Complement regulation at the molecular level: The structure of decay-accelerating factor

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Complement regulation at the molecular level: The structure of decay-accelerating factor
Published online before print January 20, 2004
P. Lukacik *, P. Roversi *, J. White , D. Esser , G. P. Smith , J. Billington * , P. A. Williams * ?, P. M. Rudd ||, M. R. Wormald ||, D. J. Harvey ||, M. D. M. Crispin ||, C. M. Radcliffe ||, R. A. Dwek ||, D. J. Evans **, B. P. Morgan , R. A. G. Smith , and S. M. Lea *
PNAS | February 3, 2004 | vol. 101 | no. 5
*Laboratory of Molecular Biophysics and ||Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, England; Adprotech Ltd., Chesterford Research Park, Saffron Walden, Essex CB10 1XL, England; **Faculty of Biomedical and Life Sciences, Division of Virology, University of Glasgow, Church Street, Glasgow G11 5JR, Scotland; and Department of Medical Biochemistry and Immunology, University of Wales College of Medicine, Health Park, Cardiff CF14 4XN, Wales
Communicated by Douglas T. Fearon, University of Cambridge, Cambridge, United Kingdom, November 5, 2003 (received for review September 18, 2003)
Abstract
The human complement regulator CD55 is a key molecule protecting self-cells from complement-mediated lysis. X-ray diffraction and analytical ultracentrifugation data reveal a rod-like arrangement of four short consensus repeat (SCR) domains in both the crystal and solution. The stalk linking the four SCR domains to the glycosylphosphatidylinositol anchor is extended by the addition of 11 highly charged O-glycans and positions the domains an estimated 177 ? above the membrane. Mutation mapping and hydrophobic potential analysis suggest that the interaction with the convertase, and thus complement regulation, depends on the burial of a hydrophobic patch centered on the linker between SCR domains 2 and 3.
CD55 | complement regulator | pathogen receptor | glycoprotein
The complement system is an important component of the immune response, consisting of more than 30 proteins that function together to provide an initial defense against invasion by pathogens. The classical, alternative, and lectin pathways (CP and AP for classical and alternative pathways, respectively), each activated by different stimuli, converge by means of a series of enzyme-linked cascades to target cells for destruction. Regulation to prevent inappropriate activation against self occurs by means of the proteins encoded in the regulators of complement activation gene clusters, which act at key points in the cascades to prevent pathological consequences. Decay-accelerating factor (CD55) is a member of the regulators of complement activation protein family (1), and its primary function is to inactivate the C3 convertases by dissociating them into their constituent proteins (reviewed in ref. 2). The importance of regulation is highlighted by the large number of human diseases that relate to inappropriate complement activation (3). Complement is also responsible for the primary rejection events in xenotransplantation; therefore, transgenic animals expressing human CD55 potentially provide a route to the generation of readily available organs that will resist rejection. Extended survival times have already been achieved for organs transplanted from human CD55-transgenic pigs to primates (4), and soluble CD55 has been demonstrated to block the Arthus reaction in vivo (5). Incorporation of CD55 into the envelope of baculovirus has proved to be a promising technology for prolonging the lifetime of virions used in genetic therapy (6). CD55 has additional roles, including acting as a binding partner for CD97 (7), a molecule whose expression is up-regulated on leukocytes during inflammatory activation. The significance of the CD97?CD55 interaction is little understood, but it has been implicated in the pathogenesis of multiple sclerosis, because CD97 and CD55, which are absent from normal white matter, are found at high levels in multiple sclerosis lesions (8). CD55 is expressed at a high level on all serum-exposed cells. Perhaps as a consequence of this expression, CD55 has been subverted by many bacterial and viral pathogens which exploit it as a receptor to facilitate cellular infection (reviewed in ref. 9).
CD55 has a C-terminal glycosylphosphatidylinositol (GPI) anchor and consists of a serine/threonine/proline-rich region and four consecutive, membrane-distal, short consensus repeat (SCR) domains characteristic of regulators of complement activation family proteins (reviewed in ref. 2). CD55 is heavily O-glycosylated in the serine/threonine/proline region and contains an N-linked glycan located between SCR domains 1 and 2. To date, our molecular understanding of the biological functions of CD55 has been derived primarily from mutagenesis studies (10, 11) and two structures of CD55 SCR domain pairs (10, 12). These structures suggested that the interactions between the regulator (CD55) and the convertases were likely to involve large areas on the surface of CD55 (the CP convertase contacting much of SCR domains 2 and 3, whereas the AP convertase contacts SCR domains 2, 3, and 4). These interactions were postulated to involve wrapping of the large convertase complexes around the smaller regulator, leading to contacts on both faces of CD55. However, mapping of the key interaction site (as defined by mutagenesis; refs. 10 and 11) for both convertases has been complicated by the fact that the site is centered on residues located at the junction between SCR domains 2 and 3. On the basis of our 1.7-? x-ray structure of SCR domains 3 and 4 (10), we proposed that many of the residues previously suggested to be the direct contacts of CD55 with the convertases were instead acting indirectly by altering the structure of CD55 at the SCR 2/3 domain interface. Uhrinova et al. (12) reached different conclusions on the basis of their solution structure of SCR domains 2 and 3, in which the key residues were seen to be solvent exposed and not structurally involved in stabilizing the SCR 2/3 domain interface. These inconsistencies and other studies that have shown that dissection of domains from their natural biological context can result in structural perturbation (13) demonstrated that a structure for the four linked SCR domains was required to fully understand the biology of CD55.
Materials and Methods
Protein Expression and Purification. Briefly, the four SCR domains of CD55 were expressed in Escherichia coli as inclusion bodies and refolded (J.W., P.L., D.E., M. Steward, N. Giddings, J. R. Bright, B.P.M., S.M.L., G.P.S., and R.A.G.S., unpublished data). Refolded CD551234 was further purified by gel filtration to separate folded and unfolded species. Activity of the pure protein was assayed with CP and AP assays (J.W., P.L., D.E., M. Steward, N. Giddings, J. R. Bright, B.P.M., S.M.L., G.P.S., and R.A.G.S., unpublished data).
Crystallization and Structure Determination. Crystals of CD551234 were grown at 4?C from mother liquor consisting of 0.2 M ammonium sulfate; 20% (wt/vol) monomethoxypolyethylene glycol 5000; 0.1 M sodium acetate, pH 4.6; and 10% (wt/vol) glycerol. The largest crystals were obtained by seeding with crushed crystals. Data were collected from flash-frozen crystals cryoprotected by addition of glycerol to a final concentration of 16%. Data were collected at the European Synchrotron Radiation Facility, Trieste Synchrotron Radiation Laboratory, and Daresbury Synchrotron Radiation Source. Data were processed with either the CCP4 suite programs MOSFLM and SCALA (14) or the HKL suite of programs (15). Molecular replacement (MOLREP; ref. 16) using the coordinates for 50% of the molecule (CD5534; ref. 10) found two copies of CD5534 in crystal forms A and B. Refinement of these partial models (BUSTER-TNT; refs. 17 and 18) in both crystal forms allowed location of Pt and Au sites and initial rebuilding to derive partial models for SCR domains 1 and 2. The sulfurs located in the partial models were added to the heavy-atom models for several rounds of phasing with SHARP (19). Eventually, a complete model was built into maps derived by combining (SIGMAA; ref. 20) experimental and model phases and averaging between the two copies of the molecule within each crystal form and between the two crystal forms (DMMULTI; ref. 21). The final model (crystal form A) obtained was used in molecular replacement (MOLREP; ref. 16) to locate four copies of CD551234 in crystal form C. Refinement in BUSTER-TNT yielded the models described. All models have been deposited in the Protein Data Bank (PBD ID codes 1OJV [PDB] , 1OJW [PDB] , and 1OJY [PDB] ).
Analytical Ultracentrifugation. The experiment was carried out in a Beckman Optima XL-A analytical ultracentrifuge equipped with absorbance optics and an An60Ti rotor essentially as described in Wallis and Drickamer (22). Briefly, serial dilutions of CD55 in 50 mM Tris/150 mM NaCl buffer, pH 7.5, were made to give three 400-?l samples with final A280 of 1, 0.5, and 0.25. Sedimentation-velocity experiments were carried out at 55,000 rpm with an Epon aluminum-filled centerpiece. Sample (400 ?l) and buffer containing no protein (425 ?l) were loaded in the sample and reference channels of a cell. The three samples in three different cells were centrifuged simultaneously. Scans were collected at 270-s intervals in the step-scan mode. The data, which allowed the calculation of sedimentation coefficient for CD55, were analyzed by the second-moment method (23) with the software supplied by Beckman Instruments. [The partial specific volume of CD55 (0.723 ml/g) was calculated from the amino acid composition.] The experiment was repeated with three additional samples of A280 0.5, 0.4, and 0.3 to give a total of six measurements.
Glycosylation Analysis and Model Building. Purified CD55 was run on SDS/10% PAGE gels, and the protein bands migrating with an apparent molecular mass of 75 kDa were excised. In situ release of N-glycans