X-ray Crystal Structure of the C4d Fragment of HumanComplement Component C4

Jean M. H. van den Elsen1†, Alberto Martin2†, Veronica Wong2

Liliana Clemenza2, David R. Rose1 and David E. Isenman2*

1Ontario Cancer Institute andDepartment of MedicalBiophysics, University ofToronto, 610 UniversityAvenue, Toronto, OntarioCanada, M5G 2M9

2Department of BiochemistryUniversity of Toronto, TorontoOntario, Canada, M5S 1A8

C4 fulfills a vital role in the propagation of the classical and lectin path-ways of the complement system. Although there are no reports to date ofa C4 functional activity that is mediated solely by the C4d region, evi-dence clearly points to it having a vital role in a number of the propertiesof native C4 and its major activation fragment, C4b. Contained withinthe C4d region are the thioester-forming residues, the four isotype-specificresidues controlling the C4A/C4B transacylation preferences, a bindingsite for nascent C3b important in assembling the classical pathway C5convertase and determinants for the Chido/Rodgers (Ch/Rg) bloodgroup antigens. In view of its functional importance, we undertook todetermine the three-dimensional structure of C4d by X-ray crystal-lography. Here we report the 2.3 A resolution structure of C4Ad, the C4dfragment derived from the human C4A isotype. Although the ,30%sequence identity between C4Ad and the corresponding fragment of C3might be expected to establish a general fold similarity between the twomolecules, C4Ad in fact displays a fold that is essentially superimposableon the structure of C3d. By contrast, the electrostatic characteristics of thevarious faces of the C4Ad molecule show marked differences from thecorresponding faces of C3d, likely reflecting the differences in functionbetween C3 and C4. Residues previously predicted to form the majorCh/Rg epitopes were proximately located and accessible on the concavesurface of C4Ad. In addition to providing further insights on the currentmodels for the covalent binding reaction, the C4Ad structure allows oneto rationalize why C4d is not a ligand for complement receptor 2. Finallythe structure allows for the visualization of the face of the moleculecontaining the binding site for C3b utilized in the assembly of classicalpathway C5 convertase.

q 2002 Elsevier Science Ltd. All rights reserved

Keywords: complement; C4; Chido/Rodgers; thioester; C3d*Corresponding author

Introduction

Complement component C4 circulates in bloodas a disulfide-linked heterotrimer consisting of a(93 kDa), b (75 kDa) and g (33 kDa) chains. C4b,the major activation product produced by C1scleavage of the a-chain, is an essential subunit ofboth the C3 convertase (C4b2a) and the C5 con-vertase (C3bC4bC2a) enzymes of the classicalcomplement pathway.1 Additionally, C4b is anopsonin capable of forming a bridge between thetarget antigen or pathogen to which it is attachedand complement receptor 1 (CR1) that among othercell types is present on phagocytes involved in theclearance of immune complexes.2 All of theseactivities depend upon the thioester-mediated

0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved

† These authors contributed equally to this work.Present addresses: J. M. H. van den Elsen, Department

of Biology and Biochemistry, University of Bath, BathBA2 7AY, UK; A. Martin, Yeshiva University, AlbertEinstein College of Medicine, New York, USA;L. Clemenza, Department of Biopathology andBiomedical Technologies, University of Palermo, CorsoTukory 211, Palermo 90134, Italy.

E-mail address of the corresponding author:d.isenman@utoronto.ca

Abbreviation used: CR1, complement receptor type 1.

doi:10.1016/S0022-2836(02)00854-9 available online at http://www.idealibrary.com onBw

J. Mol. Biol. (2002) 322, 1103–1115

transacylation of nascent C4b to a surface nucleo-phile on the target antigen/pathogen per se or onto an antibody associated with the target antigen.3,4

The complement proteins C3 and C4, as well as theprotease inhibitor a2-macroglobulin, form a proteinsuperfamily that shares the common feature ofhaving a proteolytically activatable intramolecularthioester bond that is capable of forming a covalentadduct5. The thioester bond is formed between theside-chains of cysteine and glutamine presentwithin the sequence GCGEQT/NM, a sequencethat is present at analogous positions within theprimary structures of the single chain precursormolecules of the superfamily. Phylogeneticevidence suggests that thioester-mediated trans-acylation as a mechanism of opsonizing pathogensis quite ancient and that the complement moleculeslikely arose from a monomeric a2-macroglobulin-like molecule with opsonic activity towardspathogens.5,6

The C4 locus in man has been duplicated andconsequently there are two isotypes of the protein,which are referred to as C4A and C4B, respectively.Each of these isotypes is in turn polymorphic, withmost of the amino acid variations being locatedwithin the central portion of the a-chain corre-sponding to the physiologic ,42 kDa C4d frag-ment that is generated by complement factorI-mediated cleavages of the a-chain.7,8 The C4dregion also contains the thioester-formingsequence, the four isotype-specific residues(1101PV–LD1106 in C4A and 1101LS–IH1106 in C4B),the residues giving rise to the Chido/Rodgers(Ch/Rg) blood group antigens and the site atwhich C3b covalently binds in the assembly of theclassical pathway C5 convertase. The C4A andC4B isotypes, regardless of any superimposedallelic polymorphisms, display profounddifferences in their covalent binding properties.Specifically, C4A allotypes preferentially trans-acylate onto amino group nucleophiles, whereasC4B allotypes preferentially transacylate ontohydroxyl-bearing target molecules.9,10 The elucida-tion of the biochemical basis for these transacyl-ation preferences has led to the conclusion thatthere are actually two quite different mechanismsat play for the respective isotypes.11 For C4A,upon surface exposure of the thioester bond innascent C4b fragment, there is a direct and uncata-lyzed attack by the nucleophile on the thioestercarbonyl. Since amino groups are inherently betternucleophiles than hydroxyl groups, this explainswhy amide bond formation is dominant in C4Aallotypes. For C4B (also C312) the transacylationreaction is catalyzed and involves not onlyHis1106 (H1105 in C3), i.e. the C-terminal-mostisotypic residue, but also the thiolate group of thethioester-forming cysteine residue. Specifically,upon proteolytic activation there is rapid formationof an acyl-imidazole intermediate between thethioester carbonyl and the side-chain of the iso-typic region histidine. The liberated cysteinethiolate of the thioester is then proposed to act as

a Brønsted base to increase the nucleophilicity ofhydroxyl group nucleophiles for attack on theacyl-imidazole intermediate.

Complete deficiency of C4 correlates stronglywith the immune complex disease systemic lupuserythematosus (SLE) in humans.13 Although notseen in all SLE patient population studies.14,15

there are several reports suggesting that even par-tial C4A deficiency states also correlate with anincreased risk for SLE.16 – 19 Possible reasons forthis correlation are, firstly, that the transacylationpreference for amino group nucleophiles mightmake this isotype the more important one for theclearance of IgG-containing immune complexes,20

and secondly, that the fourfold higher affinity thathas been reported for dimers of C4Ab relative tothose of C4Bb would favor the clearance by phago-cytes of immune complexes opsonized with anarray of C4Ab molecules.21 Since the isotypicsequence differences are confined to the C4d frag-ment, this suggests that the isotype-dependentCR1-binding differences reflect a contribution ofC4d to the overall C4b–CR1 interaction.

Modeling analysis of neutron scattering data ofC3 and C4 suggested that they can each bedescribed as consisting of a small and a largedomain, where the small domain corresponds tothe C3d/C4d fragment and the large one to theC3c/C4c fragment.22,23 The compact globulardomain nature of the C3d fragment has been con-firmed by the recent determination of its X-raycrystal structure.24 This structure provided the firstatomic level look at the arrangement of theresidues forming the thioester bond and mediatingthe transacylation mechanism. Despite the func-tional homology between C3d and C4d in terms ofmediating the covalent binding reaction, theamino acid residue sequence identity between thecorresponding human fragments is only about30%. Given the important contributions made bythe C4d fragment to the overall function of the C4molecule, and especially because of the isotype-related functional differences referred to above,we undertook to crystallize and determine theX-ray crystallographic structure of human C4dfragment. We now report the structure of the C4dfragment of the C4A isotype to a resolution of2.3 A. Comparison of the structure of C4Ad to thatof C3d reveals on the one hand a nearly completesuperposition of backbone conformation, but onthe other quite distinct surface chemistries.

Results and Discussion

Expression and crystallization

As detailed in Materials and Methods, webacterially expressed and purified C4d fragmentsof each human C4 isotype corresponding to eitherthe full-length fragment (residues 938–1317,delimited by the factor I cleavage sites) or to anN-terminally truncated version lacking 45 residues.

1104 Crystal Structure of C4d

In keeping with the C3 fragment nomenclature, werefer to these as C4dg and C4d, respectively. V8protease digestion of these products yieldedproteolytic limit fragments lacking 12–13 residuesat the C terminus only (see Materials and Methods)and these fragments are given the designator -V8.Of the eight permutations of C4d fragment sub-jected to crystallization trials, only the C4Adg-V8derivative yielded crystals. In this case hollowrod-like crystals were obtained within one day in28% (w/v) polyethylene glycol (PEG) 4000, 0.1 MTris–HCl (pH 7.5), 0.2 M MgCl2, 5 mM DTT.

Overall architecture of C4Adg-V8

A 2.3 A resolution structure, refined to an Rfactor of 21%, was obtained using crystals ofC4Adg-V8 (see Table 1 for X-ray data collectionand refinement statistics). The model of C4Adg-V8contains residues 977–1212 and 1237–1302 of the367 amino acid residue construct (after V8 proteasedigestion). Although it was required for crystalliza-tion, no electron density was seen for residues938–976 of the N-terminal “g” segment (aminoacid residues 938–983) and so essentially the struc-ture is that of C4Ad. There was also no electrondensity for amino acid residues 1213–1236, mostof which correspond to a large insertion relative tothe sequence of human C3d (see Figure 2). Finally,there was no electron density for the C-terminalresidues Thr1303 and Glu1304, the latter located at

the presumed V8 protease digestion site (seeMaterials and Methods).

C4Ad shares the a-a 6 barrel fold described forC3d,24 which is characterized by six parallel alphahelices forming the core of the barrel, surroundedby another set of six parallel helices running anti-parallel with the core of the C4d fragment

Table 1. Statistical data

A. Data collection statisticsResolution (A) 2.3Space group P212121

Cell dimensions (A) 56.51, 71.43,85.74

Reflections (no.) overall; shell 2.35–2.30 A 43,751; 2898Unique reflections (no.) overall; shell 2.35–2.30 A

15,703; 1057

Completeness (%) overall; shell 2.35–2.30 A 97.9; 99.4I/sI , 2 (%) overall; shell 2.35–2.30 A 24.5; 58.9Rmerge

a overall; shell 2.35–2.30 A 0.079; 0.511

B. Refinement statisticsResolution (A) 500–2.30R (%) 21.5Rfree (%) 23.3Atoms (no.) 2365Residues (no.) 301Water molecules (no.) 76Rmsd bonds (A) 0.009Rmsd angles (deg.) 1.04Rmsd improper dihedrals (deg.) 0.82Average B-factors (A2) 41.6Cross-validated sA coordinate error (A) 0.35

Rcryst ¼P

llFol 2 lFcll/P

lFol, where Fo and Fc are the observedand calculated structure factors, respectively. For Rfree, the sumis extended over a subset of reflections (,10%) excluded fromall stages of refinement.

a Rmerge ¼P

h

PilIi 2 kIll/

PiIi, where kIl is the average of

equivalent reflections and the sum is extended over all obser-vations, i, for all unique reflections, h.

Figure 1. (a) Top view superposition of the structuresof C3d and C4Ad rendered in magenta and gold,respectively, showing the a–a 6 barrel topology of themolecules with 12 helices consecutively alternating fromthe outside to the inside of the barrel. (b) Side viewribbon representations of the C4Ad structure showingthe positions of the thioester-forming residues, Cys991and Gln994, and the C-terminal isotypic residueAsp1106 (gold ball and stick), all located at the convexface of the molecule. Also shown as ball and stick arethe side-chains of the polymorphic amino acid residuesSer1157, Thr1182, Ala1188 and Arg1191. These are proxi-mately located on the concave surface and with theexception of Thr1182 contribute the major Ch/Rg epi-topes. Also indicated is a loop in the C4Ad structure(residues 1213–1236) for which no electron density wasseen. Residue Ser1217 in this loop is known to beinvolved in the assembly of the C5 convertase complex(C2a4b3b) by its interaction with C3b. All molecularimages in this and subsequent Figures were preparedusing MOLSCRIPT44 and rendered using POV-RAYe.

Crystal Structure of C4d 1105

(Figure 1). Similar to C3d, the opposing sides of theC4Ad barrel display contrasting structuralfeatures, with a convex surface containing thethioester residues at one end, and a concave sur-face at the opposite end of the barrel (Figure 1(b)).Despite only ,30% sequence identity betweenC4d and C3d, the structures of C4Ad and C3d areessentially superimposable (Figure 1(a)), withroot-mean-square-deviations between Ca atoms of0.424 A and very similar boundaries for most ofthe helices when these are depicted on the alignedsequences of C4d and C3d (Figure 2).

Thioester and isotypic residue region

The thioester-forming residues of C4 play a keyrole in opsonizing microbial agents throughcovalent attachment to the antigenic surface.Activation of C4 by C1s results in the exposure ofthe initially buried 15-membered thiolactone ringconsisting of residues Cys991-Gly992-Glu993-Gln994. As described in detail in Introduction,C4B and C3, because of the presence of histidineas the last of the isotypic residues (H1106 in C4B,H1105 in C3), transacylate onto target hydroxylgroups via a two-step catalyzed mechanism. Bycontrast, in C4A amino group nucleophiles directlyattack the nascently exposed thiolactone in anuncatalyzed manner. Figure 1 (right panel) showsthe position of thioester-residues, Cys991 andGln994, and the C4A isotype-specific residueAsp1106. A close-up of this region is presented inFigure 3, showing the thioester in an open confor-

mation, as seen in the C4Ad structure. Only aslight twist of the Gln994 side-chain around itschi-1 axis (i.e. the Ca–Cb bond, see rotation arrowin Figure 3) is required to model the conformationof the intact thioester bond. This rotation bringsthe carbonyl carbon of Gln994 to within 2 A of theCys991 sulfur group, which is typical for a S–Cbond length. Superposition of the C3d and C4Adthioester regions (Figure 3) shows that the back-bone and side-chain positions of the thioester andits flanking residues are virtually identical. Fromthis comparison it can be concluded that theabsence of Cys17 (C988 in mature C3 numbering)in the structure of the C17A mutant form ofhuman C3d that was crystallized in the earlierstudy did not affect the overall conformation ofthe thioester region. Despite substantial sequencedifferences in the isotypic segment between C4Adand C3d (PCPVLD and DAPVIH, respectively),including the presence of an extra proline residue(P1101) in C4A, no significant differences in confor-mation are found between the two structures inthis region. It is therefore unlikely that the C4B iso-typic segment will adopt a different conformationfrom that seen in the other two examples becausethe C4A isotypic sequence PCPVLD is as dissimilarfrom the corresponding DAPVIH sequence of C3das it is from the C4B sequence LSPVIH. We cannotexplain the failure of C4Bdg-V8, or an engineered938–1306 V8-like equivalent, to crystallize andthereby enable the most definitive proof on thispoint. However, we know this was not due to theabsence of Pro1101, because a 938–1306 C4Bdg

Figure 2. Sequence alignment of C4Ad (1HZF) and C3d (1C3d) with the boundaries of the respective structure-derived secondary structure elements indicated in each case. The numbering of the a-helices is in accordance withthe standard nomenclature adopted for a–a 6 barrel molecules.25 310 Helices and strict b-turns are designated as Tand TT, respectively. The alignment was performed with Clustal X and the secondary structure elements were depictedon the aligned sequences using the web-based program ESPript. 1.9.

1106 Crystal Structure of C4d

construct in which Leu1101 was mutated to Proalso did not crystallize.

On the basis of the ability (C3/C4B) or inability(C4A) to form the acylimidazole covalent inter-mediate of the two-step catalyzed mechanism, theHis/Asp substitution at position 1106 in the iso-typic region can explain most of the difference intheir respective covalent binding properties.3

There remain, however, several unresolved issuesupon which our structural comparison of C3d andC4Ad has shed some light. Whereas activated C4Bpossesses significant ability to react with themodel amino group-containing compound glycine,thus forming amide linkages, C3 shows absolutelyno reactivity towards glycine, but about the samereactivity with the model hydroxyl group-contain-ing compound glycerol as does C4B. Law &Dodds3 proposed that the binding of glycine toC4B resulted from the direct uncatalyzed attack ofthe amino group on the thioester in competitionwith acylimidazole formation. They further specu-lated that the reason that this did not occur in C3was that the rate of acylimidazole formation in C3was greater than in C4B. A possible explanationfor this rate increase suggested by the C3dstructure is that a hydrogen bond between a ringnitrogen of His133 (equivalent of C4d isotypicresidue 1106) in C3d and a negatively chargedGlu135 side-chain would render His133 a strongernucleophile.24 It was suggested at the time fromsequence alignment that such an interactionwould be missing in C4d, where the equivalentresidue to Glu135 is Ser1108. The C4Ad structurenow confirms that Ser1108 indeed occupies thesame spatial position as does Glu135 in C3d andfurther that there are no other residues in thevicinity that could mediate a similar H-bondingrole on a His residue at position 1106 (Figure 3).

As shown in Figure 4(a), whereas the convexsurface of C4Ad is predominantly lined with acidicresidues, which accordingly display an extensiveelectronegative surface potential, the correspond-ing surface of C3d displays many more positivelycharged and neutral regions. These significantdifferences in electrostatic potential may contributeto determining the transacylation target moleculepreferences (i.e. not just the nucleophilic prefer-ence) of C3 and C4. In view of the structural con-servation seen in the isotypic regions of C4Ad andC3d, we expect that the surface of C4Bd would dis-play an overall charge distribution pattern that issimilar to C4Ad.

Native C4A domain interface

In the earlier study on the structure of humanC3d, it was noted that the amino acid residuesinvolved in the transacylation mechanism formedpart of a surface patch of highly conserved andlargely apolar residues where the degree ofsequence conservation among a diverse series ofspecies was similar to what was seen for the buriedcore residues of C3d.24 On the basis of this, it wasproposed that this patch, whose boundaries aredenoted by the dotted enclosure in Figure 4(a)(left), forms a domain interface that allows thethioester residues to be sequestered from thesolvent in the native state of the intact C3 molecule.It was further noted that the residues forming thesurface patch were largely conserved in humanC4d. As can be seen in Figure 4(a) (right), the sur-face rendition of the C4Ad structure confirms thepresence of a patch of very similar chemicalcharacteristics to that of C3d. It could thereforelikewise serve as the domain interface to sequesterthe thioester and supply some of the strain energy

Figure 3. Superposition of thethioester region of C3d and C4Ad.Also shown for each molecule arethe conformations of the respectiveisotypic segments and the sequencedifferences between the two struc-tures. The color scheme is gold forC4Ad and magenta for C3d. Thethioester-contributing residuesCys991 and Gln994 of C4Ad areshown in the open conformation(in C3d the thioester cysteine resi-due had been mutated to alanine).If the side-chain of Gln994 is rotated508 about its chi-1 axis, as indicatedby the rotation arrow, the carbonylcarbon of this residue is brought towithin S–C bonding distance (2 A)of the sulfur moiety of Cys991.

Crystal Structure of C4d 1107

required to keep it in its closed conformation innative C4. One minor difference between C3d andC4Ad in this putative domain interface is that,whereas the side-chain of Tyr273 in C3d is fullyexposed on the patch, the correspondingPhe1280 residue in C4Ad is completely buriedwithin the hydrophobic core of the molecule(Figure 4(a)).

The concave end of the a–a barrel of C4Ad

Shown in Figure 4(b) is a comparison of electro-static surface potential renditions of the concaveend of the C3d and C4Ad a–a barrels. Onceagain, despite the overall similarities in shape,there are very significant differences in the distri-bution of electrostatic potential. For example,whereas the central depression in C4Ad is fairlyneutral, in C3d there is significant negative electro-static potential for this area. The a–a barrel fold

seen in C3d and C4d is also found in the enzymemolecules endoglucanase,25 glucoamylase26 andthe b-subunit of the protein farnesyl transferase,27

none of which have any detectable sequence simi-larity with C3d or C4d, or for that matter, withone another. Interestingly, the depression on theconcave surface serves as the substrate-bindingsite in the case of all three enzymes. Partly for thisreason, and partly because the highly chargednature of the depression and rim of C3d was con-sistent with the known strong ionic strengthdependence of the CR2–C3d interaction,28 – 30 thissurface was proposed as a candidate site for theinteraction of C3d with CR2.24 Indeed, recentalanine scanning experiments of residues liningthe depression have identified two clusters ofresidues (denoted in Figure 4(b), left panel) thatare important for making at least a partialcontribution to the interaction with CR2.31 Of thetwo clusters, the 160s cluster appears to be the

Figure 4 (legend opposite)

1108 Crystal Structure of C4d

Figure 4. Molecular surface representations of four faces of the respective C3d and C4Ad molecules colored forelectrostatic potential (highly negative, red; highly positive, blue). (a) The convex face of C3d (left) and C4Ad (right).Conserved residues surrounding the thioester residues are labeled in both molecules. The putative domain interfacesin intact C3 and C4A that would respectively sequester the thioester from the solvent are denoted by the dotted lineboundaries. This domain interface is inferred from the sequence and charge conservation among diverse species C3and C4 molecules as discussed.24 (b) The concave faces of the structures of C3d (left) and C4Ad (right). Labeled arethe surface-exposed residues in C3d which according to mutagenesis data are involved in interactions with CR2.31

Acidic residues are labeled in yellow, basic residues in green and hydrophobic residues in khaki. Surface-exposedpolymorphic residues of C4Ad, which in this case represent the antigenic determinants for Ch1, Ch6 and Ch3 alloanti-bodies, are marked in white. (c) A comparison of C3d (left) and C4Ad (right) on a side face of the a–a barrel which inthe case of C3d has recently been implicated by a co-crystal structure report33 as forming the binding interface for CR2.The buried surface of the C3d–CR2 interface is indicated by the dotted line and the labeled residues within thevisualized interface are ones for which the backbone carbonyl oxygen atoms are involved in hydrogen bonding (exceptfor N170 where the side-chain contributes to interactions within the interface). The thioester region for each molecule isindicated for orientation purposes. (d) Molecular surface representations of C3d (left) and C4Ad (right) showing a sideview of the a–a barrel that represents a 1808 rotation from that shown in (c). Labeled are charged surface-exposedresidues. Arrows indicate the position of the 25-residue insertion in C4Ad, relative to the C3d amino acid residuesequence, that is missing in the structure and that contains within it S1217, which is the site of covalent attachmentof C3b in forming the classical pathway C5 convertase. All molecular surface images were produced using GRASP.45

Crystal Structure of C4d 1109

more vital as even single residue mutations of side-chains protruding into the solvent, for exampleD163A, were often sufficient for either iC3b orC3dg to lose more than 90% of their respectiveCR2-binding capacities. The absence of this nega-tively charged depression in C4Ad (Figure 4(b),right panel) would be consistent with an inabilityof C4d to bind CR2. Although there is literatureshowing that intact C4 cannot bind to CR2 underconditions where some binding of intact C3 isseen,32 to the best of our knowledge, C4d hasnever been directly assessed for this bindingactivity. We have now done this and have deter-mined that C4d lacks CR2-binding activity. Specifi-cally, recombinant C4d of either isotype atconcentrations up to 20 mM showed no measurableability to inhibit rosette formation between iC3b-coated red cells and CR2-bearing Raji cells,whereas C3dg at 1 mM gave .90% inhibition ofrosette formation (data not shown).

Recently our localization of a CR2-interactingsite on the concave face of the C3d a–a barrel hasbecome controversial as a structure derived from aC3d-CR2 co-crystal identifies a region on a sideface of the a–a barrel as providing all of the con-tacts with CR2.33 This side-face is located over thetop of the rim adjacent to C3d residue E166 asdepicted in Figure 4(b) (left). A comparison of thisface of the molecule for C3d and C4Ad, is pre-sented in Figure 4(c) and here too it can be seenthat the distribution of charges and neutral areasis quite different in the two molecules. However, amost unusual feature of the CR2–C3d interfacevisualized in the co-crystal structure is that, exceptfor a direct interaction with the side-chain of C3dN170 (equivalent to Q1147 in C4d), most of theother CR2 side-chain and main-chain contacts

with C3d are to its backbone carbonyl oxygenatoms. In view of the overall similarity in backboneconformation between C4d and C3d that we havenoted above, the backbone structure in this regionwas examined in greater detail with a view tofurther rationalizing the inability of C4d to bind toCR2. Figure 5 shows a superposition of thebackbone segments of C3d and C4Ad in the CR2-binding interface area as viewed from the CR2molecule. Although several backbone segments ofC3d overlap perfectly with the corresponding seg-ments in C4Ad, others do not. For example, theH3-H4 loop, most of H6 and the more C-terminalparts of H7 overlap well. By contrast, due to aninsertion, the N-terminal part of C4Ad helix H7 isextended relative to that of C3d (see also Figure2). The different starting points of this helix, P173and P1144 in the case of C3d and C4Ad, respect-ively (indicated by asterisks in Figure 5), results ina non-superposition of the side-chain of C4AdQ1147 with C3d N170. This misorientation of theQ1147 side-chain of C4Ad would preclude for thismolecule the single direct side-chain interactionthat is seen on the C3d side of the C3d–CR2 inter-face, an interaction that is suggested by muta-genesis data to be crucial for the C3d–CR2interaction to occur.33 Finally, the superposition ofthe C-terminal-most segments of the H5 helices isless than perfect due to the sharper turn in C4AdH5 around the position of C3d residues I115 andL116. The spatial misalignment of the backbonecarbonyl groups of C4Ad residues L1089, S1090and Q1091 relative to those of C3d I115, L116 andE117 would preclude them from forming anequivalent anion hole at the C terminus of H5that according to the C3d–CR2 co-crystalstructure makes seemingly crucial H-bonds with

Figure 5. Superposition of thebackbone representations of C3d(magenta) and C4Ad (gold) in theregion corresponding to the puta-tive CR2-binding interface of C3ddepicted in the left panel of Figure4(c). The view is from the perspec-tive of the contacting residues ofthe CR2 molecule.33 The side-chainsof C3d N170, which makes thesingle significant side-chain–side-chain contact with CR2, and thecorresponding Q1147 of C4Ad, arealso depicted. Asterisked residuesP173 and P1144 denote the startingpositions of helix 7 in C3d andC4Ad, respectively. The backbonecarbonyl oxygen atoms of C3dhelix 5 residues I115, L116 andE117, which collectively form theanion hole that interacts with apositively charged arginine side-chain (R84) of CR2, are indicated,as are those of their correspondingC4Ad residues L1089, S1090 andQ1091.

1110 Crystal Structure of C4d

the side-chain of CR2 R84. Thus there are clearlysufficient differences in backbone conformation topreclude C4Ad from forming a similar bindinginterface with CR2 to that which has been observedin the C3d–CR2 co-crystal structure.

Polymorphic sites in C4d

Shown in Figure 1(b) are the positions of theside-chains of several of the polymorphic residues,including D1054, S1157, T1182 A1188 and R1191.With the exception of D1054, which is located nearthe thioester residues on the convex side of themolecule, the other polymorphic residues areproximately located and accessible on the concavesurface of the molecule (Figure 4(b), right panel).Residues 1188 and 1191 have previously beenidentified as contributing the Ch1 (A1188, R1191)or Rg1 (V1188, L1191) epitopes, residue 1157 con-tributes to the Ch6 (S1157) or Rg2 (N1157) epitopeand the combination of S1157, A1188 and R1191contribute to the Ch3 epitope.34 In general the Rgantigenic determinants segregate with C4A allo-types and the major Ch determinants with C4Ballotypes, however, allotypes C4A1 and C4B5 areexceptions to this rule in that they display so-calledreversed Ch/Rg antigenicity. In our case, althoughthe isotypic residues (i.e. 1101–1106) are those ofC4A, due to the history of the construction of theparent cDNA pSVC4A,35 our C4Ad fragmentwould be devoid of Rg antigenic determinantsand possess the antigenic epitopes for Ch1, Ch6and Ch3.

Clearance of C4b-opsonized antigens ismediated by CR1 and there have been three reportsin the literature stating that C4A allotypes bindwith higher affinity to CR1 than do C4Ballotypes.21,36,37 This has been invoked as explain-ing in part the association reported by somegroups between deficiency states of C4A andSLE.16 – 19 The study by Reilly & Mold21 is the mostquantitative of the reports on CR1 binding andthese authors claim that cysteine cross-linkedcovalent dimers of C4Ab bound to human red cellCR1 with an , fourfold higher functional affinitythan did the equivalent dimers of C4Bb. If correct,these differences are likely to reflect differences inthe respective C4d regions, but as discussed in asection above, it is improbable that there will beconformational differences between C4Ad andC4Bd that could affect a region of the moleculeremote from the isotypic residues. The isotypicresidues per se are also unlikely to be in direct con-tact with CR1, as they are close to the site ofcovalent attachment to the target and thus theiraccessibility to a macromolecule the size of CR1 isimprobable. One possibility that cannot be dis-counted at present is that the CR1-bindingdifferences in fact reflect differences in contactwith the major Ch/Rg epitopes involving residues1157, 1188 and 1191 that normally segregate withthe isotypic 1101–1106 residues. In view of thequestions raised by the present study about

the basis for the reported isotype-dependentdifferences in the C4b–CR1 interaction, we are inthe process of re-examining this issue using surfaceplasmon resonance as our method of quantifyingthe binding interaction.

Acceptor site for covalent C3b binding

As indicated in Figure 1(b), a 24-residue loopspanning S1213–P1236 is missing from the C4Admodel, as no corresponding electron density wasvisible. The N-terminal part of this loop, specifi-cally at S1217, is a transacylation target for nascentC3b in the assembly of the classical pathway C5convertase (C3bC4bC2a).38 This indicates that thisloop segment, and quite likely this whole face ofthe C4d molecule, is accessible to the solventwithin the context of the parent C4b molecule.Figure 4(d) presents a view of this side of theC4Ad a–a barrel as an electrostatic surface poten-tial rendering as well as a comparison to the equiv-alent view of C3d. The position of the insertionloop relative to C3d is shown and, as was the casefor the other three surfaces that we have renderedin Figure 4(a)–(c), this surface also demonstratesstriking differences in electrostatic surface chargebetween the C3d and C4Ad molecules. The contig-uous negatively charged surface-exposed patch,located on the side of the C4Ad barrel just abovethe insertion loop containing the C3b transacyla-tion target site, may well be a site of interactionwith the positive charges exhibited on the convexsurface of C3d. This non-covalent interaction mayin turn pre-position the thioester for transacylationonto Ser1217 of the insertion loop. In this regard,it is known that the covalent bond linking C3b toC4b Ser1217 is dispensable with respect to themaintenance of a stable and functional C3bC4bheterodimer as a subunit of the classical pathwayC5 convertase.38

Concluding remarks

Both the backbone structural similarity and sur-face chemistry conservation of the polypeptidesegments surrounding the thioester-forming resi-dues in C3d and C4Ad, respectively, confirm theprevious suggestion24 that this region may reflectthe position of a domain interface in the nativestate of C3 and C4. This interface is likely involvedin protecting the thioester from solvent hydrolysisand, in the case of C3 or C4B, preventing nucleo-philic attack by the catalytic histidine residue. Asmentioned in Introduction, molecules expressingC3-like opsonic function appear to be evolutiona-rily quite ancient. For example, the mosquito thioe-ster-containing protein TEP-1, which was shown tobe a thioester-dependent opsonin for the engulf-ment of Gram-negative bacteria by a mosquitohemocyte-like cell line, was phylogeneticallyfound to cluster with other insect and nemotodethioester-containing proteins in a clade that wasintermediate between the a2-macroglobulin clade

Crystal Structure of C4d 1111

and the C3/C4/C5 clade.6 Supporting the notionof a conserved domain interface surrounding thethioester, the sequence of the mosquito TEP-1could be successfully homology modeled intothe human C3d structure and showed that thethioester residues, the catalytic histidine residueand the conserved surrounding residues of TEP-1 were clustered on the convex surface in amanner that was very similar to that of C3d,and now by extension, C4d. Moreover, the con-servation seen on the convex surface near thesite of covalent binding did not extend to thesurface charge distribution maps for the remain-der of the modeled TEP-1 domain as these werequite distinct from those of C3d and now alsoC4d. Thus while maintaining the chemicalcharacteristics of the domain interface requiredfor transacylaton function, the surface chemistryfeatures of parts of the molecule remote fromthe covalent attachment site have evolved toenable interactions with unique binding partners.For example C3d on its own interacts with SCRs1-2 of CR2,30 with SCRs 19-20 of factor H39 andwith factor B40. We are currently assessingwhether our recombinant C4d fragments ontheir own can interact with any of the knownbinding partners of C4b.

In summary, we have found that the ,30%sequence identity between C4d and C3d is in thiscase sufficient to yield a backbone fold for C4dthat is virtually superimposable upon that of C3d.By contrast, with the exception of a generally con-served patch surrounding the thioester-formingresidues, the surface chemistries of C4d and C3dare very distinct, an observation that is consistentwith the different spectrum of protein interactionpartners for C4b and C3b, respectively. The avail-ability of the C4d structural platform will nodoubt assist future C4 functional site mappingstudies.

Materials and Methods

Protein expression and purification

As indicated in Results and Discussion, we refer to thephysiologic factor I-generated C4d fragment (938–1317,mature C4 numbering) as C4dg and the N-terminallytruncated fragment (residues 983–1317) as C4d. Seg-ments of cDNA corresponding to C4dg and to C4d ofeach isotype were amplified by PCR using as templateplasmids pSVC4A and pSVC4B, respectively.35 Theprimers used for the PCR amplification ensured that the50 end of the amplified products possessed an in-frameNco I site and the 30 end possessed a stop codonimmediately following the codon for R1317 followed byan Nde I restriction site. Restriction with these enzymesallowed insertion of the various cDNAs into the bacterialexpression vector pET15b in a manner that eliminatesthe His-tag segment and adds only the amino acidresidue sequence MG to the N termini of the respectiveC4dg and C4d proteins. Protein expression was done intransformed Escherichia coli BL21(DE3) grown in LBwith ampicillin (100 mg/ml) and induced with 0.5 mM

IPTG when the culture had reached an A600 reading of0.5–0.7. For cells harboring the C4Adg and C4Bdgconstructs, the culture was continued post-induction foran additional three hours at 37 8C, whereas for cellsharboring the C4Ad and C4Bd constructs, the post-induction growth was at 25 8C for five hours.

Harvested bacterial pellets from each liter of culturewere resuspended in 40 ml of buffer A (10 mM sodiumphosphate (pH 7.1), 50 mM NaCl, 2 mM EDTA, 0.1 mMDTT), stock PMSF was added to 2 mM and the bacteriawere lyzed in a French Press operated at 1500–2000 psi.Following centrifugation at 8000g for 20 minutes, thecleared lysate was diluted twofold with buffer A lackingNaCl and then loaded onto a 100 ml column of DEAE-Sephacel (Amersham-Pharmacia, Baie D’Urfe, Quebec)equilibrated in buffer B (10 mM sodium phosphate (pH7.1), 25 mM NaCl, 2 mM EDTA, 0.1 mM DTT). Afterovernight washing at 4 8C in buffer B, the column waseluted with buffer B in which the NaCl concentrationhad been raised to 150 mM. SDS-PAGE analysis indi-cated that induced protein (be it C4d or C4dg) eluted inthe first major peak. The pooled fractions were dialyzedback into buffer B and loaded onto a Mono Q HR 10/10FPLC column (Amersham-Pharmacia). Elution was witha linear gradient from 25 mM to 190 mM NaCl in bufferB (2 ml/minute, 14 minutes gradient duration). C4dg orC4d of either isotype eluted as a major peak at approxi-mately 120 mM NaCl. Further purification was achievedby rerunning the pooled samples on the same column,but this time at pH 8.5. The composition of the loadingbuffer was the same as buffer A, except at pH 8.5, andthe column was eluted with a linear NaCl gradient inthis buffer to a limit of 500 mM (2 ml/minute, 40minutes gradient duration). The proteins of interesteluted at approximately 275 mM NaCl. C4Adg, C4Adand C4Bdg were considered pure at this stage, whereasto remove the remaining minor contaminants in theC4Bd preparation required a further chromatographicstep on Mono S HR 5/5 at pH 6.0. The loading bufferfor the Mono S column had the same composition asbuffer B, except at pH 6.0 and the gradient limit buffercontained 500 mM NaCl (0.5 ml/minute, gradientduration 30 minutes). C4Bd, now free of minor contami-nants, eluted at approximately 350 mM NaCl. Theidentity of each product was confirmed by seven to tencycles of Edman sequencing (Biotechnology ServiceCentre, University of Toronto) and except for lackingthe initiating methionine residue, the sequence corre-sponded to the anticipated N-terminal sequence encodedby the respective cDNAs. The various C4d fragmentswere also subjected to V8 protease (endoprotease Glu-C) digestion (0.25% (w/w), 15 mM sodium phosphatebuffer (pH 7.8), 30 8C, three hours), which in each caseyielded a limit fragment showing a small shift on SDS-PAGE. The digestion product was in each case purifiedby rechromatography on Mono Q HR 10/10 under thepH 8.5 conditions described above and the variousC4dg-V8 and C4d-V8 derivatives eluted at approxi-mately 350 mM NaCl. N-terminal amino acid residuesequencing of the C4Adg-V8 product yielded exactlythe same sequence as that of intact C4Adg, thus indicat-ing that the truncation was at the C terminus. On thebasis of the known cleavage specificity of V8 protease,the magnitude of the SDS-PAGE shift and a comparisonof the amino acid residue composition data for C4Adgand C4Adg-V8, the V8 cleavage would appear to be atE1304 or E1305. The purified proteins were treated with5 mM DTT in order to ensure that the thioester sequencecysteine was in the reduced state and the proteins were

1112 Crystal Structure of C4d

concentrated using Biomax Ultrafree-4 10 kDa cut-offspin concentrators (Millipore, Bedford, MA) to at least10 mg/ml for crystallization trials. In the course of con-centrating the proteins, the buffer was exchanged to10 mM Tris–HCl (pH 8.5), 10 mM NaCl, 2 mM EDTA,5 mM DTT. The various C4d derivatives behaved asmonomers on a calibrated Superdex-200 FPLC gel fil-tration column as long as they were maintained in DTT-containing buffers. However, if the reducing agent wasomitted, disulfide-linked dimers readily formed. Sincethis phenomenon occurred with C4d derivatives ofeither isotype, and since the thioester cysteine (C991) isthe only one present in C4B isotype derivatives, we con-cluded that disulfide-linked dimer formation occurredvia C991.

Crystallization and data collection

The various C4d fragments were submitted to crystal-lization trials using vapor diffusion techniques andemploying as trial precipitants Crystal Screen and Crys-tal Screen 2 (Hampton Research, Laguna Niguel, CA).All trials were carried out in the presence of 5 mM DTT,both in the sample and in the well solution. Crystalswere obtained only for the C4Adg-V8 variant. The crys-tals belong to the orthorhombic space group P212121

with cell dimensions: a ¼ 56:51 �A; b ¼ 71:43 �A; c ¼85:74 �A; a ¼ 90; b ¼ 90; g ¼ 90: For the initial structuredetermination, C4Adg-V8 crystals were grown at roomtemperature in 28% PEG 4000, 0.1 M Tris–HCl buffer(pH 7.5), 0.2 M MgCl2, 5 mM DTT. The hollow rod-shaped crystals were mounted in a glass capillary tubeand diffraction data were collected at room temperatureto a resolution of 2.3 A on a MAR-Research ImagingPlate (J. Hendricks and A. Lenfer, Hamburg) using aRigaku Rotaflex rotating anode as X-ray source, with acopper target and Osmic focussing optics (Osmic Inc.,Troy, Michigan). Diffraction data were processed usingDENZO and SCALEPACK.41 Data collection statisticsare summarized in Table 1A.

Structure determination

The structure of the C4Adg-V8 fragment was deter-mined by molecular replacement, using the coordinatesfrom the structure of the C3d fragment of complementcomponent C3 (PDB accession code 1C3D24), and refinedto an R factor of 21% and an R-free of 23% using the pro-gram CNS.42 The structure was traced using the programO.43 Refinement and geometry statistics are listed inTable 1B.

Binding assays

The ability of recombinant C4Ad and C4Bd to interactwith CR2 was assessed in a rosette inhibition assay.31 Inthis assay Raji B lymphoblastoid cells, on which CR2 isthe only complement receptor, were pre-incubated withvariable concentrations of recombinant C4Ad or C4Bdbefore being exposed to iC3b-bearing indicator red cellsfor rosette formation. Recombinant C3dg served as thepositive control for the ability to inhibit rosette formationwith a known ligand of CR2.

Atomic coordinates

Coordinates of the C4Ad structure have beendeposited in the Protein Data Bank under accessionnumber 1HZF.

Acknowledgements

This work was supported by Canadian Institutesof Health Research Grants MOP-7081 (D. E. I.) andMOP-36397 (D. R. R.).

References

1. Rawal, N. & Pangburn, M. K. (2001). Structure/function of C5 convertases of complement. Int.Immunopharmacol. 1, 415–422.

2. Krych-Goldberg, M. & Atkinson, J. P. (2001). Struc-ture–function relationships of complement receptortype 1. Immunol. Rev. 180, 112–122.

3. Law, S. K. A. & Dodds, A. W. (1997). The internalthioester and the covalent binding properties of thecomplement proteins C3 and C4. Protein Sci. 6,263–274.

4. Campbell, R. D., Dodds, A. W. & Porter, R. R. (1980).The binding of human complement component C4 toantibody–antigen aggregates. Biochem. J. 189, 67–80.

5. Dodds, A. W. & Law, S. K. A. (1998). The phylogenyand evolution of the thioester bond-containingproteins C3, C4 and a2-macroglobulin. Immunol. Rev.166, 15–26.

6. Levashina, E. A., Moita, L. F., Blandin, S., Vriend, G.,Lagueux, M. & Kafatos, F. C. (2001). Conserved roleof a complement-like protein in phagocytosisrevealed by dsRNA knockout in cultured cells of themosquito, Anopheles gambiae. Cell, 104, 709–718.

7. Blanchong, C. A., Chung, E. K., Rupert, K. L., Yang,Y., Yang, Z., Zhou, B. et al. (2001). Genetic, structuraland functional diversities of human complementcomponents C4A and C4B and their mouse homo-logues, Slp and C4. Int. Immunopharmacol. 1, 365–392.

8. Yu, C. K., Campbell, R. D. & Porter, R. R. (1988). Astructural model for the location of the Rodgers andChido antigenic determinants and their correlationwith the human complement component C4A/C4Bisotypes. Immunogenetics, 27, 399–405.

9. Isenman, D. E. & Young, J. R. (1984). The molecularbasis for the differences in the immune hemolysisactivity of the Chido and Rodgers isotypes ofhuman complement component C4. J. Immunol. 132,3019–3027.

10. Law, S. K. A., Dodds, A. W. & Porter, R. R. (1984). Acomparison of the properties of two classes, C4Aand C4B, of the human complement component C4.EMBO J. 3, 1819–1823.

11. Dodds, A. W., Ren, X-D., Willis, A. C. & Law, S. K. A.(1996). The reaction mechanism of the internal thio-ester in the human complement component C4.Nature, 379, 177–179.

12. Gadjeva, M., Dodds, A. W., Taniguchi-Sidle, A.,Willis, A. C., Isenman, D. E. & Law, S. K. A. (1998).The covalent binding reaction of complement com-ponent C3. J. Immunol. 161, 985–990.

Crystal Structure of C4d 1113

13. Hauptmann, G., Tappeiner, G. & Schifferli, J. A.(1988). Inherited deficiency of the fourth componentof human complement. Immunodefic. Rev. 1, 3–22.

14. Dragon-Durey, M.-A., Rougier, N., Clauvel, J. P.,Callat Zuchman, S., Remy, P., Guillevin, L. et al.(2001). Lack of evidence of a specific role for C4Adeficiency in determining disease susceptibilityamong C4-deficient patients with systemic lupuserythematosus (SLE). Clin. Exp. Immunol. 123,133–139.

15. Schur, P. H., Marcus-Bagley, D., Awdeh, Z., Yunis,E. J. & Alper, C. A. (1990). The effect of ethnicity onmajor histocompatibility complex complementallotypes and extended haplotypes in patients withsystemic lupus erythematosus. Arthritis Rheum. 33,985–992.

16. Atkinson, J. P. (1989). Complement deficiency: pre-disposing factor to autoimmune syndromes. Clin.Exp. Rheumatol. (Suppl.), 3, S95–101.

17. Kemp, M. E., Atkinson, J. P., Skanes, V. M., Levine,R. P. & Chaplin, D. D. (1987). Deletion of C4A genesin patients with systemic lupus erythematosus.Arhritis Rheum. 30, 1015–1022.

18. Howard, P. F., Hochberg, M. C., Bias, W. B., Arnett,F. C. & McLean, R. H. (1986). Relationship betweenC4 null genes, HLA-D region antigens, and geneticsusceptibility to systemic lupus erythematosus incaucasian and black Americans. Am. J. Med. 81,187–193.

19. Fielder, A., Walport, M., Batchelor, J., Rynes, R.,Black, C., Dodi, I. & Hughs, G. (1983). Family studyof the major histocompatibility complex in patientswith systemic lupus erythamatosus: importance ofnull alleles of C4A and C4B in determining diseasesusceptibility. Br. Med. J. 286, 425–428.

20. Schifferli, J. A., Hauptmann, G. & Paccaud, J. P.(1987). Complement-mediated adherence of immunecomplexes to human erythrocytes. Difference in therequirements for C4A and C4B. FEBS Letters, 213,415–418.

21. Reilly, B. D. & Mold, C. (1997). Quantitative analysisof C4Ab and C4Bb binding to the C3b/C4b receptor(CR1, CD35). Clin. Exp. Immunol. 110, 310–316.

22. Perkins, S. J. & Sim, R. B. (1986). Molecular modelingof human complement component C3 and its frag-ments by solution scattering. Eur. J. Biochem. 157,155–168.

23. Perkins, S. J., Nealis, A. S. & Sim, R. B. (1990). Mol-ecular modeling of human complement componentC4 and its fragments by X-ray and neutron solutionscattering. Biochemistry, 29, 1167–1175.

24. Nagar, B., Jones, R. G., Diefenbach, R. J., Isenman,D. E. & Rini, J. M. (1998). X-ray crystal structure ofC3d: a C3 fragment and ligand for complementreceptor 2. Science, 280, 1277–1281.

25. Aleshin, A., Golubev, A., Firsov, L. M. & Honzatko,R. B. (1992). Crystal structure of glucoamylase fromAspergillus awamori var X100 to 2.2-A resolution.J. Biol. Chem. 267, 19291–19298.

26. Alzari, P. M., Souchon, H. & Dominguez, R. (1996).The crystal structure of endoglucanase CelA, afamily 8 glycosyl hydrolase from Clostridiumthermocellum. Structure, 4, 265–275.

27. Park, H. W., Boduluri, S. R., Moomaw, J. F., Casey, P. J.& Beese, L. S. (1997). Crystal structure of proteinfarnesyltransferase at 2.25 angstrom resolution.Science, 275, 1800–1804.

28. Moore, M. D., DiScipio, R. G., Cooper, N. R. &Nemorrow, G. R. (1989). Hydrodynamic, electron

microscopic and ligand binding analysis of theEpstein-Barr virus/C3dg receptor (CR2). J. Biol.Chem. 264, 20576–20582.

29. Diefenbach, R. J. & Isenman, D. E. (1995). Mutationof residues in the C3dg region of human com-plement component C3 corresponding to a proposedbinding site for complement receptor type 2 (CR2,CD21) does not abolish binding of iC3b or C3dg toCR2. J. Immunol. 154, 2303–2320.

30. Guthridge, J. M., Rakstang, J. K., Young, K. A.,Hinshelwood, J., Aslam, M., Robertson, A. et al.(2001). Structural studies in solution of the recombi-nant N-terminal pair of short consensus/com-plement repeat domains of complement receptortype 2 (CR2/CD21) and interactions with its ligandC3dg. Biochemistry, 40, 5931–5941.

31. Clemenza, L. & Isenman, D. E. (2000). Structure-guided identification of C3d residues essential forits binding to complement receptor 2 (CD21).J. Immunol. 165, 3839–3848.

32. Ross, G. D. & Polley, M. J. (1975). Specificity ofhuman lymphocyte complement receptors. J. Exp.Med. 141, 1163–1180.

33. Szakonyi, G., Guthridge, J. M., Li, D., Young, K.,Holers, V. M. & Chen, X. S. (2001). Structure of com-plement receptor 2 in complex with its C3d ligand.Science, 292, 1725–1728.

34. Yu, C. Y., Campbell, R. D. & Porter, R. R. (1988). Astructural model for the location of the Rodgers andthe Chido antigenic determinants and their corre-lation with the human complement componentC4A/C4B isotypes. Immunogenetics, 27, 399–405.

35. Ebanks, R. O., Jaikaran, A. S. I., Carroll, M. C.,Anderson, M. J., Campbell, R. D. & Isenman, D. E.(1992). A single arginine to tryptophan interchangeat b-chain residue 458 of human complement com-ponent C4 accounts for the defect in classical path-way C5 convertase subunit activity of allotypeC4A6: implications for the location of a C5 bindingsite in C4. J. Immunol. 148, 2803–2811.

36. Gatenby, P. A., Barbosa, J. E. & Lachmann, P. J.(1990). Differences between C4A and C4B in thehandling of immune complexes: the enhancement ofCR1 binding is more important than the inhibitionof immunoprecipitation. Clin. Exp. Immunol. 79,158–163.

37. Gibb, A. L., Freeman, A. M., Smith, R. A., Edmonds,S. & Sim, E. (1993). The interaction of solublehuman complement receptor type 1 (sCR1,BRL55730) with human complement component C4.Biochim. Biophys. Acta, 1180, 313–320.

38. Kim, Y. U., Carroll, M. C., Isenman, D. E., Nonaka,M., Pramoonjago, P., Takeda, J. et al. (1992). Covalentbinding of C3b to C4b within the classical com-plement pathway C5 convertase. Determination ofamino acid residues involved in ester linkage for-mation. J. Biol. Chem. 267, 4171–4176.

39. Jokiranta, T. S., Hellwage, J., Koistinen, V., Zipfel, P. F.& Meri, S. (2000). Each of the three binding sites oncomplement factor H interacts with a distinct site onC3b. J. Biol. Chem. 275, 27657–27662.

40. Jokiranta, T. S., Westin, J., Nilsson, U. R., Nilsson, B.,Hellwage, J., Lofas, S., Gordon, D. L., Ekdahl, K. N.& Meri, S. (2001). Complement C3b interactions stu-died with surface plasmon resonance technique. Int.Immunopharmacol. 1, 495–506.

41. Otwinowski, Z. & Minor, W. (1997). Processing ofX-ray diffraction data collected in oscillation mode.Methods Enzymol. 276, 307–326.

1114 Crystal Structure of C4d

42. Brunger, A. T., Adams, P. D., Clore, G. M.,DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W.et al. (1998). Crystallography and NMR system: anew software suite for macromolecular structuredetermination. Acta Crystallog. sect. D, 54,905–921.

43. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard,M. (1991). Improved methods for building proteinmodels in electron density maps and the location of

errors in these models. Acta Crystallog. sect. A, 47,110–119.

44. Kraulis, P. (1991). MOLSCRIPT: a program to pro-duce both detailed and schematic plots of proteinstructures. J. Appl. Crystallog. 24, 946–950.

45. Nicholls, A., Sharp, K. A. & Honig, B. (1991). Proteinfolding and association: insights from the interfacialand thermodynamic properties of hydrocarbons.Proteins: Struct. Funct. Genet. 11, 281–296.

Edited by R. Huber

(Received 8 April 2002; received in revised form 29 July 2002; accepted 6 August 2002)

Crystal Structure of C4d 1115