Protein Engineering vol.9 no.12 pp.1143-1149, 1996

Molecular modeling and mechanism of action of human decay-accelerating factor

Lisa Kuttner-Kondo, M.Edward Medof,William Brodbeck and Menachem Shoham1*2

Institute of Pathology and 'Department of Biochemistry, Case WesternReserve University, 10900 Euclid Avenue, Cleveland, OH 44106-4935,USA

^ o whom correspondence should be addressed

A model of the regulatory region of human decay accelerat-ing factor (DAF) was built based on the known coordinatesof a fragment of the structurally and functionally homo-logous serum protein, factor H. According to this model,the four short consensus repeats (SCRs) in DAF arearranged in a helical fashion. A positively charged surfacearea on SCRs 2 and 3, two of the three repeating unitsessential for function, is postulated to be the primaryrecognition site for the C3 convertases C4b2a and C3bBb.This area encompasses a cavity on SCR 2, as well as partof the groove on the SCR 2-SCR 3 interface. Two additionalsurface depressions are centered around the C-terminaldisulfide bridges of SCRs 3 and 4. These are likely toprovide additional ligand binding sites. Based on this modelin conjunction with sequence homology to the Ba fragmentof factor B, a mechanism of DAF's accelerated convertasedecay action is postulated.Keywords: C3 convertase/complement/DAF/decay accelera-tion/homology model building

Introduction

Decay accelerating factor (DAF) is a membrane regulatoryprotein that functions to protect host cells from injury whichcould arise from autologous complement activation on theirsurfaces (reviewed in Nicholson-Weller and Wang, 1994). Itis widely distributed, i.e. expressed on all cell types that canpotentially come into contact with the complement system(Medof et ai, 1987b). It acts by dissociating the classical andalternative pathway C3 convertases, the central amplificationenzymes of the cascade. It is freely mobile in the surfacemembrane and can inactivate these bimolecular enzymes(C4b2a and C3bBb) wherever the deposition of autologousC4b or C3b activation fragments initiates their assembly(Medof et ai, 1984). A unique feature of DAF's activity isthat it functions only intrinsically (Medof et ai, 1984), i.e. itacts only on convertase complexes assembled on the samemembrane, not on those generated on external targets, e.g.pathogens.

DAF's regulatory function derives from four contiguous~60 amino acid-long repeating units termed short consensusrepeats (SCRs), which are suspended above the surface mem-brane by an O-linked polysaccharide-rich membrane-proximalcushion (Caras et ai, 1987; Medof et ai, 1987a). DAF isattached to the membrane by a glycoinositol phospholipidanchor (GPI; Davitz et ai, 1986; Medof et ai, 1986; Figure1). It resembles other C4b/C3b regulatory proteins, as well as

a number of other complement and non-complement proteinsthat are similarly composed of SCRs (reviewed in Reid et ai,1986). The SCRs in this family of proteins exhibit varyingdegrees of homology with each other.

Among the C4b/C3b regulatory proteins that share functionalproperties with DAF, the C4b/C3b receptor (CR1 or CD35) iscomposed of 30 such repeats (Wong et ai, 1989), the C3dgreceptor (CR2 or CD21) of 15 or 16 such repeats (Mooreet ai, 1987; Weis et ai, 1988), membrane cofactor protein(MCP or CD46) of four such repeats (Lublin et ai, 1987), C4binding protein (C4bp) of 48—64 such repeats (Chung et ai,1985) and serum factor H (pMH) of 20 such repeats (Kristensenet ai, 1986; Ripoche et ai, 1988). Structure-function studiesof these different C4b/C3b regulators have shown that theirfunctional activities in each case are contained within two tofour SCR units (Klickstein et ai, 1988; Kalli et ai, 1991;Krych et ai, 1991, 1994). Studies of DAF have shown thatits regulatory activity resides within SCRs 2-4 (Coyne et ai,1992) and that its activity against C4b2a is localized withinSCRs 2 and 3, whereas its activity against C3bBb is additionallydependent upon SCR 4 (Brodbeck et ai, 1996).

Only limited information is available concerning the tertiarystructure of these important control proteins, and little or noinformation is available concerning the manner in which theyinteract with and modify their respective complement ligands.No crystallographic structures have been elucidated. However,Barlow et ai (1992, 1993) and Norman et ai (1991) havedetermined the solution NMR structures of SCR 5, SCR 16and tandem SCRs 15 and 16 of factor H (FH15-16). Thesedata, in conjunction with structure-function data, particularlyon CR1, have suggested that in the cases of factor H, C4bpand CR1, two tandem SCR units function as a 'pocket' inwhich non-contiguous interactions of the ligand with eachSCR partner provide for binding.

Molecular modeling based on the known 3-D structure of arelated protein belonging to the same family is a widely usedtechnique for predicting tertiary protein structure and probablesites of ligand interaction. DAF is homologous to and sharesconvertase-dissociating activity with factor H, C4bp and CR1.Additionally, SCR structural units contain four conservedcysteine and conserved glycine, tryptophan and phenylalanine/tyrosine residues (Reid et ai, 1986) that [in part throughdisulfide bonding of the first and third and the second andfourth cysteine residues (Janatova et ai, 1989)] are thought toprovide a common backbone upon which different amino acidsare added allowing for specific functional activities.

Here the 3-D structure of the regulatory region of DAF hasbeen modeled using the previously derived solution structureof FH15-16 [Protein Data Bank (PDB) code 1HFH], andpotential sites of its interaction with C3bBb identified. Thismodel provides explanations for some of the known functionalproperties of DAF, including the requirement of SCR 4 in itsactivity against C3bBb (Brodbeck et ai, 1996) and itsheightened avidity for C3bBb compared with C3b or C3bB

© Oxford University Press 1143

by guest on July 14, 2011peds.oxfordjournals.org

Dow

nloaded from

L.Kuttner-Kondo et at

ShortConsensus-Repeats

CHO-N-

GPI Anchor —

Serine/ Threonine" Rich Region

Glycan

Phosphatidyiinositoi

Plasma Membrane

Lipid Groups

Fig. 1. Schematic representation of DAF, consisting of four SCRs, eachwith two disulfide bonds shown as vertical bars, an N-linked carbohydratemoiety between SCRs 1 and 2, labeled CHO-N-, a serine/threonine-richregion of extensive O-glycosylation and a GPI anchor attached to the cellmembrane.

(Pangburn, 1986). Using this model in conjunction withsequence data on factor B (Mole et al, 1984) and recentlyreported sequence data on guinea-pig DAF (Nonaka et al,1995), a homolog known to downregulate human C3bBb withefficiency comparable with that of human DAF, a molecularmechanism of the action of DAF is proposed.

Materials and methodsComputationsAll computations and molecular graphics work were carriedout on a Silicon Graphics Indigo2 XZ workstation runningunder the Irix 5.3 operating system.

Sequence alignmentSequence alignment was performed using programs ALIGNand MSA (Lipman et al, 1989; Gupta et al, 1995).Model buildingModel building was carried out using program O (Jones et al,1991). Residues were mutated from the factor H sequence tothe DAF sequence within O such that the new residue hadside-chain dihedral angles conforming to the most frequentlyfound rotamer in the rotamer library supplied with O. In caseof congestion of the mutated side chain with another moiety,a different rotamer was substituted from the library. Insertionswere modeled with command lego_loop by selecting suitableloops from a library of well determined structures within O.Conformations of residues flanking the insertion were keptintact. After each of the four SCRs was built independentlyfrom either SCR 15 or SCR 16 of FH15-16 they wereassembled in the following way. The structure of SCR 1 andSCR 2 was superimposed onto the structure of SCRs 15 and16, respectively, of FH15-16. Superposition was carried outusing program LSQKAB within the CCP4 suite of crystallo-

graphic programs (Bailey, 1994) by minimizing the r.m.s.deviations of corresponding backbone atoms between the twostructures. The ligation between SCRs 1 and 2 was carriedout in program O using the regularization routine refi as wellas the command lego_loop when necessary. In a subsequentstep, the dimer SCR 1-SCR 2 was superimposed onto FH15-16 such that SCR 2 overlapped SCR 15 of FH15-16. SCR 3was then superimposed onto SCR 16 of FH 15-16 and SCR 3was ligated to the C-terminus of SCR 2 of the previously builtSCR 1-SCR 2 dimer. The process was repeated until the entirepolypeptide chain of the four SCRs was built. In this way allinter-SCR linkages were built with the same bend as the onebetween SCRs 15 and 16 in FH15-16.

Energy refinement was carried out using program X-PLOR3.1 (Brtlnger, 1992). Quality assessment of the model wasperformed with program PROCHECK (Laskowski etal, 1993)within the CCP4 suite of crystallographic programs. Secondarystructure assignments and solvent accessibility calculationswere performed using program DSSP (Kabsch and Sander,1983).

Cavities on the surface of the protein were detected andcharacterized using program VOIDOO (Kleywegt and Jones,1994) from the Uppsala suite of crystallographic programsemploying a probe radius of 1.4 A.

Electrostatic surface potentials were calculated and displayedwith program GRASP (Nicholls et al, 1991).

Results and discussion

Model buildingOur molecular modeling of DAF involved the following steps.First, each of DAF's four SCRs was built individually fromthe more homologous SCR of FH15-16. Accordingly, DAFSCRs 1, 3 and 4 were built upon SCR 16, while DAF SCR 2was built upon SCR 15. Insertions in the sequence weremodeled to loops in between secondary structural elements.Side-chain dihedral angles were selected according to themost commonly found rotamer from a rotamer library unlessprevented by steric hindrance, in which case another rotamerwas selected.

Second, DAF's individual SCRs were ligated in the computerby superimposing each tandem pair of individual SCRs, inturn, onto the structure of FH15-16. In this way the bendbetween each pair of successive SCRs was built uniformlyaccording to die bend between SCRs 15 and 16 in FH15-16.

Third, the resultant model was subjected to regularizationof the stereochemistry followed by 160 cycles of energyrefinement with program X-PLOR 3.1 (Brtlnger, 1992). Thefinal model was stereochemically reasonable, possessing r.m.s.deviations from ideality of 0.01 A and 3.1° for bond lengthsand angles, respectively. In all, 98% of non-glycine and non-proline residues were found to be in allowed regions of theRamachandran diagram.

Finally, the regions involved in ligand binding were identifiedby scanning the surface of the model for unusual features suchas cavities and outstanding electrostatic potential. This wascarried out in conjunction with sequence comparisons withguinea-pig DAF, known to function on human C3 convertasesin a manner comparable with human DAF (Nicholson-Welleret al, 1981), and with the Ba fragment of factor B (Moleet al, 1984), known to cooperate with the Bb fragment inbinding to C3b (Hourcade et al, 1995).

The degree of sequence identity between each of DAF's

1144

by guest on July 14, 2011peds.oxfordjournals.org

Dow

nloaded from

Molecular modeling of human DAF

Fig. 2. Smoothed backbone representation of the DAF model in tube form.The eight disulfide bridges are shown as sticks, and cysteine residuenumbers are indicated. N- and C-termini are indicated by the letters N andC, respectively. The SCR domains are labeled SCR 1, SCR 2, SCR 3 andSCR 4. This figure was constructed using the program GRASP (Nichollsetal., 1991).

SCRs and FH15-16 ranges from 23 to 34%. Although thesesimilarities are not striking, we based our use of FH15-16 formodel building on the functional similarity of DAF to factorH, as well as conservation of the disulfide bridges whichshould provide for a common scaffolding of the SCRs of thetwo proteins. Validation of this approach is provided by theoutcome. As will be seen below, the resultant model allowsthe development of a reaction mechanism that would accountfor many of DAF's functional properties.

Description of the structureAccording to the model derived from the above procedures(Figure 2), the secondary structure of each of DAF's SCRsconsists of five P-strands in an up-and-down fashion formingan ellipsoid with a long axis of 31 A, a short axis of 12 Aand a third axis that varies, both between different SCRs andalong the major axis of each individual SCR, from 12 to 18A. The strands are roughly parallel or antiparallel to the majoraxis of the ellipsoid. Sheet-forming hydrogen bonds existbetween strands 2, 3, 4 and 5. Within each SCR, disulfidebonds connect strand 1 with strand 4 at the N-terminal poleand strand 3 with strand 5 at the C-terminal pole. The eightdisulfide bridges in DAF are between cysteine residues 2 and47, 31 and 60, 64 and 111, 95 and 124, 129 and 170, 156 and186, 191 and 233, and 219 and 249. The linker betweensuccessive SCRs is very short, comprising two or three residues

Fig. 3. Grooves and cavities on the DAF model. The backbone is shown ina ribbon representation with N- and C-termini labeled N and C,respectively. The SCR domains are labeled SCR 1, SCR 2, SCR 3 and SCR4. The locations of the grooves at each inter-SCR interface are denoted byellipses drawn in dotted lines. SCRs 2, 3 and 4 each contain one cavitycentered around the semi-transparent density shown in yellow. Residueslining the cavities and grooves are shown in a ball-and-stick representation.Cavities on the surface of the protein were detected and characterized usingthe program VOIDOO (Kleywegt and Jones, 1994) and a probe radius of1.4 A. This figure was constructed using the program O (Jones et al., 1991)and edited with the Silicon Graphics routine showcase.

only. As measured by the intersecting angle between straightlines drawn through (J-strands 5 and 1 of successive SCRs,the bend between successive SCRs is ~90°. Strands 2 and 4contain loops that vary in size amongst the different SCRs.

As depicted in Figure 2, according to the model, DAF'sfour SCRs are arranged in a helical fashion to form a moleculethat looks S-shaped viewed perpendicular to the helical axis.The 250 residue-long polypeptide chain is contained withinan imaginary box of overall dimensions 85X50X40 A.

The accessible surface area of DAF is 16 797 A2, ascalculated using program DSSP (Kabsch and Sander, 1983).This is a high number for a 250 residue-long protein, as aglobular protein of the same size would have a much smallersurface area. For example, carbonic anhydrase (PDB codelbcd), which is 11 residues longer than DAF, has an accessiblesurface area of 11 843 A2. Thus, as predicted by the derivedmodel, DAF has a 42% larger surface area than a comparablysized globular protein.Surface features of DAF and potential convertase bindingsitesTo ascertain which parts of DAF's surface could serve aspotential sites of interaction with the convertases, the model

1145

by guest on July 14, 2011peds.oxfordjournals.org

Dow

nloaded from

L.Kuttner-Kondo et at

SCR2SCR1 s c s ijovgiKcnsr--Ri-LotQnju.RYvrpRcrYFYPvrmi—

120 125b tbab

AV--1FOC

Fig. 4. Electrostatic surface potential of the DAF model as calculated anddisplayed using the program GRASP (Nicholls el al., 1991). The orientationof the molecule is the same as in Figure 2. Red and blue surfaces representelectrostatic potentials in the range -2 to 2 kB7", where kB is the Boltzmannconstant and T is the absolute temperature. Calculations were performed fordielectric constants of 80 and 2 for water and protein, respectively. SCRs 1and 4 are largely negatively charged. However, the most striking feature is apositively charged surface encompassing a large area on SCR 2, includingthe cavity on SCR 2 as well as part of the groove on the interface betweenSCRs 2 and 3. This surface includes the three consecutive lysine residues125-127. This area is postulated to be the major interaction site of DAFwith the convertases.

BuDAFQpDAF H TVL T DQO

KJDf IQ O I RDO T PA

QuUAFQpOAF

' R H SASA S

13

HL3SAS]P PT I 6A n

136 140bb c b)

0 LV U U TTEL L C Q F D P R T E A A M K UA 0 CZ 7*1

118 200 230 2 4 0 250• d • • • • * * « dd d •

3 1 (VLT KSH 3 I R0 VR V W I T E H V E

Fig. 5. Amino acid sequence of the four SCRs of guinea-pig DAF (GpDAF)compared with those of human DAF (HuDAF). GpDAF residues which arenot conserved are shown below the corresponding residue of HuDAF.Residues lining the grooves and cavities of the model are indicated bylower-case letters as follows: 'a', SCR 2 cavity; 'b' . groove between SCRs2 and 3; 'c', SCR 3 cavity: 'd', groove between SCRs 3 and 4; and 'e',SCR 4 cavity. Numbering begins with the first residue of the mature formof HuDAF.

12& 140 160 ISO 187"bb c b b c c c c c c c b cccdKKSC mPOK«HCQIt7VTGGI L - FOATT gnCWTQTILTQtnOTCLl SOB tVQMIDTUECR

TAICD

1S8

sousou

Fig. 6. Sequence alignment of SCRs 2-4 of human DAF and SCRs 1-3 ofthe Ba fragment of human factor B. While the overall sequence identity is28%, there are two highly conserved stretches with sequence identities of 39and 41% (shown in bold). These regions contain the proposed bindingsurfaces to the convertases. Residues located in the grooves and cavities areindicated as in Figure 5. Residues of the Ba fragment of factor B that havebeen implicated in binding to C3b are underlined (Hourcade et al, 1995).

was first inspected for any binding pockets on its surfaces. Asseen in Figures 3 and 4, three such grooves, located on theinterfaces between successive SCRs 1 and 2, 2 and 3, and 3and 4, could potentially serve as ligand binding sites. Of thethree, the groove between SCRs 2 and 3 is the most likelycandidate for a specific binding pocket (Figure 3) because itcontains a number of exposed hydrophobic residues, notablyleucine 147 and phenylalanine 148. As seen in Figure 3, theseresidues are part of a turn connecting strands 2 and 3 at theN-terminal pole of SCR 3 facing the inter-SCR groove, whilevaline 121 and another phenylalanine (F123) are on SCR 2on the opposite side of the groove. Another nearby exposedhydrophobic residue is leucine 171. The distances betweeneach of these hydrophobic side chains range between 6 and12 A, distances appropriate for hydrophobic ligand moietiesto sandwich in between. Three tandem lysine residues line thebottom of this groove: K125, K126 and K127 (Figure 3).Moreover, there is an additional exposed basic arginine residue(R100) nearby. As can be seen in Figure 4, adjacent to thisgroove there is a contiguous patch of positive electrostaticsurface potential that covers much of one side of SCR 2 aswell as some of SCR 3. As shown in Figure 5, all except oneof these basic as well as the exposed hydrophobic residues areeither conserved or conservatively replaced between humanand guinea-pig DAF (Nonaka et al., 1995), which shares fullfunctional homology with human DAF (Nicholson-Welleretai, 1981).

In contrast to the SCR 2-SCR 3 interface, the SCR 1-SCR2 interface (Figures 3 and 4) lacks an adjacent cluster ofpositively charged residues, and has only two pairs of phenyl-alanine and leucine residues, F35-L115 and F59-L112(Figure 3), which could serve as ligand binding sites. Inaddition, LI 12 is not conserved in guinea-pig DAF (Figure 5)where, instead, an arginine is present. In contrast to the SCR1-SCR 2 interface, the SCR 3-SCR 4 interface (Figure 3) hasa cluster of three carboxylates, D238, E239 and E241, on oneside of the groove and two arginine residues, R187 and R212,pointing towards each other on the other side of the groove(Figure 3). An analysis of Figure 5 shows that in guinea-pigDAF some of these residues are not conserved, where in placeof R187 and E239, an isoleucine and a valine, respectively,are present. Therefore these grooves are less likely to beimportant for function than the SCR 2-SCR 3 groove.

According to the model, in addition to the grooves on DAF'ssurface, there are three cavities that could participate in ligandbinding sites. As seen in Figure 3, two of these cavities arelocated at the C-terminal end of SCR 2 and the C-terminalend of SCR 3, in both instances around the disulfide bridge

1146

by guest on July 14, 2011peds.oxfordjournals.org

Dow

nloaded from

B

Molecular modeling of human DAF

cDa,

D

Fig. 7. Proposed mechanism of action. The model proposes that DAF and the Ba fragment of factor B share common binding sites on C3b (A). Followingcleavage of the Ba fragment from factor B by factor D, release of the Ba fragment from the C3bB complex, DAF binds C3bBb (B and C). The Bb fragmentis destabilized (D) and displaced. Once Bb is displaced, DAF is released from the complex (E).

(Figure 2). In both cases there is an exposed phenylalanineresidue at the cavity entrance (Figure 3). In the case of thecavity on SCR 2, F123 (which, as indicated above, is part ofthe groove on the SCR 2-SCR 3 interface) constitutes part ofthe opening to the cavity. This SCR 2 cavity is 8 A in diameterand is lined by atoms from residues L70, A73, Y93, C95,R101, E122, F123 and C124.

The cavity on SCR 3 near the interface with SCR 4 issimilar (Figure 3). This cavity is 10 A deep and 10 A wide,and is lined by atoms from residues 1135, F154, C156,LI62, F163 (the exposed phenylalanine in the SCR 3-SCR 4interface), G164, S165, T166, P184, E185 and C186. However,not all of this SCR 3 cavity may be important for DAF'sfunction because mutagenesis of one of the cavity residues,serine 165, to leucine abolishes adhesin binding to DAF butdoes not grossly abolish DAF's regulatory activity, althoughthe effect of the change has not been studied precisely (Nowicki

et ai, 1993). In the cases of both the SCR 2 and SCR 3cavities, the role of the phenylalanine residue exposed tothe groove deserves further investigation. As hydrophobicinteractions only act over short distances, it is possible thatthis residue could serve to pull a ligand moiety into the cavityonce the ligand is bound to the groove. As shown in Figure3, a third cavity is located on SCR 4. It is 10 A long andrather shallow, with a depth of ~3 A. This depression is linedby atoms from residues 1197, Y217, C219, M225, E228, H229,S230, 1231 and P247.

The conclusion from the above inspection of the model'ssurfaces is that one recognition site on DAF for the convertasesis likely to be the SCR 2-SCR 3 interface with a portion ofits SCR 2 attached cavity. Additional contacts could beprovided by the SCR 3-SCR 4 groove and its attached cavity,as well as by the shallow depression on SCR 4. A central rolefor the SCR 2-SCR 3 interface is consistent with observations

1147

by guest on July 14, 2011peds.oxfordjournals.org

Dow

nloaded from

L.Kuttner-Kondo et al

(Brodbeck et al, 1996) that DAF's regulatory activity for boththe classical and alternative pathway is dependent on SCRs 2and 3.

Sequence homology to factor B

DAF and the Ba fragment of factor B are structurally relatedin that Ba comprises three SCRs, as shown in Figure 6. Theamino acid sequences of the SCRs in the two proteins showhomology that varies along the chain. While the overallsequence identity of Ba SCRs 1-3 with DAF SCRs 2-A is28%, there are two highly conserved stretches with sequenceidentities ~40% (shown in bold). These stretches are locatedaround the cavity on SCR 3 and the shallow depression onSCR 4. As highlighted in the figure, residues 152-184 on SCR3 of DAF share 39% sequence identity with the correspondingsequence on SCR 2 of Ba. This stretch contains eight out of11 residues lining the cavity on SCR 3. Of the 11 cavityresidues, six are conserved, two are conservatively replaced(I135F and P184A) and three are not conserved.

A second region of striking sequence homology betweenDAF and Ba engulfs DAF residues 214—247 lining the shallowdepression on SCR 4. In this stretch the degree of identitywith the corresponding sequence on SCR 3 of Ba is 41%. Ofthe nine residues lining the shallow depression on DAF SCR4, four are conserved, four are conservative replacements(M225L, E228S, H229Q and S230R) and only one is notconserved.

With respect to the cavity on SCR 2, the smallest of thethree cavities on the model's surface, residues are conservedto a lesser degree. Of the eight residues present, four areconserved, one is a conservative replacement (A73V) andthree are not conserved. In addition, the degree of sequenceconservation in the region adjacent to this cavity (23% forresidues 70-124) is not as high as that for the correspondingregion on SCR 3 or 4. Thus, to provide for an interaction ofDAF with the alternative pathway C3 convertase that couldbe shared with Ba, the cavity on SCR 3 and the shallowdepression on SCR 4 would appear to be important. Thebinding regions on the SCR 2-SCR 3 interface and on theshallow SCR 4 depression are designated in Figure 6 by thelower-case letters 'b' and 'e', respectively.

Proposed mechanism of action of DAF

Based on the above molecular modeling and the propositionthat DAF and Ba share common binding sites on C3b, aproposed reaction scheme for DAF's mechanism of action isdepicted in Figure 7. According to this model, DAF bindsweakly to cell-bound C3b or C4b (Kinoshita et al, 1986) butis unable to bind to and dissociate C3bB (Fujita et al., 1987;Hourcade et al., 1996)._Following cleavage of the bound factorB zymogen by factor D and release of the Ba fragment fromthe complex, the binding sites on C3b for DAF are uncoveredand DAF binds to the C3bBb complex. The affinity of bindingto the C3bBb complex is higher than that to C3b alone(Pangburn, 1986) because of either conformational changes inC3b induced by the presence of Bb or the interaction of DAFwith Bb. In support of the latter alternative, recent neutronand X-ray solution scattering work suggests that Ba and Bbform contacts with each other (Chamberlain et al., 1996).Upon binding of DAF to the complex, binding of the Bbfragment to C3b is destabilized (Fujita et al, 1987) and Bb isdisplaced. Following release of the Bb fragment, the affinityof interaction between DAF and C3b decreases, and DAF

dissociates so that it is able to bind to and destabilize anotherC3 convertase complex on the same cell surface.

Some but not all of the currently available data concerningDAF's structure are consistent with the above proposed modeland reaction scheme. Preliminary imaging of the full-lengthDAF protein by atomic force microscopy has yielded anelongated shape with an approximate overall length:width ratioof 2.9 (Muzic.D., Raghavachari,M., Marchant.R., Medof.M.E.,unpublished results). Comparison of human and guinea-pigDAF sequences with a recently reported mouse DAF sequence(Spicer et al., 1995) shows conservation of the KKK residuesin the SCR 2-SCR 3 groove. Site-specific mutagenesis offactor B (Hourcade et al, 1995) has shown that factor Bmutations, which correspond to SCR 2 residues 120-123 inthe SCR 2-SCR 3 groove (Figures 3 and 4), decrease bindingto C3b by 44%. However, not explained by the model, someother factor B mutations, which do not correspond to any ofthe binding regions proposed by the model, also decreasebinding. Site-specific mutation of the key residues proposedin the above model and a precise analysis of the effects onDAF's function will be required for further testing of thevalidity of the proposed structure and reaction scheme.

AcknowledgementsThe authors thank Sara Cechner for preparation of the manuscript This workwas supported in part by grants AI23598 (M.E.M ) from the National Institutesof Health and DAAH04-95-1 -03% (M.S.) from the US Army Research Office.

ReferencesBailey.S. (1994) Ada Crystallogr., D50, 760-763.Barlow.P.N., Norman,D.G., SteinkassererA. Horne.TJ., PearceJ.,

Driscoll.P.C, Sim,R.B. and Campbell.I D. (1992) Biochemistry, 31, 3626-3634.

Barlow.PN., SteinkassererA, Norman.D.C, Kieffer.B., Wiles.A.P., Sim.R.B.and Campbell.I.D. (1993) J. Mol. Bioi, 232, 268-284.

Brodbeck.W.G., Liu.D., SperryJ, Mold.C. and Medof,M.E. (1996)J. Immunol, 156, 2528-2533.

BriingerAT. (1992) X-PLOR: A System for X-ray Crystallography and NMR.Version 3.1, Yale University Press, New Haven, CT.

Caras,I.W., Davitz.M.A., Rhee.L., Weddell.G., Martin.D.W.Jr andNussenzweig.V. (1987) Nature, 325, 545-549.

Chamberlain.D., HinshclwoodJ., Ullman.C.G., Smith.K.F., Sim.R.B andPerkins.S.J. (1996) Mol. Immunol, 33, 80.

Chung.L.P, GagnonJ. and Reid.K.B. (1985) Mol. Immunol, 22, 427^135.Coyne.K.E., Hall.S.E., Thompson.E.S., Arce.MA, KinoshitaJ., Fujita.T,

Anstee.D J., Rosse,W. and Lublin,D.M. (1992)7. Immunol, 149,2906-2913.Davitz,MA, Low,M.G. and Nussenzweig.V. (1986) J. Exp. Med., 163,

1150-1161.Fujita.T., Inoue.T., Ogawa,K., Iida.K. and Tamura,N. (1987) J. Exp. Med.,

166, 1221-1228.Gupta,S.K., KececiogluJ. and Schaffer.A.A. (1995) Lecture Notes Comput.

Sci., 937, 128-145.Hourcade,D.E., Wagner.L.M. and Oglesby.TJ. (1995) / Biol Chem., 270,

19716-19722.Hourcade,D., Medof.M.E., Brodbeck.W., Wagner.L.M. and Oglesby.TJ. (1996)

Mol Immunol, 33, 57.JanatovaJ., Reid,K.B.M. and WillisAC. (1989) Biochemistry, 28,4754-^761.Jones,T.A.,ZouJ.-Y.,Cowan,S.W.andKjeldgaard,M.(1991)/4c(aCo'i'a"osr,

A47, 110-119.Kabsch.W. and Sander.C. (1983) Biopolymers, 22, 2577-2637.Kalli.K.R., Hsu.P., Bartow.TJ., AheranJ.M., Matsumoto.A.K., Klickstein.L.B.

and Fearon.D.T. (1991) J. Exp. Med, 174, 1451-1460.KinoshitaJ., Medof,M.E. and Nussenzweig.V. (1986) J. Immunol, 136,

3390-3395.Kleywegt.GJ. and Jones.T.A. (1994) Ada Crystallogr., D50, 178-185.Klickstein.L.B., Bartow.T.J., Meletic.V., Rabson.L.D., SmithJ.A. and

Fearon.D.T. (1988) J. Exp. Med., 168, 1699-1717.Kristensen.T., Wetsel.R.A. and Tack.B.F. (1986) J. Immunol, 136, 3407-3411.Krych,M., Hourcade.D. and AtkinsonJ.P. (1991) Proc. Natl Acad. Sci. USA,

88, 4353-4357.

1H8

by guest on July 14, 2011peds.oxfordjournals.org

Dow

nloaded from

Molecular modeling or human DAF

Krych,M., ClemenzaJ-., Howdeshell.D., Hauhart.R., Hourcade,D. andAtkinsonJ P. (1994) / Biol. Chem., 269, 13273-13278.

Laskowski.R.A., MacArthur.M W., Moss£>.S. and ThomtonJ.M. (1993)/ Appl. Crystallogr., 26, 283-291.

Lipman.DJ., Altschul.S.F. and Kececioglu.J.D. (1989) Proc. Natl Acad Sci.USA, 86, 4412-4415.

Lublin.D.M., Lemons,R.S., Lc Beau,M.M., Holers.V.M , Tykocinski.M.L.,Medof.M.E. and AtkinsonJ.P. (1987) / Exp. Med., 165, 1731-1736.

Medof,M.E., Kinoshita,T. and Nussenzweig.V. (1984) / Exp. Med., 160,1558-1578.

Medof.M.E., Walter,E.I., Roberts.W.L., Haas,R. and Rosenberry,T.L. (1986)Biochemistry, 25, 6740-6747.

MedoCM.E., Lublin.D.M., Holers,V.M., Ayers.DJ., Getty.R.R., LeykanU.E,AtkinsonJ.P. and Tykocinski.M.L. (1987a) Proc. Natl Acad. Sci. USA, 84,2007-2011.

Medof,M.E., Walter.E.I., RutgersJ.L., Knowles.D.M. and Nussenzweig.V.(1987b) J Exp. Med., 165, 848-864.

MoleJ.E., Anderson J.K., Davison,E.A. and Woods.D.E. (1984)7. Biol. Chem.,259, 3407-3412.

Moore.M.D., Cooper.N.R., Tack.B F and Nemerow.G.R. (1987) Proc. NatlAcad. Sci. USA, 84, 9194-9198.

Nicholls.A., Sharp,K.A. and Honig.B. (1991) Proteins, 11, 281-296.Nicholson-Weller,A. and Wang.C.E. (1994) / Lab. Clin. Med., 123, 485-^91.Nicholson-Weller,A., BurgeJ. and Austen.K.F. (1981) / Immunol, 127,

2035-2039.Nonaka,M., Miwa,T., Okada,N., Nonaka,M. and Okada,H. (1995)/ Immunol.,

155, 3037-3048.Norman,D.G., Barlow.P.N., Baron,M., DayAJ., Sim.R.B. and Campbell.I.D.

(1991) / Mol. Biol., 219, 717-725.Nowicki.B., Hart,A., Coyne.K E., Lublin.D.M and Nowicki.S. (1993) J. Exp.

Med., 178,2115-2121.Pangburn,M.K. (1986) / Immunol., 136, 2216-2221.Reid.K.B.M, Bentley.D.R., Campbell,R.D., Chung.L.P., Sim,R.B.,

Christensen.T. and Tack.B.F. (1986) Immunol. Today, 7, 230-234.RipocheJ., Day,AJ., Harris.TJ.R. and Sira.R.B. (1988) Biochem. / , 249,

593-602.Spicer.A.P., Seldin.M.F. and Gendler.S.J. (1995) / Immunol, 155, 3079-3091.WeisJJ., Toothaker.L.E., Smith J. A., WeisJ.H. and Fearon.D.T. (1988) / Exp.

Med., 167, 1047-1066.Wong/W.W., CahillJ.M., Rosen.M.D., Kennedy.C.A., Bonaccio,E.T.,

Morris.MJ., WilsonJ.G., Klickstein.L.B. and Fearon.D.T. (1989) / Exp.Med, 169, 847-863.

Received May 29, 1996; revised August 5, 1996; accepted August 14, 1996

1149

by guest on July 14, 2011peds.oxfordjournals.org

Dow

nloaded from