The Extended Multidomain Solution Structures of the Complement Protein Crry and its Chimeric...

The Extended Multidomain Solution Structures of theComplement Protein Crry and its Chimeric ConjugateCrry-Ig by Scattering, Analytical Ultracentrifugationand Constrained Modelling: Implications for Functionand Therapy

Mohammed Aslam1, Joel M. Guthridge2, Bradley K. Hack3

Richard J. Quigg3, V. Michael Holers2 and Stephen J. Perkins1*

1Department of Biochemistryand Molecular Biology, DarwinBuilding, University CollegeLondon, Gower Street, LondonWC1E 6BT, UK

2Division of RheumatologyDepartment of Medicine andImmunology, University ofColorado Health SciencesCentre, Denver, CO 80262USA

3Section of NephrologyDepartment of MedicineUniversity of Chicago, ChicagoIL 60637, USA

Complement receptor-related gene/protein y (Crry) is a cell membrane-bound regulator of complement activation found in mouse and rat. Crrycontains only short complement/consensus repeat (SCR) domains. X-rayand neutron scattering was performed on recombinant rat Crry containingthe first five SCR domains (rCrry) and mouse Crry with five SCR domainsconjugated to the Fc fragment of mouse IgG1 (mCrry-Ig) in order to deter-mine their solution structures at medium resolution. The radius of gyra-tion RG of rCrry was determined to be 4.9–5.0 nm, and the RG of thecross-section was 1.2–1.5 nm as determined by X-ray and neutron scatter-ing. The RG of mCrry-Ig was 6.6–6.7 nm, and the RG of the cross-sectionwere 2.3–2.4 nm and 1.3 nm. The maximum dimension of rCrry was18 nm and that for mCrry-Ig was 26 nm. The neutron data indicated thatrCrry and mCrry-Ig have molecular mass values of 45,000 Da and140,000 Da, respectively, in agreement with their sequences, and sedimen-tation equilibrium data supported these determinations. Time-derivativevelocity experiments gave sedimentation coefficients of 2.4 S for rCrryand 5.4 S for mCrry-Ig. A medium-resolution model of rCrry was deter-mined using homology models that were constructed for the first fiveSCR domains of Crry from known crystal and NMR structures, and linkedby randomly generated linker peptide conformations. These trial-and-error calculations revealed a small family of extended rCrry structuresthat best accounted for the scattering and ultracentrifugation data. Thesewere shorter than the most extended rCrry models as the result of minorbends in the inter-SCR orientations. The mCrry-Ig solution data weremodelled starting from a fixed structure for rCrry and the crystal structureof mouse IgG1, and was based on conformational searches of the hingepeptide joining the mCrry and Fc fragments. The best-fit models showedthat the two mCrry antennae in mCrry-Ig were extended from the Fc frag-ment. No preferred orientation of the antennae was identified, and thisindicated that the accessibility of the antennae for the molecular targetsC4b and C3b was not affected by the covalent link to Fc. A structural com-parison between Crry and complement receptor type 1 indicated that thedomain arrangement of Crry SCR 1-3 is as extended as that of the CR1SCR 15-17 NMR structure.

q 2003 Elsevier Science Ltd. All rights reserved

Keywords: X-ray scattering; neutron scattering; short consensus/complement repeat; analytical ultracentrifugation; constrained modelling*Corresponding author

0022-2836/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved

E-mail address of the corresponding author: [email protected]

Abbreviations used: Ab, antibody; b2GPI, b2 glycoprotein I; Crry, complement receptor-related gene/protein y;DAF, (CD55), decay-accelerating factor; Ig, immunoglobulin; MCP, (CD46), membrane cofactor protein; FH, factor H;PBS, phosphate-buffered saline; SCR, short consensus/complement repeat; VCP, vaccinia virus coat protein.

doi:10.1016/S0022-2836(03)00492-3 J. Mol. Biol. (2003) 329, 525–550

Introduction

In the immune system, normal host cells are pro-tected from the destructive action of complementby cell surface regulatory proteins belonging tothe “regulators of complement activation” family.These proteins are formed from chains of shortcomplement/consensus repeat (SCR) domains. Inman, complement receptor type 1 (CR1, CD35: 30SCR domains), decay-accelerating factor (DAF,CD55; four SCR domains) and membrane cofactorprotein (MCP, CD46: four SCR domains) inhibitthe C3 and C5 convertases by interacting withcomplement C3 and/or C4 via binding sites thatare contained between two and four SCRdomains.1 In mouse and rat, while DAF is wide-spread in these mammals, the role of DAF iscomplemented by the complement-related gene/protein y (Crry), and that of MCP is almost entirelyreplaced by Crry.2,3 Crry possesses five and sevenSCR domains in mouse and rat, respectively, inwhich the SCR 1-4 domains of mouse and rat Crryshow a high level of sequence identity with eachother, while mouse Crry SCR-5 shows a high levelof sequence identity with rat Crry SCR-6 (and ratCrry SCR-7 in a duplicated version). Crry hassequence and gene structure similarities to CR1.4

Crry is not considered to be a genetic homologueof MCP or DAF, but rather corresponds to thespecific activities of a single protein in rodent thatare dispersed to two separate proteins, MCP andDAF, in man. This species difference is likely to bedriven by the role of MCP and DAF as specificreceptors by human pathogens such as the measlesvirus.4,5

Crry is expressed on a wide variety of murinecells. When expressed on human K562 erythro-leukaemic cells, Crry prevents deposition ofmouse C3 fragments on the cell surface duringactivation of either the classical or alternativecomplement pathway. Thus, Crry has complementregulatory activity.5 Both human CR1 and mouseCrry have comparable cofactor activity for the fac-tor I-mediated cleavage of C3b and C4b, and bothsoluble mouse Crry and human CR1 exhibitdecay-accelerating activity in both the classicaland alternative pathways.4,5 Crry-deficient micedemonstrate a profound loss of foetal viabilitythat begins relatively early during gestation and isassociated with C3 deposition and an inflamma-tory infiltrate.6 Transgenic mice that overexpressCrry as a soluble protein are protected from anti-body-induced glomerular injury.7,8 These obser-vations led to the creation of two recombinantforms: (i) rat Crry (rCrry) and mouse Crry(mCrry) with five SCR domains; and (ii) mouseCrry in which two five-SCR molecules have beenconjugated with the Fc fragment of mouse IgG1antibody to create a chimeric structure (mCrry-Ig)(Figure 1).7,9 – 11 These recombinant products dimin-ished renal injury induced by complement-fixingnephrotoxic antibodies substantially, and mouseCrry-Ig has been shown to ameliorate intestinal

ischaemia-reperfusion injury12 and anti-phospho-lipid Ab-mediated foetal loss.13

The role of Crry as a complement regulatory pro-tein raises key questions in relation to the solutionstructure of rCrry and mCrry-Ig and their func-tional implications. Crystal and NMR structuresfor individual SCR domain pairs range from com-pletely extended to folded-back orientations (seeFigure 1 of Perkins et al.14). Crystal structures forfour SCR domains in vaccinia coat protein (VCP)and five SCR domains in b2-glycoprotein I(b2GPI) show extended arrangements of SCRdomains.15 – 17 However, an X-ray scattering studyof b2GPI showed that the crystal structurerequired remodelling for agreement with the solu-tion data, showing that this is more flexible thansuggested by the crystal structure.18 Ultracentrifu-gation studies of the complement receptor type 2SCR-1,2 domain pair showed that the SCR pair isextended in solution, even when complexed withC3d, while two crystal structures suggestotherwise.19 – 22 Constrained X-ray and neutron scat-tering, and sedimentation coefficient modelling offactor H with 20 SCR domains revealed a folded-back structure in solution.23 The combined resultsof these studies suggest that the orientationbetween two adjacent SCR domains cannot bepredicted, although the observation of folded-back

Figure 1. SCR domain arrangement in rCrry andmCrry-Ig. The location of the presumed N-linked glyco-sylation sites are denoted by c symbols, and the numberof linker residues between adjacent domains is denotedby the numbers next to the linker in question.

526 Solution structure of Crry

structures appear to be correlated with the pre-sence of long inter-SCR linkers.

In order to determine whether Crry exhibits anextended or folded-back structure in solution, weapplied our joint X-ray and neutron scattering,and sedimentation modelling approach to deter-mine a medium-resolution structure for rat Crry(rCrry). As all the interdomain linkers are four orfive residues in length, the structural interest ofrCrry lies in the prediction that its inter-SCRarrangement will not be folded-back, unlike factorH. The scattering study of mCrry-Ig is relevant tothe high potential that bivalent molecules of thistype have as anti-inflammatory therapeuticreagents that suppress complement activation. Thepractical application of molecules such as mCrry-Ig depends on the assumption that the two mCrryantennae in mCrry-Ig behave independently insolution. We show that both rCrry and mCrry-Igpossess extended structures as predicted, and thatthe mCrry antennae are relatively independent ofthe Fc fragment in solution. We discuss theimmunological and therapeutic implications ofthese structures in terms of their role in comp-lement regulation.

Results and Discussion

X-ray scattering analyses for rCrry andmCrry-Ig

The preparations of rCrry and mCrry-Ig proteinsfor scattering and ultracentrifugation studiesresulted in single bands when analysed by SDS-PAGE, and homogeneous peaks when analysed bysize-exclusion chromatography. X-ray scatteringexperiments were performed on rCrry and mCrry-Ig (Materials and Methods). In Guinier analyses todetermine the overall shape of the proteins fromthe radius of gyration RG (a measure ofelongation), the fits in Guinier plots were linear inan appropriate Q·RG range up to 1.0 for rCrry and1.1 for mCrry-Ig (Figure 2(a) and b), where Q ¼4p sin u=l (2u ¼ scattering angle; l ¼ wavelength).Even though radiation damage was observed inone preliminary session with mCrry-Ig, in all thefinal data sessions, the ten successive time-framesobtained during each X-ray data acquisition foreach sample were unchanged, and thereforeshowed that no radiation damage effects hadoccurred. The mean final X-ray RG of rCrry was5.0(^0.4) nm, and that for the X-ray RG of mCrry-Ig was 6.6(^0.2) nm.

In cross-sectional Guinier analyses to determinethe shorter axes of the two proteins from thecross-sectional radius of gyration RXS; a single linearfit range at Q values higher than those used forthe Guinier RG plots above was identified foreach of rCrry and mCrry-Ig (Figure 3(a) and (b)).These Q ranges resulted in mean cross-sectionalradii of gyration RXS of 1.5(^0.1) nm for rCrryand 2.3(^0.2) nm for mCrry-Ig. For rCrry, no

second linear region was identified from the RXS

analyses that resembled that found for factor H:23

This indicated that the SCR domains in rCrry donot form higher-order folded-back structures thatresemble those seen for factor H; and that the RXS

value of 1.5 nm monitors the interdomain orien-tation between adjacent SCR domains. For mCrry-Ig, a second linear region was identified in theRXS plots at large Q values. Fits in the Q range of0.44 nm21 to 0.80 nm21 resulted in an RXS value of1.3(^0.1) nm (not shown). This RXS value is simi-lar in magnitude to that of rCrry of 1.5 nm andthat of factor H of 1.7 nm, suggesting that it moni-tors the interdomain orientations between adjacentSCR domains. Even though the scattering contri-bution from the Fc fragment will complicate thisinterpretation, it can be inferred that this inter-SCR orientation is similar between rCrry andmCrry. If the solution structure of rCrry isapproximated as an elongated elliptical cylinder,the expression:

L ¼ ½12ðR2G 2 R2

XSÞ�1=2

applies, in which L is the length of the cylinder.24

The RG and RXS values resulted in a length of16(^2) nm for rCrry and an equivalent length esti-mate of 27(^3) nm for mCrry-Ig. The length L isalso given by the intensity ratio:

L ¼ pIð0Þ=½IðQÞ·Q�Q!0

in which Ið0Þ and ½IðQÞ·Q�Q!0 are obtained fromthe two Guinier fits.25 This expression gave similarvalues of 17(^1) nm for rCrry. This shape assump-tion is not a good one for mCrry-Ig (Figure 1), butlengths of about 23 nm are obtained using this.

The indirect transformation of the scatteringcurve IðQÞ (measured in reciprocal space) into realspace gives the distance distribution function PðrÞ(Figure 4). This represents the summation of allthe distances r between every pair of atoms withinthe protein. The RG; Ið0Þ and L values that are cal-culated from the PðrÞ curve are now based on thefull scattering curve in the Q range between0.013 nm21 and 2.2 nm21, and give another deter-mination of L that is now independent of shapeassumptions. The RG values calculated from thePðrÞ curves were in good agreement with theGuinier values. The most frequently occurring dis-tance for rCrry is 2.7 nm from the maximum M inthe PðrÞ curve, and its length L was determined tobe 18(^1) nm at the point when PðrÞ becomeszero at large r: The PðrÞ curve for mCrry-Igsuggests a separation of two peaks M1 and M2,where M1 occurs at 4 nm and M2 occurs at 9 nm.This indicates that the two mCrry fragments andthe Fc fragment of the IgG1 structure may beconformationally independent of each other, byanalogy with the similar result obtained for theT-shaped antibody structure of IgA1. The positionsof M1 and M2 are similar to those seen in IgA1,although the two peaks are less clearly resolvedcompared to IgA1.26 The M1 peak is assigned to

Solution structure of Crry 527

the sum of all the interatomic distances within eachof the mCrry and Fc fragments, while the M2 peakcorresponds to the most frequently occurring dis-tances within the entire mCrry-Ig protein. Thelength of mCrry-Ig was 26(^1) nm (Figure 4(b)).

Neutron-scattering analyses for rCrryand mCrry-Ig

Neutron-scattering experiments were performedon rCrry and mCrry-Ig in 2H2O buffer (Materialsand Methods). The rationale for the use of neutronscattering included: (i) the verification of theabsence of radiation damage effects on the X-rayscattering curves; (ii) the opportunity to measurethe glycoprotein scattering curves in conditions ofhigh negative solute–solvent scattering contrastsin distinction to the positive contrast obtainedwith X-ray scattering, this being a control ofinternal density inhomogeneities in the two glyco-proteins; (iii) the ability to calculate molecularmass; and (iv) the absence of measurable hydrationshells in the neutron data, which facilitates the

modelling of the two glycoproteins.27 InstrumentLOQ at the ISIS facility with a less extensive Qrange was used for measuring rCrry and mCrry-Ig. Instrument D11 at the Institut Laue Langevinwas used to confirm the LOQ data analyses, sincethe minimum effective Q value reached with LOQwas only 0.1 nm21 while D11 can reach lower Qvalues (Figure 2(d)). In Guinier fits to determinethe RG values, linear fit regions were observed(Figure 2(d)) for both the LOQ data (upper plot)and the D11 data (lower plot), which indicated theabsence of aggregation sometimes induced in pro-teins by the use of 2H2O buffers. On extrapolationto zero concentration, the mean neutron RG valuedetermined to be 4.9(^0.2) nm for rCrry and6.7(^0.2) nm for mCrry-Ig. The RXS values weredetermined using the same Q ranges as thoseused in the X-ray analyses, and were 1.2(^0.2) nmfor rCrry and 2.4(^0.2) nm for mCrry-Ig. Thesecond RXS value for mCrry-Ig at large Q wasdetermined to be 1.3(^0.2) nm. The length L wasdetermined to be 16(^2) nm for rCrry and28(^2) nm for mCrry-Ig from the RG and RXS

Figure 2. Guinier RG analyses of rCrry and mCrry-Ig. The Guinier plots are displaced arbitrarily on the intensity axisfor reason of clarity. The IðQÞ data used to obtain RG values are denoted by filled circles in the Q·RG range enclosed byarrows depicted on the curves. (a) X-ray RG fits using a Q range of 0.08–0.2 nm21 for rCrry at a concentration of3.9 mg/ml. (b) X-ray RG fits using a Q range of 0.07–0.17 nm21 for mCrry-Ig at a concentration of 5.6 mg/ml. (c) Neu-tron RG fits using a Q range of 0.08–0.26 nm21 for rCrry at concentrations of 7.0 mg/ml and 8.2 mg/ml. (d) Neutron RG

fits using a Q range of 0.12–0.18 nm21 for mCrry-Ig at a concentration of 11.3 mg/ml on LOQ (upper graph) and 0.09–0.18 nm21 at 2.5 mg/ml.


values. L was estimated to be 20(^2) nm for rCrryand 27(^3) nm for mCrry-Ig from the Guinierintensities ratio. The PðrÞ curves gave lengths of19(^1) nm for rCrry and 24(^2) nm for mCrry-Ig.The consistency of these neutron RG and RXS

values with the X-ray data validates these twoindependent data sets.

The molecular mass values of rCrry and mCrry-Ig were obtained from their Guinier Ið0Þ valuesnormalized by their protein concentrations c: Amean Ið0Þ=c value of 0.0499 ^ 0.0010 was calcu-lated for rCrry at 4.5 mg/ml and 6.8 mg/ml, andone of 0.155 ^ 0.040 was calculated for mCrry-Igbetween 2.5 mg/ml and 11.3 mg/ml. The linearrelationship between protein Ið0Þ=c valuesmeasured in 2H2O buffers and normalized againststandard polymers and the molecular mass forLOQ ðIð0Þ=c £ 9:105Þ26 resulted in values of45,000 ^ 1000 for rCrry and 140,000 ^ 35,000 formCrry-Ig. Both determinations are consistent withthe sequence-derived molecular mass of 46,600 forrCrry and 158,600 for mCrry-Ig assuming that thefour N-linked glycosylation sites on rCrry and the12 N-linked glycosylation sites on mCrry-Ig con-tained triantennary complex-type oligosaccharidestructures.

Comparison of the X-ray and neutron scatteringresults with the mean overall length of a singleSCR domain of 3.6(^0.2) nm indicated how theSCR domains were arranged in rCrry. This meanlength was calculated for 32 SCR domains foundin 15 NMR and crystal structures (Materials andMethods), where the length is defined as the a-car-bon separation between residue 22 preceding thefirst Cys residue of the SCR and residue þ2 follow-ing the last Cys residue of the SCR. If the five SCRdomains of rCrry were fully extended in solution,the predicted value of L for rCrry would be18(^1) nm. This agrees well with the experimentalL values of 16–17 nm from the X-ray and neutronRG and RXS values, 16–19 nm from the Guinierintensity ratios, and 18 nm from the PðrÞ curves. Ifthe five SCR domains in mCrry-Ig are fullyextended, the rearrangement of the Y-shaped struc-ture in Figure 1 into a T-shaped structure wouldresult in a predicted L value of 18 nm þ 18nm ¼ 36 nm. If alternatively the Y-shaped structurein Figure 1 was rearranged so that the two Crryfragments are parallel with and adjacent to eachother, this would result in a predicted L value of18 nm þ 6 nm ¼ 24 nm (where the length of the Fcfragment is 6.3 nm). The second of these two

Figure 3. Guinier RXS analyses of rCrry and mCrry-Ig. The same scattering curves shown in Figure 2 were used. (a)X-ray Guinier RXS fits using a Q range of 0.30–0.60 nm21 for rCrry. (b) X-ray Guinier RXS fits using a Q range of0.20–0.34 nm21 for mCrry-Ig. (c) Neutron Guinier RXS fits using a Q range of 0.30–0.60 nm21 for rCrry. (d) NeutronGuinier RXS fits using a Q range of 0.20–0.34 nm21 for mCrry-Ig.


estimates agrees well with the experimentallyobserved L values of 24 nm to 26 nm from the PðrÞcurves. These initial considerations suggest thatthe solution arrangement of the Crry antennae inmCrry-Ig resembles a more compact structure,rather than an extended one.

Analytical ultracentrifugation data for rCrryand mCrry-Ig

Analytical ultracentrifugation provided an inde-pendent monitor of the solution scattering results.Molecular masses were determined for rCrryusing sedimentation equilibrium experiments(Materials and Methods). Curve fits assumed thepresence of a single species, and this resulted inexcellent fits with random small residuals (Figure5(a)–(d)). A systematic decrease in the molecularmass of rCrry was observed with increasing rotorspeed (Figure 5(e)). A small concentration depen-dence was observed (Figure 5(e)). Extrapolation tozero concentration gave molecular mass values of51,500(^300) Da for a rotor speed of 14,000 rpm,47,000(^500) Da for a rotor speed of 17,000 rpmand 43,300(^100) Da for a rotor speed of20,000 rpm for the interference data (Figure 5(e)).

A similar speed dependence was seen in the absor-bance data of rCrry at 0.5 mg/ml, from whichmolecular mass values of 52,000(^4000) Da,47,000(^7000) Da and 43,000(^1000) Da wereobtained for rotor speeds of 14,000 rpm,17,000 rpm and 20,000 rpm, respectively. Thisdependence on rotor speed is attributable tosample polydispersity.28 The most likely cause ofthis is if rCrry is heterogeneously glycosylated atits four N-linked glycosylation sites (Figure 1).Thus, if rCrry possesses four biantennary com-plex-type oligosaccharides, the total molecularmass is predicted to be 44,000 Da, while, if rCrrypossesses four tetraantennary complex-type oligo-saccharides, the total molecular mass is 49,200 Da.This range agrees with that observed in Figure 5(e).

The sedimentation equilibrium experiments formCrry-Ig resulted also in good curve fits withsmall residuals (Figure 5(e)–(g)). This time, basedon curve fits for rotor speeds of 11,000 rpm,14,000 rpm and 17,000 rpm, the molecular massesof mCrry-Ig were similar at 165,000(^6000) Da,166,000(^10,000) Da and 172,000(^10,000) Da,respectively, for both the absorbance and inter-ference data when extrapolated to zero concen-tration (not shown). The mean value of

Figure 4. Distance distribution functions PðrÞ for rCrry and mCrry-Ig. The maximum of the PðrÞ curve is denoted byM, the most frequently occurring distance within rCrry, and by M1 and M2 for mCrry-Ig. The maximum dimension ofCrry-Ig is denoted by L, which is taken to be where the PðrÞ curve intersects PðrÞ ¼ 0.


171,000(^8000) Da (Figure 5(h)) is in good agree-ment with the range of sequence-derived molecu-lar masses of 149,700 Da, 158,600 Da and165,000 Da for mCrry-Ig when calculated on theassumption of 12 biantennary, triantennary or tetra-antennary complex-type oligosaccharides, respect-ively (Figure 1). This time, there was littleevidence for polydispersity effects in mCrry-Ig.

The sedimentation coefficient s020;w from sedi-

mentation velocity experiments monitors macro-molecular elongation and complements the RG

determination. Time-derivative analysis using thegðspÞ method to analyse pairs of sedimentationboundaries resulted in s0

20;w values of 2.4 S and5.4 S from the peak centres in Figure 6(a) and (b).The mean s0

20;w value was determined to be

2.4(^0.1) S from a total of 15 determinations forrCrry, and 5.4(^0.1) S from a total of 18 determi-nations for mCrry-Ig. Since the Gaussian peakwidth in Figure 6 gives the diffusion coefficient iftime-broadening effects are negligible, molecularmasses were determined from this and the s0

20;wvalue by the Svedberg equation as a control, andwere found to be close to the expected values forrCrry and mCrry-Ig.

Homology modelling of the SCR domains ofrCrry and mCrry-Ig

The constrained modelling of the scatteringand sedimentation data for rCrry and mCrry-Igbased on known domain structures resulted in a

Figure 5 (legend opposite)


Figure 5. Sedimentation equilibrium analyses for rCrryand mCrry-Ig. (a) and (b) Best-fit curves for rCrry at0.46 mg/ml for rotor speeds of 14,000 rpm and20,000 rpm using absorbance optics. The fits result inmolecular mass of 51,500 Da and 43,600 Da, respectively.(c) and (d) Best-fit curves for rCrry at 2.3 mg/ml forrotor speeds of 14,000 rpm and 20,000 rpm using inter-ference optics. The residuals of the four curve fits areshown in the upper panels. (e) Observed interference-derived molecular mass as a function of concentrationand rotor speed (X, 14,000 rpm; W, 17,000 rpm; K,20,000 rpm). Regressions to zero concentrations gavemolecular mass values of 52,000(^260) Da,47,000(^480) Da and 43,000(^20) Da, respectively. Themolecular mass values based on absorbance data aredepicted by A, in which the error bar represents the stan-dard deviation of the molecular mass from three scans at

equilibrium. (f) and (g) The curve fits for mCrry-Ig at0.21 mg/ml for a rotor speed of 14,000 rpm using absor-bance and interference optics, respectively. (h) Themean molecular mass values of mCrry-Ig are shown asa function of concentration based on the interferencedata (X) from three rotor speeds (11,000 rpm,14,000 rpm and 17,000 rpm), and regression gave a mol-ecular mass of 171,000(^4000) Da at zero concentration.In comparison, the molecular mass values of mCrry-Igfrom absorbance data are depicted by A.

Figure 6. Sedimentation velocity fits of the gðspÞ distri-bution for rCrry and mCrry-Ig. In (a) and (b), ten scansrecorded at eight minute intervals were recorded usingabsorbance optics at 280 nm and a rotor speed of35,000 rpm for rCrry at 1.47 mg/ml and mCrry-Ig at0.15 mg/ml. Analyses gave s0

20;w values of 2.4 S and5.4 S, respectively, as arrowed.


medium-resolution structure for both these pro-teins. Initially, this procedure involved the con-struction of homology models for the fiveindividual SCR domains of rat and mouse Crryfrom experimental structures. Five of these wereselected from 32 available crystal and NMR struc-tures for single SCR domains on the criterion thatthese required the least number of residue inser-tions and deletions in the conformationally variablesurface loops in the SCR superfamily (Figure 7). Todetermine the best template to use, the rCrry andmCrry SCR sequences were aligned with thesequences for known SCR structures on the basisof: (i) the fully conserved residues Cys4, Cys32,Cys46, Trp52, and Cys59; (ii) the conservation ofresidues that constitute the six antiparallel b-strands in the structural core of the SCR domain;and (iii) the conservation of the side-chain solventaccessibilities. From this analysis, the crystal struc-tures of b2GPI and MCP, and the NMR structuresof SCR domains in VCP and CR1 were selected asmodelling template for Crry.16,29 – 31 The structurevalidation program PROCHECK showed that 81%to 94% of the residues appeared in the mostfavoured regions in the two crystal structures,while 42% to 70% did so likewise in several SCRNMR structures, indicating a preference for theuse of crystal structures when possible. All tenmodels for rCrry and mCrry were constructedusing standard homology modelling methods(Materials and Methods), using as template thestructures specified in Figure 7. Even though theVCP SCR-4 and CR1 SCR-17 NMR structures wereless accurate than the crystal structures, theyprovided an almost complete insertion-free anddeletion-free template for the modelling of theCrry SCR-3 and SCR-4 domains, and were used inthe modelling (Figure 7). Final examination withPROCHECK showed that 48% to 83% of the resi-dues in the rCrry models occurred in the mostfavoured regions of the Ramachandran plot (theactual value reflected that for the reference tem-plate structure, being the same or slightlyreduced), and likewise 41% to 80% of the mCrryresidues occurred in the most favoured regions.The Crry models satisfied the remaining stereo-chemical checks in PROCHECK.

There are four N-glycosylation sites in Crry, twoof which occur in topologically identical positionsin SCR-1 and SCR-4 of rCrry and mCrry (Figures1 and 7). Individual complex-type oligosaccharidesrange in molecular mass from 2200 Da (biantenn-ary) to 3500 Da (tetraantennary). The scatteringand ultracentrifugation molecular masses do notconsistently favour one oligosaccharide structure.Accordingly, the oligosaccharide observed in thecrystal structure of the Fc fragment of murineIgG132 was adapted and attached to the four N-gly-cosylation sites to create homology models forbiantennary and triantennary Crry.

A total of 20 inter-SCR linker conformationshave been observed in seven crystal structuresand eight NMR structures to date. Fifteen of these

are four residues long with a mean inter-Cys59-Cys4 a-carbon separation of 1.52(^0.12) nm, twoare three residues long with an a-carbon separ-ation of 1.29 nm, and three are eight residues longwith an a-carbon separation of 1.73 nm (these arefolded back). Molecular graphics inspections sum-marized in the two alternative views of Figure 8show that all the four-residue linkers are extendedand many are conformationally similar. Four ofthe 20 linkers (a, b, c, and d in Figure 8) deviate sig-nificantly from the average. For two of these (b andd in Figure 8), this deviation is explained by theunique occurrence of a Pro residue immediatelybefore the first Cys residue. One of these (b inFigure 8) is the FH SCR-15/SCR-16 linker, whichhas been used previously to model many pairedSCR structures. Another set of four linkers deviatesslightly from the average (w, x, y, and z in Figure 8).Three linkers in three CR2 SCR-1/SCR-2 structuresare eight residues in length and are folded back atthe centre; however, the two views of Figure 8show that each one can be considered as two four-residue linkers, each of which shows a confor-mation that is similar to the majority conformationseen for the four-residue linkers. In summary,even though gross deviations occur from a consen-sus structure and their conformation cannotusually be predicted,14 Figure 8 indicates that thefour-residue linker conformations can be similarto each other.

Randomized linker modelling of rCrry byconstrained scattering fits

Modelling was performed to determine the sol-ution arrangement of the five SCR domains withinrCrry. Starting models for rCrry were created fromthe 20 known linker structures, based on four iden-tical copies of each one to connect the five SCRdomains. Only eight linkers resulted in extendedrCrry models that were at least 15 nm in length,and possessed RG values of 5.0 nm to 5.6 nm, inagreement with the experimental RG value of5.0(^0.4) nm, and s0

20;w values of 2.2 S to 2.4 S, inagreement with the experimental s0

20;w value of2.4(^0.1) S (Table 1). The other 12 linkers resultedin rCrry models with significantly reduced overalllengths that were incompatible with the exper-imental values. The X-ray and neutron goodness-of-fit R-factors for the eight better models rangedfrom 7.1% to 8.8% for the X-ray data and 4.9% to5.6% for the neutron data, which compare wellwith other modelling analyses.33 These trial calcu-lations showed that the RXS values were stronglymodel-dependent, in which the eight better start-ing models had RXS values of 0.1 nm to 0.9 nmthat were smaller than the experimental value of1.5 nm (X-rays) and 1.2 nm (neutrons). The effectof replacing the biantennary oligosaccharide chainswith triantennary ones did not alter the calculatedRG and R-factors; but the s0

20;w values worsenedfrom 2.2–2.4 S to 2.7–2.8 S. Accordingly, biantennarystructures were used in all subsequent calculations.




Figure 7. Sequence and structure alignments used to model the SCR domains of rCrry and mCrry. This is based on34 NMR and crystal SCR structures, which are identified by their PDB codes at the left. Eight putative N-glycosylationsites in rCrry and mCrry are underlined. The consensus length of an SCR domain is 60 residues as shown. The fullyconserved C and W residues (Cys4, Cys32, Cys46, Trp52 and Cys58) are identified by vertical strokes. The observedsecondary structures in the NMR structures were identified using DSSP, where b-strand residues are denoted byE. The six consensus b-strands (B1 to B6) that were consistently identified in these structures are shown above thealignment. The observed side-chain accessibilities in these structures were identified using COMPARER, where 0denotes 0–9% accessibility, 1 denotes 10–19% accessibility, and so on up to 9. Residues with accessibilities up to 19%are denoted as buried, and the consensus exposed or buried positions are shown above the alignment (e, exposed; b,buried).


The rCrry curve fits were improved by refine-ment of the inter-SCR orientation to obtain betteragreement with the RXS values. Three automatedmodelling strategies based on the generation ofrandomly-selected linker structures were applied.23

In these, the four rCrry linker sequences (Figure 7)were modelled as extended b-strands, then the lin-ker conformations were randomized using molecu-lar dynamics.

(i) In the Randomized-1 modelling (Table 1),no distance restraint was used in generating10,000 linker conformations for each of the fourrCrry linkers. A total of 2000 rCrry models wasassembled randomly from the 40,000 confor-mers. Even with the use of generous 20% filters(Table 1), only 28 models were compatible withthe experimental data. The tip-to-tip distancesand RG values for these best models were signifi-cantly lower than the experimental values. Thismodelling showed that only extended rCrrystructures would fit the solution data.

(ii) In the Randomized-2 modelling, the linkerlengths were set to be 95% of the maximallyextended length when generating the 40,000 lin-ker conformations. The use of tighter 10% filtersfor the RG; RXS and s0

20;w values identified 111good fit models from the 2000 rCrry modelsthat were created. Since the tip-to-tip distancesand RG values were still less than the experimen-tally observed values, this suggested that therCrry models should be even more extended inorder to fit the solution data.

(iii) In the Randomized-3 modelling, the mini-mum linker distance was set to be 100% maxi-mally extended, meaning that the rCrry modelscorresponded to reorientations of the five SCRdomains about the four linkers. The rCrrymodels had a tip-to-tip range between 2 nm and19 nm, resulting in RG values that rangedbetween 1 nm and 5 nm (Table 1). The 10% filters

now resulted in 217 good fit models. Narrowerfilters, which also specified a minimum length Lof 15 nm (cf. Figure 4), resulted in 12 finalextended rCrry structures (green cluster inFigure 9(a)) that gave low R-factors (Figure 9(b))and accounted for the experimental RG; RXS ands0

20;w values. In these, the tip-to-tip distance isnot extended maximally, reflecting the need forslightly bent inter-SCR orientations. A represen-tative curve fit is shown in Figure 10(a) for abest-fit final model. In comparison to 500 of the2000 starting models (Figure 11(a)), all 12 finalmodels showed extended structures, even forthose that appeared at first sight to be bent back(Figure 11(b)). The most extended rCrry modelof all (in red in Figure 9; Figure 11(c)) gave RG;RXS and s0

20;w values similar to those of the eightbest-fit starting models of Table 1 and a slightlyworsened curve fit (Figure 10(b)). No preferredinter-SCR orientation was identified, exceptpossibly at the SCR-2/SCR-3 linker where a Proresidue is immediately adjacent to the Cys residue.

To ensure that all possible SCR arrangementshad been considered, a systematic Rotationalsearch (Table 1) explored all the tilt and twistangles between two SCR domains in 158 incre-ments. Each of the four linkers had the same tiltand twist angle as the b2GPI SCR1/2 linker, andthe long axis was set as the X-axis: The Y-axis andZ-axis rotations were between 1208 and 1208 in 158increments when the X-axis rotation is fixed at 08,and the X-axis was stepped between 08 and 3308in 308 steps, resulting in 12 £ 17 £ 17 ¼ 3468models. The RG values ranged between 5.3 nmand 1.9 nm, and the R-factors ranged between7.4% and 34%. The filters resulted in only threebest-fit structures from the 3468 starting structures,and confirmed again that only highly extendedrCrry models fit the data.

Figure 8. Ribbon views of 20 lin-ker structures seen in crystal andNMR structures of pairs of SCRdomains. The most common linkerconformations are shown in yellow.Four linker conformations thatdeviate from the most commonones are denoted by green ribbonsand labelled a to d. Four linkersthat show less conformational devi-ations are labelled w to z. The eightresidue and three residue linkersare shown in purple and red,respectively. The left-hand viewcorresponds to the SCR-1 structureof b2GPI, in which the linkers areshown superimposed at the C ter-minus at Cys58. The right-handview corresponds to the SCR-2structure of b2GPI, in which thelinkers are superimposed at the Nterminus at Cys4.


Randomized linker modelling of mCrry-Ig byconstrained scattering fits

The aim of the modelling for mCrry-Ig was toelucidate the position of the two mCrry structuresrelative to the Fc structure. The mCrry structurewas taken to be the same as that of rCrry, thisbeing justified from their sequence similarities,and the similarity of their RXS values (see above).Since the only variable in the modelling fit wasthe hinge peptide between SCR-5 of mCrry andthe N terminus of the Fc fragment, this modelling

is quite different from that performed for rCrry.The hinge has two parts, firstly an eight residuelinker between SCR-5 and the start of the IgG1hinge sequence, and secondly the 15 residue hingeof IgG1 to the N terminus of the Fc fragment (fullhinge sequence: (C)VSRLADPE-VPRDCGCKPCIC-TVPEV(S)).

The four major parameters that were explored inthe mCrry-Ig modelling are now summarized(Table 1; Materials and Methods). The VPRDCG-CKPCICTVPEV hinge peptide was left in theasymmetric static conformation seen in the crystal

Table 1. Summary of modelling searches for SCR domain arrangements for rCrry and mCrry-Ig in solution

Orientation oflinker

Minimum length of ofeach linker (nm)

Number ofmodels

Good-fitmodelsa

Tip-to-tipdistanceb

X-rayRG (nm)

X-rayRXS-1

(nm)R-factor

(%) s020;w (S)

Experimentalvalues for rCrry

18 ^ 1 5.0 ^ 0.4 1.5 ^ 0.1 n.a. 2.4 ^ 0.1

CD46 SCR 1/2 1 0 17.0 5.0 0.8 7.1 2.35b2GPI SCR 2/3c 1 0 17.2 5.1 0.8 7.3 2.33VCP SCR 2/3 1 0 17.2 5.3 0.4 8.1 2.34CR1 SCR 15/16 1 0 18.0 5.3 0.7 7.4 2.30b2GPI SCR 1/2c 1 0 18.1 5.4 0.3 8.8 2.34CR1 SCR 16/17 1 0 19.1 5.6 0.4 8.0 2.24Randomized-1 No linker restraints 2000 28 13 ^ 2 4.2 ^ 0.2 1.3 ^ 0.1 7.1 ^ 0.4 2.70 ^ 0.07Randomized-2 95% of linker lengths 2000 111 14 ^ 2 4.6 ^ 0.1 1.5 ^ 0.1 7.1 ^ 0.5 2.54 ^ 0.05Randomized-3 100% of linker lengths 2000 217 14 ^ 2 4.6 ^ 0.1 1.5 ^ 0.1 7.3 ^ 0.6 2.51 ^ 0.03Rotational 3468 3 12 ^ 2 4.6 ^ 0.1 1.4 ^ 0.1 7.6 ^ 0.2 2.63 ^ 0.03

Experimentalvalues formCrry-Ig

n.a. 6.6 ^ 0.2 2.3 ^ 0.2 n.a. 5.4 ^ 0.1

Randomized-1(A)

95% of hinge lengthd 2000 16 10.3 ^ 1.6,8.1 ^ 2.5

6.9 ^ 0.2 2.3 ^ 0.1 11 ^ 1 5.75 ^ 0.2

Randomized-2(A)

100% of hinge lengthd 2000 11 9.3 ^ 2.2,11.0 ^ 3.1

7.0 ^ 0.1 2.2 ^ 0.1 11 ^ 1 5.60 ^ 0.2

Randomized-3(S)

95% of hinge lengthd 2000 4 8.2 ^ 0.5,12.1 ^ 2.6

7.0 ^ 0.1 2.2 ^ 0.1 11 ^ 1 6.04 ^ 0.2

Randomized-4(S)

100% of hinge lengthd 2000 9 7.8 ^ 0.5,12.6 ^ 2.3

6.9 ^ 0.2 2.3 ^ 0.1 14 ^ 1 5.96 ^ 0.2

Rotational-5 (A) 1350 16 10.8 ^ 1.1,9.6 ^ 1.7

6.9 ^ 0.2 2.3 ^ 0.2 12 ^ 2 5.64 ^ 0.2

Rotational-6 (S) 1350 7 11.1 ^ 0.7,10.6 ^ 1.8

7.1 ^ 0.1 2.3 ^ 0.1 11 ^ 1 6.14 ^ 0.3

Randomized-7(A)

95% of hinge lengthd 2000 72 9.2 ^ 2.0,9.0 ^ 3.4

6.9 ^ 0.2 2.3 ^ 0.1 10 ^ 1 5.72 ^ 0.3

Randomized-8(A)

100% of hinge lengthd 2000 45 7.9 ^ 2.4,10.8 ^ 2.2

6.9 ^ 0.2 2.3 ^ 0.1 11 ^ 1 5.67 ^ 0.3

Randomized-9(S)

95% of hinge lengthd 2000 5 8.7 ^ 0.8,15.4 ^ 1.6

6.9 ^ 0.2 2.2 ^ 0.2 11 ^ 1 5.73 ^ 0.3

Randomized-10(S)

100% of hinge lengthd 2000 1 9.9, 18.1 7.0 2.5 11 6.10

Rotational-11 (A) 1350 11 8.1 ^ 2.7,15.1 ^ 4.0

7.1 ^ 0.1 2.4 ^ 0.1 11 ^ 1 5.88 ^ 0.4

Rotational-12 (S) 1350 1 13.5, 7.5 7.2 2.1 10 6.00

n.a., not applicable.a For rCrry, the good fit models were defined as those that satisfied three filters of ^20% for the RG; RXS and s0

20;w values in the Ran-domized-1 search, and those that satisfied three filters of ^10% for the RG; RXS and s0

20;w values for the eight trial models and the Ran-domized-2, Randomized-3 and Rotational searches (see text for more details). For mCrry-Ig, the good fit models were defined by^10% filters for the RG and RXS values and a ^15% filter for the s0

20;w value in all the Randomized and Rotational searches asshown. The fourth filter of ^5% of the wet and dry volumes was used in all the searches (values not shown).

b For rCrry, the tip-to-tip distance is measured between the Ca atoms of the N-terminal Thr1 residue in the SCR-1 domain and theC-terminal Glu60 in the SCR-5 domain (Figure 7). For mCrry-Ig, the first distance corresponds to that between the centre of mass ofSCR-3 in one antennae and the Fc fragment, and the second to that between the centres of mass of the two SCR-3 domains on thetwo Crry antennae.

c Mean of two determinations from two crystal structures.d The asymmetric and symmetric hinge peptides in the mCrry-Ig models are denoted by A and S, respectively, and the searches

were constrained by hinge lengths of either 95% or 100% as shown. The mCrry-Ig searches were performed with two mCrry modelsthat were derived from the most extended rCrry model (searches 1–6) and the best-fit rCrry model (searches 7–12).


structure,32 so that all the resulting mCrry-Igmodels were asymmetric. Alternatively, the hingepeptide was remodelled into a 2-fold symmetricstructure by means of molecular dynamics simu-lations based on tethering the three hinge disul-phide bridges to a 2-fold symmetry axis passingthrough the long axis of the Fc fragment. The poss-ible antennae conformations in mCrry-Ig were sur-veyed either by the randomized moleculardynamics modelling of the hinge peptide or bythe use of systematic rotations of the hinge peptide.The length of the hinge peptide in the randomizedsearches was restrained to either 95% or 100% ofits starting value based on an extended b-strandstructure. Either the best-fit rCrry structure of

Figure 10(a) or the most extended rCrry structureof Figure 10(b) were used to represent the mCrryantennae.

The outcome of the 12 searches is summarized inTable 1. The searches were effective in generatingrandom conformations of the mCrry antennae, asexemplified by Figure 14(a). The Randomized-7search was the most successful in giving 72 best-fit models, summarized in Figure 12:

(i) The volume filter of ^5% eliminatedmodels in which two or more SCR domainsoverlapped with other parts of mCrry-Ig, mostlyin the bottom left-hand corner of Figure 12(a).Overlap between a single SCR domain and thereminder of mCrry-Ig was often missed becauseof the extended nature of its structure. Hence,the final mCrry-Ig models had to be checkedboth visually and computationally for bumps.

(ii) The RG filter of ^10% favoured the 870more compact arrangements of the mCrry anten-nae relative to the Fc fragment in the lowerleft-hand half of Figure 12(b). Either the mCrrystructure was not extended maximally from the

Figure 9. Structural analysis of the 2000 rCrry modelsfrom the Randomized-3 search with 100% linkers(Table 1). (a) Comparison of the RG values for the 2000rCrry models (yellow circles) with the distance betweenthe a-carbon atoms of Glu1 in the SCR-1 domain andGlu60 in the SCR-5 domain. The 12 best-fit final rCrrymodels (Figure 11(b)) are indicated by the green circles,and the most extended rCrry model (Figure 11(c)) is indi-cated by the red circle. (b) Comparison of the R-factorðR2:0Þ values for the 2000 rCrry models with their RG

values. Note that the 12 best-fit final rCrry models(green) are within the set of lowest R-factor values withvalues between 6.6% and 7.8%. The most extendedrCrry model (red) has an R-factor of 6.8%.

Figure 10. X-ray and neutron curve fits for (a) the best-fit rCrry model and (b) the most extended rCrry modelobtained from the Randomized-3 search. These modelscorresponded to the best of the 12 best-fit final modelsfor rCrry, and that denoted by the red circle in Figure 9.The points correspond to the experimental data and thelines correspond to the calculated curve from eachmodel. In each panel, an a-carbon trace of each best fitmodel is shown with the carbohydrate moiety attached.SCR-1 is at the left. The upper curve fit corresponds tothe X-ray data, while the lower curve fit corresponds tothe D11 neutron data. The Q ranges used in the RG andRXS analyses of Figures 2 and 3 are indicated by thearrowed ranges.


Fc fragment, or the hinge was asymmetric tocause the full structure to be less extended. Thiswas why the Randomized-7 and -8 searcheswere more successful compared to the Random-ized-1, -2, -3, -4, -9 and -10 searches. The meanseparations between the centres of one mCrryantenna and the Fc fragment ranged between7.8 nm and 13.5 nm, in contrast to the maximumseparation of 23 nm in Figure 12. The mean sep-arations between the centres of the two mCrryantennae ranged between 7.5 nm and 18.3 nm,in contrast to the maximum separation of 17 nmin Figure 12. Use of the most extended or mostsymmetric mCrry structures was less successful.This was confirmed by Figure 13(b), in which amuch extended mCrry-Ig model with an RG of9.9 nm resulted in wide deviations in the RG

region at low Q.(iii) The RXS filter of ^10% corresponded to

the large-order separation of the two antennaein the mCrry-Ig structure. Figure 12(c) identified737 compatible models but did not discriminatebetween models in which either the two anten-nae were close together and parallel with eachother (Figure 14(b)), or were extended awayfrom the central Fc fragment in a distortedT-shaped structure (Figure 14(c)).

(iv) The best R-factors corresponded to 222models at the centre of Figure 12(d).

The two best-fit models of Figures 14(b) and (c)from the Randomized-7 and Randomized-8searches gave similar curve fits that extended to Qvalues of about 1 nm21 in Figure 13(a), this beinga better fit range than that seen for factor H:23 Thefinal mCrry-Ig R-factors were 9.6% (X-rays) and6.6–6.8% (neutrons). In contrast, the poor fit forFigure 13(b) corresponded to R-factors of 15.3%(X-rays) and 9.6% (neutrons). Most of the best-fitmodels were consistent with the experimental sedi-mentation coefficient of 5.4 S to within^0.3 S. These corresponded to the Randomized-2,-7, -8 and -9 and the Rotational-5 searches (Table 1).

Conclusions

SCR domains are the most widespread domaintype in the complement proteins, and an under-standing of the solution conformation betweenadjacent SCR pairs is central to the molecularmechanism of complement activation. Our newmethod of scattering curve modelling constrainedby rigid body SCR domain structures connectedby flexible linker structures has provided infor-mation on inter-SCR domain arrangements inCrry that complements our earlier study of the lin-kers found in the folded-back structure of factor Hof complement.23 It was hypothesized that, if theinter-SCR linkers are as long as eight residues, asfound in factor H and complement receptor type2, significant flexibility and movement can result.23

While the inter-SCR linker conformations exhibit abroad range of structures in solution,14 if the inter-SCR linkers are four or five residues in length,only a modest degree of bending occurs (Figure 8).This hypothesis was verified by the present Crrymodelling studies, which demonstrated extendedSCR arrangements. In fact, an inspection of the 12best-fit models in Figure 11(b) shows that thedegree of conformational variability between SCR1-2 and SCR 2-3 (four residue linkers) is noticeablyless than that between SCR 3-4 and SCR 4-5 (fiveresidue linkers). These solution studies will con-tinue to be of great relevance for the elucidation ofstructure–function relationship in SCR proteinswhere intrinsic segmental flexibility is importantfor function.

rCrry is composed of six or seven SCR domainsbut only the N-terminal five SCR domains werestudied here, the first four of which are equivalentto those in mCrry, which contains only five SCRdomains. The SCR-1, SCR-2, SCR-3 and SCR-4domains in rCrry and mCrry show 70% to 85%sequence identity with each other, while the SCR-5domain of mCrry shows 82% sequence identitywith the SCR-6 and SCR-7 domains of rCrry (row1 of Table 2). Fifteen of the 18 residues in the fourinter-SCR linkers are identical (83% identity).These statistics indicate that the overall structuresof rCrry and mCrry will be similar. Our structuredeterminations for rCrry and mCrry-Ig show thatthe five SCR domains are found in extended con-formations with a small degree of bend betweenadjacent SCR domains. The sequence similaritybetween Crry and CR1 implies that the presentstructural determination for rCrry and mCrry-Ig isrelevant to the solution structure of CR1. Highlevels of sequence identity of 50% to 70% areobserved between Crry SCR1-3 and CR1 SCR1-3,CR1 SCR 8-10, CR1 SCR 15-17 and CR1 SCR 22-24(Table 2), while these levels of identity fall tobetween about 20% and 30% for the remainingSCR domains. The recent NMR structure determi-nations of the SCR 15-16 and SCR 16-17 domainpairs in CR1 that bind to C3b and C4b show highly

Table 2. Sequence identities between selected SCRdomains in rCrry, mCrry and human CR1

rCrry SCR-1 SCR-2 SCR-3 SCR-4 SCR-6/7

mCrry 70 74 85 75a 82a

CR1 SCR 1-3 60 64 69CR1 SCR 8-10 61 57 69CR1 SCR 15-17 61 57 70CR1 SCR 22-24 54 57 67

mCrry SCR-1 SCR-2 SCR-3CR1 SCR 1-3 63 62 66CR1 SCR 8-10 49 59 66CR1 SCR 15-17 49 59 67CR1 SCR 22-24 46 52 61

a rCrry SCR-4 is compared with mCrry SCR-4, and rCrrySCR-6 and SCR-7 are compared with mCrry SCR-5.




extended linker structures (Figure 15).31 Aftersuperimposition of the two SCR-16 domains, thedistance between the a-carbon atoms of the N-terminal Cys897 in SCR-15 to the C-terminalCys1091 in SCR-17 in CR1 is 9.4 nm. This is withinerror of the corresponding distance of8.9(^0.6) nm in the 12 best-fit rCrry structures in

Figure 11(b). The overall structures of CR1 SCR-15/SCR-17 and Crry SCR-1/SCR-3 exhibit similardegrees of elongation (Figure 15). As shown, theinter-SCR orientation in Crry SCR-1/SCR-3 differsfrom that in CR1, and this results from the lowerresolution of the scattering modelling. Theextended SCR arrangement found with these three

Figure 11. Summary of the conformational search to identify best-fit models for rCrry. (a) The first 500 unfilteredrCrry models from the total of 2000 obtained for Randomized-3 search are superimposed on SCR-3, shown as a blueribbon at the centre with the red spheres denoting the Cys residues in the two disulphide bridges. (b) The 12 filteredbest-fit final rCrry models from the Randomized-3 search are superimposed on SCR-3, shown as a blue ribbon at thecentre. Nine of the models are in green, with three apparent outliers in yellow, purple, and cyan. These are shown incomparison with the most extended rCrry model in dark blue. (c) The most extended rCrry model is shown in darkblue with its extended carbohydrate chains in red.

Figure 12. Structural analysis of the 2000 mCrry-Ig models generated in the Randomized-7 search. This search isbased on the 95% hinge length for the asymmetric IgG1 structure and the best-fit rCrry model of Figure 10(a). Allfour panels depict the calculated parameters of the models as a function of the distance between the centres of massof the two mCrry antennae and those between one of the two mCrry antennae and the Fc fragment. The red circlesdenote the 11 best-fit models identified by all the filters, while the green circles indicate all the models in each panelthat satisfy the filter in use. The mean values of the 72 best-fit models in this search are summarized in Table 1. (a)The 1921 green and red circles show the models that exhibit no steric overlap between the mCrry and Fc fragments(volume filter of ^5% from the sequence-calculated value), while the models that do overlap sterically are shown inyellow. (b) The 870 green and red circles show the models whose RG values satisfy ^10% filters from the experimentalRG value of 6.6 nm, while the models that show poor agreement with the experimental RG value are shown in yellow.(c) The 737 green and red circles show the models whose RXS values satisfy ^10% filters from the experimental RXS

value of 2.3 nm, while the models that show poor agreement with the experimental RXS value are shown in yellow.(d) The 222 green and red circles show the models with R-factor values less than 10.0%, while the models withR-factors larger than 10.0% are shown in yellow.


domains may result from the shorter four-residuelinkers between these three SCR domains, andmay lead to a better-defined solution arrangementthat is optimal for binding to C3b and C4b ligands.It is concluded that the present determinations ofextended rCrry and mCrry solution structures isconsistent with the NMR structures for two SCRpairs in CR1, and support the postulate that Crryand CR1 exhibit functional identity.

Chimeric antibody molecules in which the Fabfragments are replaced by other biologically reac-tive species are important for biotechnology andtherapeutic applications through their ability todouble the effective concentration of a proteinligand and to couple this with antibody effector

function.34 The mouse IgG1 Fc fragment was incor-porated into mCrry-Ig because it is a non-comp-lement-activating isotype. When fused to mCrry,this results in a complement inhibitor that shouldnot be recognized as a foreign molecule whenused chronically in murine models. Studies withmCrry-Ig have demonstrated that this is clinicallyeffective in mouse models, where it has a circulat-ing half-life of about 40 hours,10,12,35 and likewisefree recombinant mCrry is effective.36 Up to now,no structural data have been presented on the sol-ution conformations of these complement inhibi-tors when covalently coupled to a Fc fragment.The best-fit models for mCrry-Ig demonstrate sev-eral features that are relevant to the developmentof these molecules. Most importantly, the structureof the inhibitor molecule is as extended both whenfree (rCrry) and when conjugated (mCrry-Ig), andthis implies that mCrry-Ig is functionally accessiblefor interactions with its complement ligands.

The mCrry-Ig modelling posed questions aboutthe degree of functional access of ligands to thetwo separate Crry molecules when conjugated tothe Fc fragment. Both C3b and C4b are largemacromolecules with molecular masses of184,000 Da and 188,000 Da, and with dimensionsof 18 nm by 10 nm.37,38 mCrry-Ig and mCrry arereported to be equally effective as inhibitors.7 Themodelling indicated that the mCrry antennae inmCrry-Ig can adopt a range of semi-extended con-formations relative to the Fc fragment and yet beconsistent with the scattering and ultracentrifuga-tion data. The reasons for diverse arrangementsmay include the flexibility of the 23 residue hingesequence with at least three disulphide bridges init that may tether the Crry structures together attheir C terminus (Figure 1), together with thegreater flexibility of the two five-residue linkersbetween SCR 3-4 and SCR 4-5. The possibility ofhydrogen bond interactions between the extendedcarbohydrate chains on the two separate Crryantennae if these come into contact with eachother cannot be discounted, and this factor wouldfavour models such as that in Figure 14(b). Whatis clear from the modelling is that the most highlyextended conformations of mCrry antennae do notyield good curve fits and can be ruled out (Figure13(b)). The similar functional effectiveness ofmCrry and mCrry-Ig is most likely to result fromthe flexibility of the macromolecule. After the firstmCrry antenna has bound to its ligand, the secondmCrry antenna would remain mobile and be able

Figure 13. X-ray and neutron curve fits for one of the11 best-fit models for mCrry-Ig from the Randomized-7search. Its representation follows that of Figure 10. (a)The best-fit asymmetric model was taken from one ofthe 11 best-fit final fits for mCrry-Ig (see the text), inwhich both the X-ray and neutron curves give good fitswith the data at low and medium Q values and there isno steric conflict. (b) A curve fit with a highly extendedT-shaped symmetric mCrry-Ig model is shown to illus-trate the class of extended models that do not fit theexperimental scattering curve at all Q values.

Figure 14. Summary of the conformational search to identify best-fit models for mCrry-Ig. (a) The first 100 unfilteredmCrry-Ig models from the 2000 models of the Randomized-1 search are each superimposed on the Fc fragment, shownas a blue ribbon at the centre. The mCrry antennae are shown as green main-chain traces. (b) One of the 11 best-fit finalmCrry-Ig models from the Randomized-7 search with a minimum hinge length constraint of 95% is shown with the Fcfragment shown in blue outline with red carbohydrate chains at the centre (see Figure 13(a)). The RG value is 6.9 nm,the RXS value is 2.3 nm, the R-factor is 9.6% and s0

20;w is 5.58 S. (c) One of the six best-fit final mCrry-Ig models fromthe Randomized-8 search with a minimum hinge length constraint of 100% is shown. The RG value is 7.2 nm, the RXS

value is 2.3 nm, the R-factor is 9.6% and s020;w is 5.73 S.




to access a second ligand. In this context, a com-parison with antibody solution structures is rel-evant. Thus bovine IgG1 and IgG2 (with 19residue and 12 residue hinges, respectively) wasshown to be Y-shaped by neutron scattering fits,while human IgA1 with an O-glycosylated 23 resi-due hinge was shown to be T-shaped by X-rayand neutron scattering fits. These antibodies differin that IgG1 possesses interchain disulphidebridges in its hinge sequence while these areabsent from IgA1, thus rationalizing the observedY-shape and T-shape arrangements.26,39 It is poss-ible that more effective chimeric molecules mightbe generated by appropriate modifications of thehinge carbohydrate, disulphide bridges andsequence lengths in order to optimise the separ-ation of the Crry antennae for its C3b and C4bligands.

Materials and Methods

Purification of rCrry and mCrry-Ig

The SCR-1 to SCR-5 domains of rCrry were expressedand purified from the supernatant of Pichia pastoris cellsstably transformed with a construct directing the syn-thesis of soluble rCrry.9 Briefly, the GS115-pPIC9-Crry-transfected strain of Pichia was inoculated into 10 ml ofbuffered minimal glycerol medium (100 mM potassiumphosphate (pH 6.0), 13.4 g/l of yeast nitrogen base,0.4 mg/l of biotin, 1% (v/v) glycerol). These cultureswere grown at 30 8C in a shaking incubator until the cul-tures reached an absorbance at 600 nm ðA600Þ of 6. Thecultures were expanded into 400 ml of buffered minimalglycerol medium which were grown under the sameconditions. This culture was used to inoculate a Bioflo3000 fermentor containing minimal media for fermenta-tion and glycerol was used to feed the fermentor culturesgrowth until an A600 of 200 was attained. The productionof soluble rCrry was induced by the mixed-feed method

by the addition of methanol. The culture was monitoredby measuring cell density as well as the continuousmeasurement of dissolved oxygen. The fermentationcontinued for 96 hours after the introduction of methanolor until oxygen consumption ended as indicated by100% dissolved oxygen levels. The cell-free culturesupernatant from the fermentation cultures was clarifiedby centrifugation at 10,000g, followed by filtrationthrough 0.45 mm filters. The amount of initial proteinproduced was determined to be approximately 10 mg ofrCrry/l. Approximately four litres of clarified super-natant was concentrated 50-fold by ultrafiltration usinga 10,000 Da cut-off membrane and dialysed against25 mM Bis-Tris, pH 6.0. This material was then loadedonto a 1.5 cm £ 20 cm MonoQ column (Pharmacia, Pis-cataway, NJ). Bound proteins were eluted with a lineargradient to 1.0 M NaCl, 25 mM Bis-Tris (pH 6.0) at aflow-rate of 3 ml/minute. Fractions containing rCrrywere identified by SDS-PAGE and Western blotting.These fractions were pooled and dialysed against25 mM Bis-Tris (pH 7.1) and loaded onto a MonoPHR5/20 column (Pharmacia) and the bound proteinswere eluted with a linear gradient of polybuffer 74 (pH5.0) at a flow-rate of 0.75 ml/minute. Positive fractionswere identified by SDS-PAGE. These fractions werepooled, dialysed against phosphate-buffered saline(PBS) (pH 7.4) and concentrated by ultrafiltration to1 ml using a 10,000 Da cut-off concentrator. This wasapplied to a Superose 12 gel-filtration column (Pharma-cia) equilibrated in PBS (pH 7.4) and separated at aflow-rate of 0.5 ml/minutes. Positive fractions wereidentified by SDS-PAGE, pooled and concentrated toyield a total of 20 mg of rCrry (SCR15).

Two molecules of the SCR-1 to SCR-5 domains ofmCrry were conjugated with the N termini of the Fcregion of mouse IgG1 antibody (two CH2 and two CH3domains) at its hinge to create a recombinant chimericstructure that was expressed in NS/0 plasmacytomacells. mCrry-Ig was purified from the supernatant ofNS/0 cells stably transformed with a plasmid directingthe synthesis of an IgG1 chimeric form of Crry.7 Seed cul-tures of mCrry-Ig producing NS/0 cells were grown inIscove’s Modified Dulbecco’s medium without phenol

Figure 15. Comparison of the NMR structures for CR1 SCR-15/SCR-16 (red) and CR1 SCR-16/SCR-17 (purple) withthe best-fit solution scattering structure for rat Crry (blue). The two pairs of Cys residues in each SCR are colouredyellow to permit topological comparisons.


red, and supplemented with 10% (v/v) fetal calf serum,1 mM sodium pyruvate, 100 U/ml penicillin, 1 mg/mlstreptomycin and 2 mM L-glutamine until approximately1 £ 108 viable cells could be isolated. These cells werethen transferred into 1 l spinner flasks containing 500 mlof the same Iscove’s medium and the cultures allowedto expand. Once these cultures reached a density ofapproximately 5 £ 106 viable cells/ml, they were finallyexpanded into two ten-litre spinner flasks containingfive litres of Iscove’s medium with 1% fetal calf serum.These cultures were cultured to exhaustion (approxi-mately seven days), during which the secretion ofmCrry-Ig into the culture medium reached a maximumof 80 mg/l. These culture supernatants were thenclarified by centrifugation at 2000g for 20 minutes. Thisclarified supernatant was passed through17 cm £ 6.3 cm Pall Gelman Pleated Capsule filters(0.2 mm) with an effective filtration area of 1380 cm2

(Pall Corp., East Hills, NY), and concentrated 20-fold byultrafiltration using a 10,000 Da cut-off filter in anAmicon ultrafiltration system. This material was thenpurified in two purification runs. First, the sample wasdialysed against three successive four litre volumes of10 mM potassium phosphate (pH 7.4). This materialwas then passed over a 1.5 cm £ 20 cm Q-SepharoseFast Flow (Pharmacia) column at a flow-rate of 4 ml/minute. The bound material was eluted using a 100 min-ute gradient to 10 mM potassium phosphate (pH 7.4),250 mM NaCl. The mCrry-Ig positive fractions wereidentified by SDS-PAGE and Western blotting. ThemCrry-Ig fractions were pooled and concentrated toapproximately 10 ml, then applied to a 2.5 cm £ 60 cmHigh-Load S-200 column (Pharmacia) equilibrated in20 mM potassium phosphate (pH 7.4), 0.5 M NaCl. Theprotein was separated at 2 ml/minute and positive frac-tions were identified by SDS-PAGE. The overall proteinyield was 4 mg of mCrry-Ig from one litre of cell culture.

Immediately prior to data acquisition, non-specificaggregates of rCrry and mCrry-Ig were removed bysize-exclusion chromatography on a Superdex 75HiLoad 16/60 column and Superdex 200 HiLoad 16/60column (Amersham Biosciences, Amersham, UK),respectively. The gel filtrate was concentrated under N2

pressure using a YM10 membrane in a pressure cell(Millipore UK Ltd, Watford, UK). rCrry and mCrry-Igconcentrations were determined from absorption coeffi-cients of 9.4 and 11.4 (1%, 280 nm, 1 cm path-length),respectively, calculated from its sequence (NCBI acces-sion numbers; NP_062174 and XP_129683 respectively)after the addition of four and 12 biantennary complex-type oligosaccharide chains. The X-ray scattering andanalytical ultracentrifugation data were measured usingPBS (137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4,1.5 mM KH2PO4; Sigma D-5652) at pH 7.5, to which0.5 mM EDTA, 0.02% (w/v) NaN3 and 0.1% (w/v) Pefa-bloc SC were added as preservatives or protease inhibi-tors. The neutron scattering data were measured usingPBS in 99.9% 2H2O. Proteins were dialysed into PBS in99.9% 2H2O at 6 8C for 48 hours with three changes ofdialysate. The rCrry and mCrry-Ig samples were routi-nely analysed by SDS-PAGE before and after exper-iments to confirm their integrity.

X-ray scattering data acquisition at the SynchrotronRadiation Source

X-ray scattering data was measured in three indepen-dent sessions at the Synchrotron Radiation Source at

Daresbury, Warrington, UK at the solution scatteringcameras Station 2.1 and 8.2, both equipped with a 500-channel quadrant detector.40 – 42 Experiments were per-formed with beam currents in a range between 90 and252 mA and a ring energy of 2.0 GeV. Sample-to-detectordistances of 3.5 m and 4.3 m yielded an accessible Qrange of 0.05 nm21 to 2.2 nm21, calibrated using fresh,wet, slightly stretched rat tail collagen (diffraction spa-cing of 67.0 nm). rCrry between 2.0 and 15 mg/ml andmCrry-Ig between 1.1 and 23 mg/ml were measured at15 8C with checks for radiation damage and the minimiz-ation of buffer subtraction errors as described.23,26 Datareduction was performed using the standard Daresburysoftware package OTOKO after normalization using auniform 55Fe radioactive source.43

Neutron scattering data acquisition at ISIS andthe ILL

Neutron scattering data were obtained in three differ-ent beam sessions on the LOQ instrument equippedwith a 3He ORDELA wire detector at the pulsed neutronsource ISIS at the Rutherford Appleton Laboratory, Did-cot, UK.42,44 Proton beam currents of 140–190 mA wereused. Data acquisitions at 15 8C were for 50 £ 106–400 £ 106 monitor counts in runs lasting one to six hoursfor rCrry and mCrry-Ig concentrations of 4.5–6.8 mg/ml and 0.9–16 mg/ml, respectively. Other details wereas described.26 Neutron scattering data were obtained inone beam session on Instrument D11 using the high-fluxreactor at the Institut Laue-Langevin (ILL) in Grenoble,France.42,45 rCrry and mCrry-Ig in 2H2O buffers wasmeasured at 8.2 mg/ml and 2.5 mg/ml, respectively,with acquisition times of 5–20 minutes. Sample to detec-tor distances of 2.0 m and 10.0 m, a wavelength l of1.00 nm Dl/l of 10%) and a rectangular beam apertureof 7 mm £ 10 mm were used, resulting in a combined Qrange of 0.05 nm21 to 1.12 nm21. Data reduction wasbased on standard ILL software using the DETEC,RNILS, SPOLLY, RGUIM and RPLOT routines†.

Analysis of reduced X-ray and neutron data

In a given solute-solvent contrast, the radius of gyra-tion RG is a measure of structural elongation if theinternal inhomogeneity of scattering densities has noeffect. Guinier analyses at low Q give the RG and the for-ward scattering at zero angle Ið0Þ :24

ln IðQÞ ¼ ln Ið0Þ2 R2GQ2=3

This expression is valid in a Q RG range up to 0.7 forextended rod-like particles, and is approximate in a QRG range up to 1.5 in which it slightly underestimatesthe true RG value. The relative Ið0Þ=c values ðc ¼sample concentrationÞ for samples measured in thesame buffer during a data session gives the relative mol-ecular masses Mr of the proteins when referencedagainst a suitable standard.46,47 If the structure iselongated, the mean radius of gyration of the cross-sec-tional structure RXS and the mean cross-sectional inten-sity at zero angle ðIðQÞ·QÞQ!0 is obtained from:48

lnðIðQÞQÞ ¼ lnðIðQÞQÞQ!0 2 R2XSQ2=2

† Ghosh, R. E. (1989). A computing guide for smallangle scattering experiments. Institut Laue Langevininternal publication 89GH02T.


The RG and RXS analyses lead to the triaxial dimensionsof the macromolecule. If the structure can be representedby an elongated elliptical cylinder:

L ¼ ð12ðR2G 2 R2

XSÞÞ1=2

where L is its length.24 Alternatively, L is given bypIð0Þ=ðIðQÞ·QÞQ!0:

25 The two semi-axes, A and B; of theelliptical cylinder are calculated by combining the dryor hydrated volume V ðV ¼ pABLÞ with the RXS valueðR2

XS ¼ ðA2 þ B2Þ=4Þ: The hydrated volume is obtainedon the basis of a hydration of 0.3 g of water/g of glyco-protein and 0.0245 nm3 per water molecule.49 Data ana-lyses employed an interactive graphics programSCTPL5 (A. S. Nealis & S.J.P., unpublished software) ona Silicon Graphics 4D35S Workstation and a Perl scriptprogram SCTPL7 (J. T. Eaton & S.J.P., unpublished soft-ware) on Silicon Graphics Indy and O2 Workstations.

Indirect transformation of the scattering data IðQÞ inreciprocal space into real space to give the distance dis-tribution function PðrÞ was performed using GNOM.50,51

PðrÞ corresponds to the distribution of distances rbetween any two volume elements within one particleweighted by the product of their respective electron ornuclear densities relative to the solvent density. Thisoffers an alternative calculation of the RG and Ið0Þ that isbased on the full scattering curve, and gives a model-independent maximum dimension of the macromoleculeL: The rCrry X-ray curve contained 466 data pointsbetween Q values of 0.08 nm21 to 2.2 nm21 and was fitwith Dmax set as 18 nm, while the mCrry-Ig X-ray curvecontained 456 data points between Q values of0.10 nm21 and 1.8 nm21 and was fit with Dmax set as34 nm (Figure 4(a) and (b)). The rCrry LOQ neutroncurve contained 67 data points between Q values of0.15 nm21 and 2.0 nm21 and was fit with Dmax set as18 nm, while the mCrry-Ig LOQ neutron curve contained75 data points between Q values of 0.12 nm21 and2.0 nm21 and was fit with Dmax set as 34 nm (Figure 4(c)and (d)). Other details of the PðrÞ analyses are reportedelsewhere.26

Analytical ultracentrifugation data acquisitionand analysis

Analytical ultracentrifugation was performed on aBeckman XLi instrument in which the protein concen-tration distribution within the cell was monitored usingits absorbance monitored at a single wavelength setbetween 280 nm and 295 nm, and its refractive indexmeasured by interferometry. Sedimentation equilibriumexperiments were performed for rCrry at three concen-trations between 0.5 mg/ml and 2.3 mg/ml in PBS andfor mCrry-Ig at three concentrations between 0.05 mg/ml and 2.1 mg/ml in PBS. Data were acquired over 45hours using six-sector cells in an AnTi 50 rotor withcolumn heights of 2 mm at rotor speeds of 11,000 rpm,14,000 rpm, 17,000 rpm and 20,000 rpm until equilibriumhad been reached at each speed, as shown by the perfectoverlay of runs measured at five hour intervals. The mol-ecular mass ðMÞ was analysed on the basis of a singlespecies using Beckman software provided as an add-onto Origin Version 4.1 (Microcal Inc.), for which the partialspecific volume v for rCrry and mCrry-Ig were calcu-lated to be 0.710 ml/g and 0.713 ml/g, respectively,from its sequence:49

cr ¼ croexp½ðv2=2RTÞMð1 2 �vrÞðr2 2 r2

oÞ�

where cr is the concentration at radius r; crois the concen-

tration of the monomer at the reference radius ro; v is theangular velocity, R is the gas constant, T is the tempera-ture in Kelvin, and r is the solvent density.

Sedimentation velocity data were acquired over 16hours at rotor speeds of 35,000 rpm and 42,000 r.m intwo-sector cells in an AnTi 50 rotor with solutioncolumn heights of 12 mm. The rCrry concentrationwas 1.5 mg/ml, and that for mCrry-Ig was 0.15 g/ml,both being in PBS. For the gðspÞ analyses, successiveabsorbance and interference scans were recorded ateight minute intervals, the shortest interval possibleunder standard measurement conditions. In time-derivative analyses, the subtraction of pairs of concen-tration scans versus radius in the cell eliminates sys-tematic errors from baseline distortions in the cellwindows and permits the averaging of many pairs ofsubtractions. The extrapolation of individual subtrac-tions to the start time gives the gðspÞ function, whichwas computed using the DCDT þ program,52 fromwhich the sedimentation and diffusion coefficientswere determined from the peak position and width,respectively (Figure 6). The fits were determined inconditions when the maximum permissible measurablemolecular mass was over 16 times the expected value,meaning that time broadening effects were negligible.

Homology modelling of the SCR domains in rCrryand mCrry-Ig

A total of 32 SCR domains from seven crystal struc-tures and eight NMR structures were analysed in orderto model the SCR domains of rCrry and mCrry (thePDB codes are given in Figure 7). In order to align the32 sequences, their secondary structures were identifiedusing DSSP53 and their side-chain solvent accessibilitieswere calculated using a probe of radius 0.14 nm to rep-resent a water molecule using COMPARER.54,55 Theresulting alignment was verified visually by the super-imposition of the SCR domains on the basis of the con-served b-strand residues using Biosym CommandLanguage script files within INSIGHT II moleculargraphics software (Accelrys, San Diego) in conjunctionwith the use of Crystal Eyes stereo glasses.

The rCrry and mCrry SCR domains were modelledusing INSIGHT II, HOMOLOGY, DISCOVERY and BIO-POLYMER software (Accelrys, San Diego). The crystalstructures of SCR-1 and SCR-2 in b2GPI and SCR-1 inCD46 and the NMR structures of SCR-4 in VCP andSCR-17 in CR1 were selected as reference structures onthe ideal criteria that each possessed six b-strands, hadno residues in disallowed regions in a Ramachandranplot, and required the fewest insertions or deletions inthe sequence alignment. The sequence identities rangedfrom 17% to 38% between the reference and the model.Other modelling details are reported elsewhere.23 Thefour cis-peptides found in the reference structures wereremoved by searching for new loops to bridge each one.Distorted geometries at splice regions at loop insertsand residues in disallowed regions in Ramachandranplots were corrected using DISCOVER refinement. Bian-tennary complex-type carbohydrate structures (Man3-

GlcNAc4Gal2NeuNAc2; Mr 2200) or triantennary ones(with an extra GlcNAc.Gal.NeuNAc chain) were addedto each of the four N-linked glycosylation sites in anextended conformation from the protein surface, basedon the structure found in the crystal structure of the Fcfragment of murine IgG1 (PDB code 1igy)32 (Figures 1and 7).


Modelling of rCrry by constrained scattering fits

A strategy based on randomized arrangements of SCRdomains in five-domain rCrry models was developed inwhich the SCR domains were connected by libraries of10,000 linker peptides generated by molecular dynamicssimulations to follow method 1 as described by Aslam& Perkins.23 Each linker peptide sequence included theCys59 and Cys4 residues of the preceding and followingSCR domains respectively (Figure 7). Using a BiosymCommand Language script within INSIGHT II software,each rCrry model was created by randomly selecting alinker conformation from each linker library in turn,each of which was superimposed onto the precedingand following SCR domains by use of these Cys59 andCys4 coordinates. Duplicate atoms were then removed.By this approach, three sets of 2000 rCrry models werecreated, in which either no restraint was imposed on thesimulations, or a distance restraint of 95% or of 100% ofthe exact extended b-strand length between the Ca atomsof Cys59 and Cys4 was imposed on the simulations.Note that this Cys superimposition procedure was alsoused to generate the 20 trial models of rCrry structuresusing the 20 known structures for the inter-SCR linkersfrom crystallography and NMR. These rCrry linkerswere identical in sequence and structures with thoseseen in the experimental structures, but were accountedfor in the use of the rCrry volume in the modelling.

A rotational search modelling strategy was also usedto create rCrry models, which corresponds to method 2as described.23 The interdomain orientation of the fourlinkers in rCrry was set to be the same as that betweenSCR-1 and SCR-2 in b2GPI, one of the most lineardomain pairs.23 The five SCR domains were then rotatedsystematically about each other by applying a given setof the same X; Y and Z-axes rotations to all four SCRdomains, in which the X-axis corresponded to the long-est axis of the rCrry model. The four sets of rotationalaxes were set with their origins defined at the Ca atomof residue 35 in each domain, namely Gly35 or Asp35(Figure 7), since topologically this residue is the most C-terminally located one in each SCR domain. The Y-axeswere directed towards the Ca atom of Cys4 in each SCR.The linker sequences were not included in these models,meaning that the rCrry models possessed 18 feweramino acid residues; however, this was rectified duringthe subsequent coordinate conversion to small spheres,which was based on the full volume of all 320 aminoacid residues and 44 carbohydrate residues. The finalsearch entailed rotations between 08 and 3308 in 308steps about the X-axis; and between 21208 and 1208 in158 steps about the Y and Z-axes to generate12 £ 17 £ 17 ¼ 3468 models.

Modelling of mCrry-Ig by constrained scattering fits

The solution structure modelling of mCrry-Ig wasbased on combining our best-fit solution structure forrCrry with the Fc fragment found in the crystal structureof mouse IgG1 (PDB code 1igy).32 The homology modelsof the five mCrry domains were superimposed on theirequivalents in either the best-fit or the most extendedrCrry structure. The four rCrry linker sequences areidentical in length with those in mCrry and were usedunchanged even though there were three residuechanges out of 18 in the mCrry linkers. The Fc crystalstructure displayed an asymmetric hinge structure atresidues VPRDCGCKPCICTVPEV (£2). These residues

as observed crystallographically were connected to SCR-5 of mCrry with the residues VSRLADPE that were mod-elled initially as an extended b-strand conformation. Atotal of 14 £ 2 residue changes were made to the CH2and CH3 domains in the Fc crystal structure in orderthat this corresponded to the mCrry-Ig sequence, andfour missing residues SPGK were appended to the twoC termini in extended b-strand conformations. Mostnotably, the residue changes introduced a second pair ofglycosylation sites in the CH3 domains that are not presentin the mouse IgG1 crystal structure (Figures 1 and 7).Biantennary complex-type oligosaccharides wereattached to these described above for rCrry.

In a randomized linker modelling strategy, thesequence of the 27 residue linker and hinge peptidebetween SCR-5 and the Fc fragment, which included theN-terminal Cys and C-terminal Ser residues to allow forthe subsequent superimpositions with the SCR-5 and Fcdomains, respectively (CVSRLADPEVPRDCGCKPCIC-TVPEVS), was subjected to molecular dynamics simu-lations using DISCOVER3. A conformational library of10,000 structures for the linker and hinge was generatedfollowing the same method 1 as described.23 In the simu-lations, exact distance and torsional restraints on themain-chain atoms were applied to the C-terminal Serresidue in order to permit its subsequent superimposi-tion with the Fc fragment. Distance restraints wereapplied to the second, third and fourth intercysteine dis-ulphide bonds in the hinge sequence in order that thesewere maintained in all the linker-hinge models (the firstdisulphide bridge is not observed in the crystal struc-ture). The length of the entire peptide was restrained to95% or 100% of its original separation between the start-ing and terminal Ca atoms. A total of 2000 models withasymmetric hinge structures was selected randomlyfrom this library for the modelling of the mCrry-Igstructure.

To introduce symmetry into the mCrry-Ig models inthe randomized linker modelling analysis, the 2-foldsymmetry axis of the Fc fragment was computed byreference to the eight Ca atoms of the four conserved dis-ulphide bridges in each of the CH2 and CH3 domains.This 2-fold dyad symmetry axis enabled the three disul-phide bonds in the hinge region to be tethered to it suchthat the distance restraint between a sulphur atomwithin each disulphide bond and the symmetry axiswas fixed to half the length of the disulphide bond. TheFc fragment is not completely symmetric about this axis(see Table 1 of Harris et al.32). Accordingly one-half ofthe Fc fragment (the CH2 and CH3 domains and thehinge peptide VPRDCGCKPCICTVPEV) was superim-posed via a rotation about the Fc symmetry axis to theother half of the Fc fragment using the direct superimpo-sition of the eight Ca atoms of the four conserved disul-phide bridges in each of the CH2 and CH3 domains. Thiswas achieved with an rms deviation of 0.03 nm. Thisresulted in an initial 2-fold symmetric structure inwhich energy minimization was used to recreate thethree disulphide bonds in the hinge. From this startingmodel, a library of 10,000 symmetric hinge structureswas generated using the procedure followed above forthe asymmetric hinge structures. A total of 2000 modelswith symmetric hinge structures was selected randomlyfrom this library for the modelling of the mCrry-Igstructure.

Two other searches performed systematic rotationalsearches of the five-SCR Crry antennae about the asym-metric and symmetric Fc fragment models used in therandomized searches. The mCrry model was held rigid


as for the randomized searches. The X-axis correspondedto the longest axis of the Crry SCR domains, and theother axes were set so that the origin for the rotationsoccurred at the Ca atom of Cys59 of SCR-5 and theY-axis corresponded to the direction towards the Ca

atom of Gly17 in SCR-5. The rotational search wasbased on two sets of identical rotations of the Crry anten-nae between 08 and 3308 in 608 steps about the X-axis;and between 21058 and 1058 in 158 steps about the Yand Z-axes to generate 6 £ 15 £ 15 ¼ 1350 models.

Debye scattering curve modelling of rCrryand mCrry-Ig

In order to calculate the scattering curves, Debyesphere models were created by placing each coordinatemodel within a three-dimensional grid of cubes, whereeach cube side is of length 0.70 nm and 0.52 nm forrCrry and mCrry-Ig, respectively. This length is muchless than the nominal resolution of the scattering curves.Provided that the number of atoms within a given cubeexceeded a user-defined cut-off, a sphere of the samevolume as the cube (sphere diameter 0.868 nm and0.645 nm for rCrry and mCrry-Ig, respectively) wasplaced at the centre of the cube. This cut-off was deter-mined by the requirement that the total volume ofspheres in each model was within 1% of the dry volumeof 51.7 nm3 and 177.8 nm3 for rCrry and mCrry-Ig,respectively, calculated from its amino acid and carbo-hydrate composition.49 The totals of 151 and 1267 dryspheres for rCrry and mCrry-Ig, respectively, were usedto calculate the neutron scattering curves. X-ray scatter-ing modelling requires the addition of a hydration shell.A hydration of 0.3 g of H2O/g of glycoprotein and anelectrostricted volume of 0.0245 nm3/bound watermolecule27,49 gave hydrated volumes of 68.7 nm3 and178.7 nm3 for rCrry and mCrry-Ig, respectively. Thisshell was modelled using the HYPRO procedure,56 inwhich excess spheres was added to the surface of thedry sphere model, and these were removed sequentiallyuntil the desired hydration volume of 200 and 1685spheres had been reached for rCrry and mCrry-Ig,respectively.

The X-ray and neutron scattering curve IðQÞ was cal-culated assuming a uniform scattering density for thespheres using the Debye equation as adapted tospheres.26,57 The X-ray curves were calculated from thehydrated sphere models without corrections for wave-length spread or beam divergence, as these are con-sidered to be negligible for synchrotron X-ray data. Afull-width/half-height wavelength spread Dl/l of 10%for l of 0.6 nm and a beam divergence of 0.016 radianwere used to correct the calculated curve for the LOQneutron data fits.56 The number of spheres N in the dryand hydrated models after grid transformation wasused to assess any potential steric overlap between theSCR domains, where models showing less than 95% ofthe required total were discarded (a 5% filter). The mod-elled scattering curves were assessed by calculation ofthe RG and RXS-1 values from the modelled curve in thesame Q ranges used for the experimental Guinier fits, inwhich filters corresponding to ^5% or ^10% of theexperimental values were applied. One other filter wasapplied based on the s0

20;w values calculated from thesphere models (see below). In the favourable case of therCrry model, the length L of the models was determinedfrom the tip-to-tip distance between the N terminus andC terminus of the rCrry models for comparison with the

L value determined from the PðrÞ curve. Models thatpassed these filters were then ranked using a goodness-of-fit R-factor defined by analogy with protein crystallo-graphy and based on the experimental curves in the Qrange extending to 2.0 nm21 (denoted as R2:0).58 A 2000model search for mCrry-Ig required two days of R12000central processor unit time on a Silicon Graphics Octane(270 MHz, with 1280 Mb memory).

Sedimentation coefficient modelling of rCrryand mCrry-Ig

Procedures for hydrodynamic simulations based onsphere models have been tested.57,59 The GENDIA pro-gram was used to calculate the s0

20;w values startingfrom the hydrated rCrry and mCrry-Ig models. The out-come of these modelling searches was confirmed withthe slower but more exact program HYDRO.

Protein Data Bank accession numbers

The best-fit Ca coordinate models for rCrry (Figure10(a)) and mCrry-Ig (Figure 14(b) and (c)) have beendeposited in the RCSB Protein Data Bank with the acces-sion codes 1ntj and 1ntl.

Acknowledgements

We thank the Biotechnology and BiologicalSciences Research Council for a Special Student-ship Award, and the Wellcome Trust for an equip-ment grant for the Beckman XLi analyticalultracentrifuge. R.J.Q was supported by NIH grantR01DK41873 and V.M.H was supported by NIHgrant R01CA53615. We thank Mrs S. Slawson, MrA. Gleeson, and Dr J. G. Grossmann (SRS, Dares-bury), Dr P. A. Timmins (ILL, Grenoble), and DrR. K. Heenan and Dr S. M. King (ISIS, Rutherford-Appleton Laboratory) for instrumental support,and Dr J. T. Eaton for assistance with the PDB filesubmission.

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Edited by R. Huber

(Received 4 February 2003; received in revised form 24 March 2003; accepted 9 April 2003)


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The Extended Multidomain Solution Structures of the Complement Protein Crry and its Chimeric...

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