, such a site would have to be pentameric and
symmetrical, probably requiring a similarly symmetrical array of
contact residues on the C1q globular head. This seems unlikely. It
seems more likely that there are five possible C1q binding sites per
pentamer, but that only one of these sites, possibly formed by two
adjacent protomers, is able to engage. This alternative possibility
would offer much more flexibility and would match size requirements
more closely. Thus, an array of CRP molecules bound flat on the surface
of a pathogen or damaged cell would present a wide array of possible
C1q-binding site locations and orientations. This would allow binding
of multiple C1q heads to multiple pentamers on a one-to-one basis,
without the need for significant structural rearrangement in either
molecule. The pentameric symmetry of CRP enhances this flexibility, but
may also be required to provide the correct interprotomer geometry for
C1q binding at the protomer interface.
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Acknowledgments |
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Footnotes |
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2 Current address: Division of Rheumatology, Department of Medicine, MetroHealth Medical Center, Cleveland, OH 44109.
3 Address correspondence and reprint requests to Dr. John E. Volanakis, Biomedical Sciences Research Center "Alexander Fleming," P.O. Box 74145, 166 02 Varkiza, Greece.
4 Abbreviations used in this paper: CRP, C-reactive protein; PCh, phosphocholine; PnC, pneumococcal C-polysaccharide; wt, wild type; NHS, normal human serum.
Received for publication May 22, 2000. Accepted for publication January 4, 2001.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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