Structures of the rat complement regulator CrrY

The structure of rat CrrY1–4 determined in two distinct crystal forms shows a pronounced bend at the interface between domains 3 and 4.


Introduction
Complement constitutes the most ancient arm of the immune system, providing a first line of defence against blood infection by pathogens, and links innate to cellular immunity (Ricklin et al., 2010). Prevention of activation on self-surfaces is achieved by complement regulatory proteins (Liszewski et al., 1996). The two main complement regulation mechanisms that protect self-tissue from unwanted complement activation are decay-acceleration activity (DAA), in which the regulator dissociates the complement-activating C3 and C4 convertases, and factor I cofactor activity (CA), in which the regulator assists the serine protease factor I in cleaving and degrading the same C3 and C4 convertase precursors, C4b and C3b (Walport, 2001a,b).
Inappropriate or unregulated activation of complement has been implicated in a wide range of human inflammatory conditions (Szebeni, 2004). In order to investigate these conditions in vivo in mouse and rat models, the murine complement system and its regulators have been studied and characterized. These studies revealed that in addition to homologues of the human complement regulators CD46 (which has CA) and CD55 (which has only DAA), rats and mice possess an exclusively murine complement regulator: complement receptor 1-related protein Y (CrrY; Wong & Fearon, 1985;Foley et al., 1993). CrrY is a membrane-bound protein comprised of five complement control protein (CCP) domains [also known as short consensus repeat (SCR) domains or Sushi domains] in mice and six or seven CCP domains in rats (owing to alternative splicing), followed by a transmembrane and cytoplasmic region in both rats and mice. The protein possesses both DAA and CA (Kim et al., 1995;Li et al., 1993).
CrrY is expressed across a broad range of tissues both in rats and in mice, while rat and mouse homologues of the membrane-bound regulators CD46 and CD55 (which are ubiquitously expressed in humans) are more tissue-specific. In mice CD46 is only expressed in the testis, while in rats expression is restricted to the testis and the acrosome of developing and mature spermatozoa (Mizuno et al., 2004). The CD55 homologue in rats is widely expressed, with a tissue distribution similar to that of human CD55 (Spiller et al., 1999). In mice, one CD55 homologue encodes a protein with wide tissue expression, while a second is only expressed in germ cells. Both CA and DAA are required to maintain homeostasis of the complement system and given the expression patterns of the CD46 and CD55 homologues CrrY is central to this process . The importance of CrrY in controlling complement activation is high-lighted by the embryonic lethal phenotype of CrrY À/À mice (Xu et al., 2000).
The first four CCP domains of rat CrrY (CrrY 1-4 ) have been shown to have full complement regulatory activity (Fraser et al., 2002). In this manuscript, we describe two crystal structures of a construct consisting of these first four CCP domains of rat CrrY and compare it with its functional human homologues CD55 and CD46.

Cloning, expression and purification
A construct spanning the first four CCP domains of rat CrrY (CrrY 1-4 ) was cloned and expressed in Escherichia coli BL21 (DE3) cells and refolded to allow formation of the eight disulfide bonds, as reported by Fraser et al. (2002). The sample was further purified by size-exclusion chromatography on an S200 16/60 column (GE Healthcare) in Tris-buffered saline (TBS). This material yielded crystals belonging to space group P2 1 2 1 2 (PDB entry 2xrd).
Unfortunately, the initial cloned construct was lost and cloning was reperformed without significant differences in the sequences (the new construct has one fewer Gly residue at the N-terminus and one extra Ser residue at the C-terminus). The purification protocol was also optimized to improve the sample purity and the newly cloned and purified protein yielded crystals belonging to space group P2 1 2 1 2 1 (PDB entry 2xrb). The novel construct and protein-purification protocol are described in the following.
A cDNA clone of the rat CrrY gene was obtained from the mammalian gene collection (IMAGE ID 5599318) and a PCR insert encoding domains 1-4 was produced using the following primers (Sigma-Aldrich): 5 0 -GCCATCTACTCATATGCAGTGCCCAGC-3 0 and 5 0 -GCGCTCGAGCTAGGATTTCACCTTGAAGCAGC-3 0 . This insert was ligated between the NdeI and XhoI sites of a modified pET-15b vector (Novagen) that has the NcoI site replaced with an NdeI site. The sequence was verified by sequencing.
CrrY 1-4 was expressed in E. coli strain B834 (DE3) using Luria-Bertani broth in the presence of 100 mg ml À1 ampicillin. Large cultures were inoculated using a small overnight culture and were incubated at 310 K with rapid shaking. Protein expression was induced with 1 mM IPTG when A 600 reached 0.6 and the cells were harvested by centrifugation (20 min, 5000g, 277 K) after 4 h of induction.
The supernatant was refolded by dropwise addition to 2 l 20 mM ethanolamine, 1 mM EDTA, 1 mM cysteine, 2 mM cystine at pH 11.0 at 277 K with constant stirring. The protein was then left overnight at 277 K to refold.
The volume of the refolded protein solution was reduced to 50 ml using a 10 000 MWCO Vivaflow concentrator (Sartorius) and the sample was then dialysed using 10 000 MWCO Snakeskin dialysis tubing (Pierce) against 25 mM Tris, 10 mM NaCl pH 7.4 overnight at 277 K. The protein was then concentrated to 10 ml in a 10 000 MWCO Vivaspin concentrator (Sartorius) and purified using an S75 16/60 size-exclusion column (GE Healthcare) equilibrated in 25 mM Tris, 10 mM NaCl pH 7.4. A final purification using a Mono Q 5/5 ionexchange column (GE Healthcare) was performed using a protocol that started with binding in 25 mM Tris, 10 mM NaCl pH 7.4 buffer followed by a gradient reaching 25 mM Tris, 0.3 M NaCl pH 7.4 over 45 column volumes. CrrY 1-4 eluted at 9.99 mS cm À1 .

Structure determination and refinement
The structure was initially solved in the lower resolution P2 1 2 1 2 form (PDB entry 2xrd) by sequential molecular replacement with the CCP4 ( (Stein, 2008). This model was then used to determine the structure of the higher resolution P2 1 2 1 2 1 form (PDB entry 2xrb) by molecular replacement, again using Phaser. The structures were built and refined iteratively using BUSTER-TNT (Blanc et al., 2004) and Coot (Emsley et al., 2010), using secondary-structure target restraints in the lower resolution form (Table 2). Stereo images of a representative volume of the crystal electron density and of the entire protein main chain are shown in Fig. 1. The structures were deposited in the PDB with accession codes 2xrb and 2xrd.

Results and discussion
The two crystal structures of rat CrrY 1-4 show a hockey-stick-shaped molecule with the elongated handle comprising CCP domains 1-3, with approximate dimensions 25 Â 25 Â 120 Å , and the blade made by CCP domain 4 (Fig. 1a). The electron density of the crystal form diffracting to 2.5 Å resolution allowed unambiguous tracing of residues 37-290 (Fig. 1b). The loop 52-54 is poorly ordered in the lower resolution structure. The domains are standard CCP domains and are organized in -sheets held together by two disulfide bridges (Fig. 2a).
In both crystal forms rat CrrY 1-4 retains the same overall conformation and secondary-structure elements, which can be taken to be representative of its solution structure. Small changes at the CCP1-CCP2 and CCP3-CCP4 interdomain junctions induced by the different crystal environments in the two crystal forms cause the two models to superpose with an overall r.m.s.d. on C atoms of 1.3 Å over 239 residues. The structural agreement within the individual CCP modules is closer, with r.m.s.d.s on C atoms of 0.53, 0.12, 0.29 and 0.60 Å for CCP1, CCP2, CCP3 and CCP4, respectively, over the $60 residues of each CCP domain between the two crystal forms.
The overall arrangement of CrrY 1-4 is very similar to those observed in the crystal structures of other two factor I (fI) cofactors,     and CD46 1-4 (Persson et al., 2010). The fH 1-4 cofactor, as seen in the crystal structure of the C3b-fH 1-4 complex , overlays with CrrY 1-4 particularly well in the first three domains, which are predicted to form the bulk of the contacts with fI (28% sequence identity, r.m.s.d. on C atoms of 1.6 Å over 180 residues). Fig. 2(b) shows both our crystal structures of CrrY 1-4 overlaid on this structure of fH 1-4 . Similarly, the first three rat CrrY CCP domains, CrrY 1-3 , overlay with CD46 1-3 with an r.m.s.d. on C atoms of 1.92 Å (34% sequence identity). Fig. 2(c) shows the overlay of CrrY 1-4 with fH 1-4 and CD46 in the context of the cofactor-C3b complex on the basis of alignment of domains 2 and 3. All three cofactors also share a patch of negatively charged residues on the surface that could contact factor I (Roversi et al., 2011;see Fig. 3).
The rat CrrY 1-4 CCP3-CCP4 interdomain interface, where the hockey-stick handle meets the blade, buries $300 Å 2 and is centred on specific contacts between hydrophobic patches on CCP3 (residues 162-167) and CCP4 (residues 228-234 and 278-279), forming a kink in the structure. A kink was predicted at this interface by a solutionscattering study (Aslam et al., 2003). The fH 1-4 molecule in the crystal structure of domains 1-4 of fH (fH 1-4 ) in complex with C3b also bends between CCP3 and CCP4 and this has been proposed to be crucial to function . The structure of CD46 1-4 , recently determined in complex with a viral receptor also shows a pronounced bend at the same domain interface (Persson et al., 2010). Thus, a kink at the interface between domains CCP3 and CCP4 in CrrY 1-4 , fH 1-4 and CD46 1-4 is likely to be a shared feature of all fI cofactors.
In the central portion of the regulator, the structure of fH 2-3 overlays onto CrrY 2-3 and CD46 2-3 with r.m.s.d.s of 1.4 and 1.8 Å 2 , respectively, showing that CCP2 and CCP3 are not likely to change much upon going from free to bound regulator. The major differences between the free (CrrY 1-4 , CD46 1-4 ) and C3b-bound (fH 1-4 ) regulators localize at the CCP1-CCP2 and CCP3-CCP4 interfaces, which appear to be remodelled in forming the regulatory complex, bringing domains CCP1 and CCP4 closer to C3b. The change in orientation of CCP4 with respect to CCP2 and CCP3 is particularly striking when comparing the bound and unbound regulators, with CCP4 rotated more than 70 away from C3b in both the unbound structures Factor I cofactors. (a) The crystal structure of the fI cofactor rat CrrY 1-4 (PDB entry 2xrb) as a cartoon representation rainbow coloured from the N-terminus (red) to the C-terminus (blue). (b) Overlay of the crystal structures of CrrY 1-4 (PDB entries 2xrb and 2xrd; rainbow coloured) on the structure of fH 1-4 from the C3b-fH 1-4 binary complex (PDB entry 2wii; Wu et al., 2009;grey). (c) Overlay of CrrY 1-4 (blue, this work) and CD46 1-4 (orange; PDB entry 3o8e; Persson et al., 2010) on the crystal structure of the complex between fH 1-4 and C3b (PDB entry 2wii). This figure was made using PyMOL (DeLano, 2002).
(overlaid as described above) compared with the bound fH. In this context it is interesting to note that CD55, a regulator that does not possess cofactor activity, lacks a functional and structural equivalent of this fourth domain. We propose that the structural rearrangements at the CCP1-CCP2 and CCP3-CCP4 junctions upon binding to the C3b, as illustrated in Fig. 4, are key to formation of the cofactor-C3b regulatory complex.
PR and SJ were funded by the Wellcome Trust (#083599) and the MRC (#G0400775) Project Grants to SML. The staff at the ESRF ID14-4 and Diamond I04 synchrotron beamlines assisted with data collection. Graeme Winter, Claus Flensburg, Clemens Vonrhein and Oliver Smart helped with data processing and refinement. We are grateful to Gé rard Bricogne and the members of the Global Phasing Consortium for access to a beta version of the autoBUSTER software.