Structural Analysis of Engineered Bb Fragment of Complement Factor B

Molecular Cell, Vol. 14, 17–28, April 9, 2004, Copyright 2004 by Cell Press

Structural Analysis of Engineered Bb Fragment of Complement Factor B: Insights into the Activation Mechanism of the Alternative Pathway C3-Convertase Karthe Ponnuraj,1 Yuanyuan Xu,2 Kevin Macon,1 Dwight Moore,1 John E. Volanakis,3 and Sthanam V.L. Narayana1,* 1 Center for Biophysical Sciences and Engineering School of Optometry 2 Division of Clinical Immunology and Rheumatology and 3 Department of Medicine University of Alabama at Birmingham Birmingham, Alabama 35294

Summary The C-terminal fragment, Bb, of factor B combines with C3b to form the pivotal C3-convertase, C3bBb, of alternative complement pathway. Bb consists of a von Willebrand factor type A (vWFA) domain that is structurally similar to the I domains of integrins and a serine protease (SP) domain that is in inactive conformation. The structure of the C3bBb complex would be important in deciphering the activation mechanism of the SP domain. However, C3bBb is labile and not amenable to X-ray diffraction studies. We engineered a disulfide bond in the vWFA domain of Bb homologous to that shown to lock I domains in active conformation. The crystal structures of BbC428-C435 and its inhibitor complexes reveal that the adoption of the “active” conformation by the vWFA domain is not sufficient to activate the C3-convertase catalytic apparatus and also provide insights into the possible mode of C3-convertase activation. Introduction The complement system is an important component of innate immunity; it participates in the regulation of adaptive immunity and mediates acute inflammatory and cytolytic reactions, playing a major role in host defense against invading pathogens. Activation of the alternative pathway culminates in the assembly of the C3-convertase, the protease that catalyzes the cleavage of the central complement protein C3. Formation of the C3-convertase is initiated by the association of C3b, a large multifunctional fragment of C3, with factor B. Binding to C3b induces conformational changes in factor B, which is then cleaved by factor D leading to the release of its N-terminal region, Ba (Figure 1). The resultant C3bBb bimolecular complex is the C3-convertase, the catalytic center of which is contributed by the Bb fragment. Large numbers of C3b generated by the C3-convertase deposit onto the activating microbial surfaces, marking them for immune clearance and/or lysis. Surface-bound C3b also initiates the generation of additional C3-convertase, thus amplifying complement activation. Regulation of C3bBb is achieved by its inherent *Correspondence: [email protected]

lability resulting in its dissociation, which is further promoted by regulatory proteins. Dissociated Bb is proteolytically inactive and cannot reassociate with C3b to form new C3-convertase (reviewed in Xu et al., 2001). Factor B is a modular chymotrypsin-like serine protease (SP) comprising an N-terminal region composed of three complement control protein (CCP) domains, followed by a von Willebrand factor type A (vWFA) domain and an SP domain. Activation of SPs typically occurs with the cleavage of an Arg peptide bond, resulting in the generation of a new charged N terminus. The latter is inserted into a hydrophobic region adjacent to the primary substrate binding site (S1) and forms an ion bond with the highly conserved Asp194 (chymotrypsinogen numbering) forcing the catalytic site to adopt its active conformation (Khan and James, 1998). The SP domain of factor B cannot follow this activation mechanism, since expression of its proteolytic activity is not associated with the formation of a free N terminus. Instead, the SP domain remains connected to the preceding vWFA domain, suggesting not only a novel activation mechanism but also the likely involvement of the vWFA domain in the activation process. The vWFA domain of factor B is structurally similar to the I domain of integrins, whose ligand binding is mediated by Mg2⫹ ions. Similarly, Mg2⫹ is essential for the formation of the active C3bBb complex, and the metal binding site is localized on the Bb segment (Fishelson et al., 1983). The crystal structure of the SP domain of factor B (Jing et al., 2000) revealed a pro-enzyme-like active site, further suggesting the possible involvement of the vWFA domain in the activation process. Crystal structures of integrin I domains have revealed two different conformations, the ligand-bound “open” conformation and the ligand-free “closed” conformation. The major difference between these two conformations is found at the C-terminal ␣7 helix, which upon ligand binding undergoes a 10 A˚ shift away from the ligand binding surface. This movement is associated with changes of the Mg2⫹ binding site (Emsley et al., 2000) and is thought to convey the “outside-in” signaling to the intracellular region of the receptor. The ligandbound conformational changes of the I domain could be reproduced by an engineered disulfide bond at the C-terminal region of the domain (Lu et al., 2001; Takagi et al., 2001; Shimaoka et al., 2002, 2003). In light of the above insights into the integrin I domains and their putative role in activation, we (Xu et al., 2001) and others (Hinshelwood and Perkins, 2000) proposed that binding of C3b to the Mg ion-dependent adhesion site (MIDAS) motif of the vWFA domain of Bb causes a shift of its C-terminal ␣7 helix to induce the active conformation of the SP domain. The C3bBb(Mg) complex (239 kDa) is large and inherently unstable, dissociating into C3b and inactive Bb with a half-life of 90 s at 37⬚C (Pangburn and MullerEberhard, 1986). Hence, it is not suitable for structural characterization. Given this constraint, we chose to adopt the innovative structural approach used to understand the activation mechanism of integrins and intro-

Molecular Cell 18

Figure 1. Cartoon Representation of C3-Convertase Formation C3b attached to an activating surface binds factor B, which undergoes conformational changes suitable for the cleavage by factor D, resulting in C3-convertase formation.

duced a pair of Cys residues in the vWFA domain of Bb to generate recombinant Bb in the putative ligandbound/open conformation (BbC428-C435). In this report, we present the crystal structure and functional features of BbC428-C435 and the structures of two BbC428-C435 inhibitor complexes. The results provide insights into the activation mechanism of cofactor-bound Bb.

Overall Structure of BbC428-C435 The three-dimensional structure of the BbC428-C435 molecule is shown in Figure 2A. The overall dimensions of the molecule are 90 ⫻ 50 ⫻ 50 A˚; it consists of two compact domains, namely vWFA and SP, and has the shape of a distorted dumbbell, a close match to its appearance by electron microscopy (Smith et al., 1984). The two domains are joined by a 13 residue (444-456) connecting segment, which forms a one-turn 310 helix and contains a Cys residue at position 453, disulfide bonded to Cys571 of the SP domain (Figure 2A). The vWFA domain contains 202 residues (242-443) comprising six ␣ helices, six ␤ strands, and four 310 helices. As has already been suggested on the basis of molecular modeling (Tuckwell et al., 1997), the domain adopts the dinucleotide binding or Rossman fold with a central ␤ sheet surrounded by ␣ helices. On the surface of the domain, opposite the face that interacts with the SP domain, a shallow crevice at the top of the ␤ sheet contains the MIDAS motif (Figure 2A). The two putative glycosylation sites at Asn260 and Asn353 are well exposed to the solvent, and a shapeless electron density extending from the side chain of Asn260 in the final difference map indicates that this residue is glycosylated; no such density is observed for Asn353. The C-terminal SP domain has 283 residues (457-739) and, like all other SPs, comprises twelve ␤ strands grouped into two ␤ sheets surrounded by ␣-helical and 310-helical segments. As noted previously, the core ␤ barrels of the SP domain are decorated by a number of new and unique structural motifs (Jing et al., 2000). The catalytic center is situated topologically at the opposite pole of the “distorted dumbbell” from the MIDAS site of the vWFA domain (Figure 2A).

Results and Discussion Engineering a Disulfide Bond in the vWFA Domain and Crystal Structure of the Mutant In ␣L integrin, the active conformation of the I domain could be stabilized by a single disulfide bond (Lu et al., 2001). To test the hypothesis that similar conformational changes can be induced in the vWFA domain of factor B by the introduction of a homologous disulfide bond and that, in turn, these changes can induce the active conformation of the catalytic center of the SP domain, we introduced a pair of Cys residues (C428-C435) at the corresponding positions (Figure 2A). In addition, a single Cys residue present in the vWFA domain at position 267 was mutated to Val to prevent unwanted disulfide bond formation and aggregation of the recombinant BbC428-C435. The crystal structure of BbC428-C435 was determined at a resolution of 2.0 A˚, and the initial phases were obtained by molecular replacement methods using the available crystal structure of the factor B SP domain (1DLE). After solvent flattening, the 2Fo ⫺ Fc map clearly revealed the electron density for the vWFA domain and allowed the tracing of more than 80% of its structure in the first round of model building. The current R factor/Rfree for the structure is 22.0/24.5 for all reflections observed at a resolution of 2.0 A˚. The statistics for the structure determination and refinement are summarized in Table 1.

Structure of the vWFA Domain The vWFA domain of BbC428-C435 adopts a conformation very similar to that of I domain structures. The most similar structure found by DALI (Holm and Sander, 1994) is the I domain of ␣M in open conformation (1IDO). The structural superposition (Lu, 2000) of vWFA domain of BbC428-C435 with 1IDO resulted in an rms deviation of 1.3 A˚ for 138 common C␣ atoms (Figure 3A). A similar comparison with the ␣2 I domain yielded an rms deviation of 1.5 A˚ for 133 common C␣ atoms in ligand-bound/open conformation (1DZI) and of 1.5 A˚ for 122 common C␣ atoms in ligand-free/closed conformation (1AOX). Two loops, ␣4-␤D and ␣5-␤E, located at the bottom of the vWFA domain opposite to the face that interacts with the SP domain exhibit major variations in both conformation and length compared to I-domains (Figure 3A). Similarly, there are some differences in the conformational disposition of certain ␣-helical segments. For example, the ␣2 helix of the ␣M I domain structure is replaced by a 310 helix in the vWFA domain of BbC428-C435 and by a random coil in the I domain of ␣2. Also, the ␣6 helix in the ␣2 I domain is extended further at the N-terminal end than that of the ␣M I domain and the BbC428-C435 vWFA domain. Most significantly, however, the position of the C-terminal ␣7 helix of the vWFA domain of BbC428-C435 is identical to that of the ␣7 helix of ␣2 and ␣M I domains in ligand-bound or “pseudo-liganded” (ligand mimetic

Structure of Engineered Bb Segment of Factor B 19

Figure 2. Structure and Functional Assessment of BbC428-C435 (A) Ribbon representation of BbC428-C435 crystal structure. The serine protease (SP) domain is represented in gold, the vWFA domain in cyan, and the linker joining them in deep blue. The small panel on the right depicts the engineered disulfide bond in the vWFA domain, and the one on left the disulfide bond joining the linker to the SP domain. (B) Three different views of electrostatic surface representation of BbC428-C435. Surface charge distribution was calculated using GRASP (Nicholls et al., 1991). Positively charged regions are represented in blue, negatively charged regions in red, and both polar and nonpolar regions are in white. BbC428-C435 shows three electropositive patches. Region 1 represents the L2 loop of SP domain, and regions 2 and 3 are at the intersection of SP and vWFA domains. (C) Binding of factor B and Bb to CVF. Factor B, factor B plus factor D, wt Bb, or BbC428-C435 was added to microwells precoated with CVF (10 ␮g/ ml) and incubated at 37⬚C for 2 hr. Bound Bb was detected by rabbit anti-Bb IgG and horseradish peroxidase-conjugated goat anti-rabbit IgG.

crystal lattice contact)/open conformation (Figures 3A and 3B). In the latter two structures the C-terminal ␣7 helix is shifted downward by about 10 A˚ compared to its position in the ligand-free/closed conformation. The

position of the ␣7 helix in BbC428-C435 indicates that the introduced disulfide bond induces conformational changes similar to those resulting from ligand binding to I domains and supports our hypothesis that the con-

Molecular Cell 20

Table 1. Crystallographic and Refinement Data for BbC428-C435, BbC428-C435-BCX583, and BbC428-C435-DIP Structures

Unit cell a, b, c (A˚) ␣, ␤, ␥ (⬚) Space group Resolution (A˚) Reflections total/unique I/␴ (I) Completeness (%) Rsyma (%) No. molecules in the asymmetric unit

BbC428-C435

BbC428-C435-BCX583

BbC428-C435-DIP

98.98, 98.98, 126.43 90, 90, 120 P61 2.0 273,770/47,020 26.7 99.4 6.1 1

98.36, 98.36, 125.66 90, 90, 120 P61 2.3 113,478/30,559 18.6 99.6 7.2 1

98.45, 98.45, 125.68 90, 90, 120 P61 2.6 92,682/21,183 16.9 99.4 9.0 1

20–2.0 22.0/24.5 35.15 0.006 1.26 3740 260 1 89.7/0.2 1RRK

20–2.3 20.8/24.4 30.91 0.007 1.26 3763 272 1 88.7/0.5 1RTK

20–2.6 21.2/25.1 34.49 0.007 1.26 3721 198 1 87.1/0.5 1RS0

Refinement Resolution range (A˚) R factor/Rfree (all data with 0 sigma cutoff ) Average B value (A˚2) Rmsd for bonds (A˚) Rmsd for angles (⬚) No. of protein atoms No. of water molecules No. of metal atoms Ramachandran plot (core/disallowed ) PDB code a

Rsym ⫽ ⌺|Ih ⫺ ⬍Ih⬎|/⌺Ih, where ⬍Ih⬎ is the average intensity over symmetry equivalents.

formation of the vWFA domain of BbC428-C435 is comparable to that in C3b-bound Bb. The MIDAS Site The BbC428-C435 crystals were grown in solutions lacking added divalent cations. However, the finding of an electron density with octahedral coordination geometry in the MIDAS site suggested the presence of a bound metal ion. The bound metal atom was probably Co2⫹, since it exhibited an average bond length of 2.37 A˚, a distance that compares well with that reported for Co2⫹ (2.3 A˚) in the ␣2 I domain structure (Emsley et al., 2000). In contrast, the average bond length for Mg2⫹ would be expected to be 2.1 A˚. Co2⫹ and Ni2⫹ have been shown to be suitable substitutes for Mg2⫹ in C3-convertase assembly and function (Fishelson and Muller-Eberhard, 1982; Acevedo and Vesterberg, 2003). Residues Asp251, Ser253, Ser255, Thr328, and Asp364 constitute the MIDAS binding site of the vWFA domain. Side chain hydroxyl oxygen atoms of Ser253, Ser255, and Thr328 and three water molecules complete the six coordinating ligands of Co2⫹. Asp251, Ser253, and Ser255 represent the conserved sequence motif DXSXS on the ␤A-␣1 loop, one of the three loops that surround the MIDAS motif. Two other loops, ␣3-␣4 and ␤D-␣5, donate Thr328 and Asp364, respectively. The two aspartates, Asp251 and Asp364, do not coordinate the metal ion directly but serve as a secondary coordination sphere. However, one of the carboxylate oxygens of Asp251 and one of Asp364 make hydrogen bonds to a common water molecule, which is coordinated to Co2⫹. The other carboxylate oxygen of Asp251 and Asp364 make hydrogen bonds to Ser253 and Ser255, respectively. These MIDAS site features of the vWFA domain are identical to those of the ligandbound/open I domain of ␣2 (Emsley et al., 2000) and pseudo-liganded/open I domain of ␣M (Lee et al., 1995) with the only difference coming from the coordination

of the sixth ligand. In BbC428-C435 a water molecule occupies the sixth coordination site of Co2⫹, whereas in ␣M that site is occupied by a carboxylate oxygen of a glutamate from the neighboring molecule in the crystal lattice and in the ␣2 I domain by a carboxylate oxygen from the glutamate of the collagen ligand (Figure 3C). Of the four recently determined crystal structures of ␣L I-domains with engineered disulfide bonds (Shimaoka et al., 2003), two represent high-affinity ligandbound/open conformations while the other two have only intermediate affinity. Among the two high affinity structures, one is pseudo liganded and the other one is ligand free. Similarly, one of the two intermediate affinity structures represents ligand free and the other one is in complex with the ligand intercellular adhesion molecule-1 (ICAM-1). In both the ␣L-ICAM-1 complex and the pseudo-liganded structures, two Ser, one Thr, and two water molecules participate in the octahedral coordination of the metal ion at the MIDAS site. In the ICAM-1 complex structure, the sixth coordination is from the Glu residue of the ICAM-1 molecule, while in the pseudoliganded structure it is from the Glu residue of the neighboring molecule in the crystal lattice. Thus, all MIDAS features of liganded and pseudo-liganded ␣L mutant structures are identical to those observed for the open conformation of the I domains of ␣2 (Emsley et al., 2000) and ␣M (Lee et al., 1995) (Figure 3C). These findings clearly demonstrate that the MIDAS coordination of the vWFA of BbC428-C435 and of the mutant ␣L I domain differ only in the sixth coordination with metal ion, as observed in the comparison with the I domains of ␣2 and ␣M. The question whether the I domain in the open conformation could be stabilized in the absence of ligand was answered in the affirmative by the crystal structure determination of the “disulfide-locked open” ␣L I domain (Shimaoka et al., 2003), since the previous two (␣M and ␣2) open conformation structures were determined with the ligand-like lattice contact or bound ligand (Lee et

Structure of Engineered Bb Segment of Factor B 21

Figure 3. Structure and Comparison of the vWFA Domain of BbC428-C435 with Integrin I Domain Structures (A) Overall comparison of vWFA domain of BbC428-C435 (cyan) with integrin I domain open conformation structures ␣2 (yellow) and ␣M (blue). The two disordered regions of BbC428-C435 are indicated by dotted lines. (B) (Top left) Superposition of ␣7 helix in ligand-bound/open (yellow) and ligand-free/closed (red) crystal structures of ␣2 I domain. (Top right) Superposition of Bb-vWFA domain ␣7 helix (cyan) with the corresponding helix of ligand-bound/open ␣2 I domain (yellow). (Bottom left) Comparison of MIDAS site residues observed in ligand-bound/open (yellow) and ligand-free/closed (red) crystal structures of ␣2 I domain. (Bottom right) Superposition of MIDAS site residues of BbC428-C435-vWFA domain (cyan; residue numbers indicated by asterisk) and ligandbound/open ␣2 I domain (yellow). For clarity, the water molecules that interact with the metal atom (M) are not included in the bottom left and right panels. (C) Detailed comparison of MIDAS structures of BbC428-C435 (top left), ␣2 I-collagen complex (top right), ␣L I–ICAM-1 complex (bottom left), and pseudo-liganded ␣L I domain (bottom right). The metal atom is shown as a magenta ball. Black solid and dotted lines indicate the metal coordination and hydrogen bonds, respectively. Residue E in the top right and bottom left panels corresponds to the glutamate of collagen and ICAM-1 molecules, respectively. In the bottom right panel residue E corresponds to the neighboring I domain.

Molecular Cell 22

al., 1995; Emsley et al., 2000). The open conformation observed for the vWFA domain in BbC428-C435 also supports the above observation. The combined data further support our hypothesis that the BbC428-C435 structure, specifically the vWFA module, bears similarity to C3bbound Bb. Structure of the SP Domain The SP domain of BbC428-C435 adopts a structural fold identical to that of the isolated SP domain of factor B (Jing et al., 2000). When the two structures are superposed, the observed rms deviation is 0.7 A˚ for 170 common C␣ atoms. Perona and Craik (1997) defined eight conserved loops of the chymotrypsin fold that determine the geometry of the S1 pocket and subsite specificity. Of these, loops D and E are substantially shorter in BbC428-C435 with 7 and 9 fewer residues, respectively, while loops A, B, and C as well as L1, L2, and L3 are longer. Also, the highly conserved loop 16 (Jing et al., 2000) at the N-terminal region is deleted in BbC428-C435. Interestingly, loop 16 and the shortened loops (D and E) are located toward the SP-vWFA interface while the extended loops A, B, C, L1, L2, and L3 are distributed around the S1 pocket (Figure 4A). In addition, a unique 20 residue loop segment is inserted in the middle of the molecule, adopting a helix-loop-helix conformation with a disulfide bond linking the N-terminal ends of the two helices. Arg583 located on one of these helices makes a salt bridge with Glu273 of the neighboring vWFA domain. As in the isolated SP crystal structure, no electron density is present for residues 701(220a) to 708(220h) (corresponding chymotrypsinogen numbering is given in parentheses to facilitate comparisons) in the L2 loop. The other major difference between the SP domain of BbC428-C435 and typical SP structures is obviously at the N-terminal end. The N-terminal end of Bb-SP is connected to the vWFA domain through a 13 residue linker, while in most other SPs a free NH3⫹ terminus forms an ion pair with the carboxylate side chain of Asp194, forcing the 192-194 segment to adopt an active “oxyanion hole” conformation. In the SP domain of BbC428-C435 although the overall backbone conformation of the Asn668(189)-Asp673(194) segment matches the corresponding segments of activated serine proteases (Figure 4B), it is still considered as zymogen due to the inward orientation of the carbonyl oxygen of Arg671(192) and its interactions with the hydroxyl group of Ser674(195). As a result, the Arg671(192)-Asp673(194) segment adopts a 310 helix conformation (Figure 4C). Because of the immature 310 helix conformation of the oxyanion hole, the active center exhibits a strikingly different charge distribution than that of other SPs. In contrast to the inactive conformation of the oxyanion hole, the catalytic triad residues Asp551(102), His501(57), and Ser674(195) display typical active site disposition. The S1 pocket is composed of three polypeptide segments: Asn668(189)-Ser674(195), Ser693(214)-Asp698(218a), and Arg714(225)-His717(228). Notable differences from SPs with trypsin-like specificity occur at the bottom of the S1 pocket, where Asn668(189) and Asp715(226) replace the highly conserved Asp and Gly, respectively (Figure 4D). A loss of crucial negative charge in the S1 pocket was anticipated due to the presence of Asn instead Asp at the 189 position. However, using site-directed mutagenesis,

we have previously shown that this loss is compensated by the presence of an Asp at the 226 position, which is the primary structural determinant for P1-Arg binding and catalysis (Xu et al., 2000). Domain Interface The buried surface area at the interface between the vWFA and SP domains was calculated to be 1280 A˚2. For this analysis the linker residues Ile444–Ser449 were included in the vWFA domain and Leu450–Val456 in the SP domain. The interface is constituted by a total of 22 residues from both domains, half of which are charged. The residues of the two domains make 29 contacts measuring less than 4 A˚, including three salt bridges and ten hydrogen bonds. Specifically the ␣7 helix of the vWFA domain, which is situated at the domain interface, makes five hydrogen bonds and one salt bridge with the 458-462 loop segment of the SP domain. In the isolated SP structure this segment is disordered presumably as a result of lack of interactions with the ␣7 helix. Functional Assessment of BbC428-C435 We next examined the impact of the engineered disulfide bond on cofactor binding and catalytic activity against C3 and C3-like esters. The results demonstrated about 3-fold increase of BbC428-C435 binding to the homolog of C3b, cobra venom factor (CVF) as compared to wt Bb (Figure 2C). Compared to intact wt factor B, binding of BbC428-C435 increased about 20-fold, despite the loss of the Ba binding site. However, compared to wt Bb produced by factor D cleavage of CVF-bound factor B, BbC428-C435 bound about 24-fold less efficiently. This result is in sharp contrast to the dramatic increase in ligand binding affinity displayed by ␣L integrin locked in the “active” form by a disulfide bond (Lu et al., 2001, Shimaoka et al., 2001). The difference between integrin I domain and factor B vWFA domain cannot be readily explained by available data, but it seems reasonable to suggest that the conformational change of the Bb binding site that is triggered by factor D cleavage of CVFbound factor B is more extensive than indicated by the structure of BbC428-C435. This hypothesis is supported by the observed 500-fold increased binding of newly formed Bb in the presence of cofactor over factor B, as compared to the 20-fold increased binding of BbC428-C435, despite the open-like conformation of its MIDAS motif. It would thus appear that there are at least four conformational states of the Bb binding site: a low-affinity state in intact factor B, a high-affinity one in freshly formed cofactor-bound Bb, and intermediate-affinity states in BbC428-C435 and Bb. This implies that a critical component of the “high-affinity” conformation is perhaps metastable, requiring direct contact with the cofactor to be stabilized. The rapid spontaneous dissociation of the C3bBb complex suggests further that even in the presence of cofactor the high-affinity state of the binding site has a short half-life, a property not shared by the integrin I domains but important for the regulation of the proteolytic activity of the convertase. Additional experiments supported this interpretation as BbC428-C435 like wt Bb failed to cleave C3 under conditions favoring the cleavage of C3 by the C3-convertase (data not shown). Fi-

Structure of Engineered Bb Segment of Factor B 23

Figure 4. Structure of BbC428-C435-SP Domain (A) Stereo diagram of BbC428-C435-SP domain showing the surface loops. (B) Superposition of the 668(189)-673(194) segment of BbC428-C435 (gold) with the corresponding regions of active serine proteases trypsin (dark gray, 1TRN), protein C (red, 1AUT), and factor Xa (green, 1FAX). The N-terminal ends of all these molecules (except BbC428-C435) are also shown. (C) Stereo diagram showing the detailed comparison of the C670(191)-G675(196) segment of BbC428-C435 with the corresponding segment of trypsin. The boxed area indicates the orientational difference of the carbonyl oxygen of residue 671(192). (D) Stereo diagram showing the superposition of S1 pocket of BbC428-C435 and trypsin.

nally, BbC428-C435 and wt Bb displayed equivalent catalytic efficiencies against the C3-like thioester substrate BocGly-Leu-Ala-Arg-SBzl (Table 2). These results are consistent with the finding of inactive conformation of the active center in BbC428-C435 (Figure 4C). The combined results strongly indicate that the downward shift of the ␣7 helix presumably induced by the cofactor C3b or the C428-C435 disulfide bond is not transmitted to the SP domain and does not trigger the conformational change(s) required for activation of the zymogen-like oxyanion hole.

Table 2. Hydrolysis of C3-like Thioester by Recombinant Wt and Mutant Bb Fragment Boc-Gly-Leu-Ala-Arg-SBzl Protein

kcat (s⫺1)

KM (mM)

kcat/KM (M⫺1s⫺1)

Wild-type Bb BbC428-C435

0.65 0.28

5.63 2.75

115.0 99.6

Surface Charge Distribution of BbC428-C435 and Possible C3/C3b Binding Sites Taniguchi-Sidle and Isenman (1994) suggested the N-terminal region of the ␣⬘ chain of C3b, which contains the negatively charged residues Asp730-Glu731-Asp732 and Glu736-Glu737, as the binding site for factor B and complement receptor I. Because the latter protein is made up almost entirely of CCP domains, it seems likely that the Ba region of factor B which is also made up of CCP domains binds to that site of C3b. The surface charge distribution of BbC428-C435 shows that there are three electropositive patches that also might interact with the electronegative ␣⬘/␣ chain region of C3b or C3 (Figure 2B). The first electropositive region is the part of the L2 loop, residues 701-708(220a-220h) that defines part of the S1 pocket. This loop contains a number of positively charged residues most suitably positioned to interact with the negatively charged N terminus of cofactor C3b or of substrate C3. The other two regions, located at the intersection of the two domains of Bb, are exposed

Molecular Cell 24

to the solvent and might also be accessible to the C3/ C3b electronegative region. Structure of the BbC428-C435-BCX583 Inhibitor Complex BCX583 (6-amidino-2-naphthyl-4-guanidinobenzoate), same as Nafamostat (Fujii and Hitomi, 1981), is an active site inhibitor of factor B with IC50 value of 7.0 ␮M, in hemolytic assays (Narayana et al., 1999). Interaction of BCX583 with BbC428-C435 resulted in hydrolysis of the central ester bond of the inhibitor and covalent linkage of the guanidinobenzoate to Ser674(195) (Figure 5A). The overall structure of the BbC428-C435-BCX583 complex is very similar to that of native BbC428-C435 (rms deviation of 0.2 A˚ for 479 common C␣ atoms). The main difference between the two structures is found in the L2 loop (residues 696713 [217-224]) of the SP domain. Eight residues of the L2 loop (701-708 [220a-220h]) are disordered in both BbC428-C435 and isolated SP, showing no electron density. By comparison, this loop is more ordered in the BbC428-C435-BCX583 complex, with only three residues (701-703 [220a-220c]) not showing electron density. The BCX583 molecule fits very tightly into the S1 pocket of the SP domain, with its guanidinobenzoate moiety sandwiched between the two walls. This binding is similar to that observed in the structure of guanidinobenzoyl trypsin (Mangel et al., 1990). However, in guanidinobenzoyl trypsin, the terminal guanidino group points into the S1 pocket where the primary amine and imine nitrogens make hydrogen bonds with the side chain of Asp189, whereas in the BbC428-C435-BCX583 complex, the guanidino group points outward into the solvent, where the secondary amine nitrogen interacts with Asp715(226) through a water molecule. Due to this “outward” orientation of the guanidino group and its interactions with the L2 loop, the 696-700 (217-220) segment of the loop exhibits a downward shift of about 1 A˚ compared to uncomplexed BbC428-C435 (Figure 5A). At this position, the primary amine nitrogen of the guanidino group makes three close contacts at distances of 2.8, 3.5, and 3.3 A˚ with the backbone nitrogen and oxygen of Val697(218) and backbone nitrogen of Asp698(218a), respectively. In addition, both primary amine and imine nitrogens of BCX583 are also involved in water-mediated interactions with backbone oxygen atoms of Val696(217) and Gly695(216), respectively. Presumably, these interactions are responsible for stabilizing the L2 loop. The binding of the inhibitor in the S1 pocket had no effect on the zymogen-like conformation of the oxyanion hole. In the BbC428-C435 structure the O␥ atom of Ser674(195) forms hydrogen bonds with the backbone oxygen of Ser693(214), whereas in the BCX583 complex structure, upon acylation, the hydrogen bond is broken and the O␥ atom points downward forming a covalent bond with the C␦ atom of the inhibitor molecule. Instead of pointing toward the oxyanion hole as observed in the guanidinobenzoyl trypsin complex, the carbonyl oxygen of the inhibitor projects outward and interacts with the N⑀2 atom of the catalytic triad residue His501(57) and with the carboxylate side chain of Glu484(40), respectively (Figure 5A). One interesting aspect of this complex structure is the position of Arg705(220e) of the L2 loop. The side chain of this residue assumes an extended conformation and interacts with Asp673(194), occupying the space of the salt-

Figure 5. Structures of BbC428-C435 Inhibitor Complexes (A) Superposition of the active sites of BbC428-C435 (gold) and BbC428-C435BCX583 (light green) crystal structures. The inhibitor (BCX583) covalently linked to Ser195 is held in position by hydrogen bonding to Asp715(226) through a water molecule (lavender). The L2 loop (red) is seen only in the inhibitor complex structure. (B) Comparison of the active sites of BbC428-C435-DIP and BbC428-C435. The backbone of the inhibitor-bound BbC428-C435 is shown in light green color, while the inhibitor-free BbC428-C435 is shown in gold color. Covalently bound DIP molecule points its phosphoryl oxygen into the putative oxyanion hole (marked with black star) and forces it to acquire a tight ␤ turn from a zymogen-like 310 helix (transparent red color) conformation. (C) Stereo close-up of the superposition of the 670(191)-674(194) segment of BbC428-C435 (gold) and BbC428-C435-DIP complex (light green). The orientational difference of carbonyl oxygen of the residue 671(192) is indicated in the boxed area.

bridged N terminus of trypsin-like SPs (Figure 5A). Despite this contact, the oxyanion hole displays zymogenlike 310 helix conformation. The BbC428-C435-DIP Complex The overall structure of the BbC428-C435-di-isopropyl-phosphate (DIP) complex is quite similar to the crystal structures of BbC428-C435 and BbC428-C435-BCX583 with rms devia-

Structure of Engineered Bb Segment of Factor B 25

tion of 0.2 A˚ for 479 and 480 common C␣ atoms, respectively. The inhibitor, covalently bound to Ser674(195), has well-defined electron density. The side chain of Ser674(195) is pointed downward and the side chain conformations of the other two catalytic residues, Asp551(102) and His501(57), as well as the loop segments defining the S1 pocket, are similar to those observed in the BbC428-C435 structure. The significant differences are observed in the conformation of the oxyanion hole. As noted, the oxyanion hole of BbC428-C435 and BbC428-C435BCX583 complex have a zymogen-like 310 helix conformation due to the orientation of Arg671(192). In the BbC428-C435-DIP complex, however, the carbonyl oxygen of Arg671(192) is pushed outward, due to the orientation of the phosphoryl group of the covalently bound tetrahedral DIP molecule. This movement forces the Cys670(191)Asp673(193) segment to assume a tight ␤ turn conformation similar to that observed in all active serine proteases (Figures 5B and 5C). However, unlike the trypsin-DIP complex, where due to hydrolysis only one isopropyl group is present buried in the active site, both isopropyl groups are present in the BbC428-C435-DIP structure indicating inactivity of the catalytic apparatus. Neither of the isopropyl groups interacts with any residue in the BbC428-C435 S1 pocket; consequently, the L2 loop exhibits disorder similar to that observed in the BbC428-C435 structure. Implications for the Activation Mechanism of Cofactor-Bound Bb C3-convertases are the key enzymes of complement activation responsible for the production of protein fragments and complexes with biological activity. The C3-convertase of the alternative pathway is an effective protease with KM of 5.86 ⫻ 10⫺6 M and kcat/KM of 3.1 ⫻ 105 M⫺1s⫺1 measured for the cleavage of C3 (Pangburn and Muller-Eberhard, 1986). In contrast, uncomplexed Bb, which provides the catalytic site of the convertase expresses no or very low proteolytic activity against C3 (Fishelson and Muller-Eberhard, 1984). These findings indicate that the active state of the serine protease domain of Bb is induced by a mechanism which includes its association with C3b. Solution of the three-dimensional structure of the C3bBb complex would perhaps provide valuable information about the activation mechanism, but due to its lability, the complex is not readily amenable to characterization by X-ray diffraction or other biophysical methods. Considering the structural and functional similarity between the I domains of integrins and the vWFA domain of factor B, and the demonstration that introduction of a single disulfide bond at the C-terminal end of the I domain of ␣2, ␣L, and ␣M induces conformational changes identical to those observed in the ligand-bound domain (Lu et al., 2001; Takagi et al., 2001; Shimaoka et al., 2002, 2003), we substituted Cys residues at homologous positions in Bb and generated a recombinant mutant, BbC428-C435. The crystal structure of BbC428-C435 revealed that the disposition of the cofactor binding MIDAS site is identical to that of the ligand-bound I domain of ␣2 and ␣L (Figure 3C). Most significantly, the structural comparison also demonstrated that the ␣7 helix of the vWFA domain exactly matches the position of the downshifted ␣7 helix of ligand-bound I domains.

These structural features, compatible with an active conformation of the vWFA domain, were not reflected in the structure of the catalytic site of the SP domain or the function of BbC428-C435. A structural basis for this outcome is provided by the observation that the ␣7 helix ends at the backside of the serine protease domain and is distant from the catalytic center, including the oxyanion hole. In addition, the disulfide bond C453(1)-C571(122) at the N-terminal end of the SP domain is located immediately after the ␣7 helix (Figure 2A) and it would prevent the downward movement from being transmitted beyond this knot. Therefore, activation of the catalytic center does not appear to be induced by transmission of a signal from the vWFA to the SP domain through the 10 A˚ shift of the ␣7 helix and must be achieved by some other mechanism. We have proposed previously (Kam et al., 1987) that the active conformation of the catalytic center of factor D, the enzyme that converts the C3bB complex to the C3-convertase C3bBb, is induced by its single natural substrate C3b-bound factor B. This proposal has been supported by structural data demonstrating an inactive conformation of key elements of the catalytic center of factor D (Narayana et al., 1994; Jing et al., 1998) and by mutational (Kim et al., 1995) and functional data (Taylor et al., 1999). We now wish to propose the hypothesis that the active conformation of the catalytic center of C3bBb is also induced by its substrate C3, with the cofactor C3b providing crucial “docking” sites for proper positioning of C3. Accordingly, substrate-induced catalysis may represent a paradigm for alternative pathway activation. Our hypothesis is based on the finding of an inactive conformation of the catalytic site, specifically the oxyanion hole, of BbC428-C435 despite the active-like conformation of the vWFA domain. Crucial support for the hypothesis is also provided by a previous report indicating that the catalytic activity of the CVFBb complex for synthetic esters is low and equivalent to that of Bb (Ikari, et al., 1983). Apparently, small peptide esters used as substrates in esterolytic assays lack binding sites present on C3 and essential for inducing the putative conformational changes of the active center. Support for inducible conformational activation is also provided by the plasticity of the catalytic apparatus of factor B, evidenced by the structures of the BbC428-C435 inhibitor complexes. The plasticity of the active center of Bb is clearly demonstrated by the crystal structure of the BbC428-C435DIP complex, which provides a clear view of the reoriented ␤ turn conformation of the oxyanion hole instead of the zymogen-like 310 helical conformation observed in the uncomplexed structure (Figure 5C). The bound inhibitor mimics the acylated transition tetrahedral intermediate of the substrate with its free phosphoryl group pointing into the oxyanion hole, forcing it to adopt an active conformation. The crystal structure of the BbC428-C435-BCX583 complex revealed the position of the L2 loop, which in the BbC428-C435 structure is completely disordered. The complex structure also allowed us to suggest that the terminal guanidinium group of the P1-Arg of C3 can be properly positioned in the S1 pocket by interacting with Asp715(226) through a water molecule and residues in the L2 loop. In addition, the interactions between the inhibitor and the L2 loop make the S1 pocket slightly larger

Molecular Cell 26

in volume compared to that of BbC428-C435. We suggest that the inherent flexibility of the L2 loop facilitates the expansion of the S1 pocket and that enlargement of the pocket is necessary for accommodating the bulky side chain of the P1-Arg of C3. In conclusion, the present structure clearly demonstrates that the adoption of the open conformation of the vWFA domain does not have a direct role in the activation of the SP domain. This finding suggests two possible activation models. In the first model, in addition to the binding to the MIDAS motif, C3b also binds to the SP domain and assists in the activation process. In light of the suggested C3b binding site on the SP domain of factor B (Lambris and Muller-Eberhard, 1984), one might speculate that the acidic C3b ␣⬘ chain N terminus interacts with the positively charged L2 loop, facilitating the expansion and stabilization of the S1 pocket for accommodating the P1-Arg residue of the substrate. Such an interaction could explain the absolute requirement for the presence of C3b for expression of proteolytic activity by Bb. However, we favor a second model that proposes substrate-induced activation of the catalytic site. According to this model the L2 loop interacts with the acidic site on the ␣ chain of the substrate C3. Such an interaction would not only lead to enlargement of the S1 pocket, but it may also contribute to the proper positioning of the scissile P1-Arg bond, given its vicinity to the acidic residues. If that were the case, the absolute requirement for the association of Bb with C3b for expression of proteolytic activity could be attributed to the presence of binding or docking sites on C3b for substrate C3, which in turn interacts with Bb to induce the active conformation of the catalytic center. Experimental Procedures Construction and Expression of Recombinant Bb The cDNA sequence coding for the Bb fragment was amplified by PCR using the factor B cDNA BHL4-1 (Horiuchi et al., 1993) as template. The PCR product was subcloned into the transfer vector pAcGP67A (BD PharMingen, San Diego, CA). Two Cys residues were substituted for Phe and Asn at positions 428 and 435 of the vWFA domain by using the QuikChange Mutagenesis Kit (Stratagene, La Jolla, CA) as described previously (Xu et al., 2000). In addition, a single free Cys at position 267 of vWFA was mutated to Val to avoid undesirable disulfide bridging or aggregation of recombinant Bb. Coinfection of Sf9 insect cells with the wild-type or mutant Bb construct and the linearized baculovirus AcNPV DNA was carried out by following the manufacturer’s protocols. Recombinant Bb and BbC428-C435 were expressed as secreted soluble proteins consisting of a vector-derived tripeptide Ala-Asp-Pro at the N terminus and the C-terminal 498 residues of factor B. For large-scale production of recombinant BbC428-C435, High Five cells were infected with high titer of recombinant baculovirus DNA and cultured at 26⬚C in ExCell 401 serum-free media for 3–4 days, and the supernatant was used for purification of the recombinant protein.

Purification of Recombinant Bb Recombinant Bb was purified by ion exchange chromatography using a Bio-Rex 70 (BioRad, Hercules, CA) and a Mono S HR 10/ 10 column (Amersham/Pharmacia, Piscataway, NJ). The starting buffer used for both columns was 25 mM KH2PO4 (pH 6.8). Recombinant Bb and BbC428-C435 were eluted with a linear gradient to 1 M NaCl in starting buffer. In a final purification step, trace contaminants were removed by passing through a Sephacryl S-100 HR 16/60 exclusion column equilibrated in 25 mM PO4, 100 mM NaCl (pH 6.8).

Crystallization and Data Collection Crystallization of BbC428-C435 and two inhibitor complexes was carried out by the vapor-diffusion method using the hanging drop technique at 22⬚C. Initial crystallization trials were performed using various crystallization kits, and crystals were obtained from the PEG/Ion screen (Hampton Research). The best crystals were grown from 20% PEG MME 2000, 0.1 M NaI using 1–2 ␮l protein drops mixed with an equal volume of reservoir solution. For the BCX583 complex the protein was incubated with molar excess of inhibitor, dissolved in DMSO, overnight at room temperature. The synthesis and chemical modification reaction of BCX583 is based on the procedure reported previously (Fujii and Hitomi, 1981). The crystals of BbC428-C435-DIP complex were obtained from BbC428-C435 protein pretreated with DFP during the final stages of the purification process. Both native and complex crystals grew after 3–5 days and belonged to the hexagonal space group P61. The crystal volume per protein mass (Vm) was calculated to be 3.1 A˚3 Da⫺1 (Matthews, 1968), and the solvent content was 60%, which suggested the asymmetric unit contained one molecule of BbC428-C435. High-resolution 2.0 A˚ diffraction data for BbC428-C435 crystals were collected on a 3 ⫻ 3 mosaic CCD detector at the SBC-CAT 19 BM beamline of the Advance Photon Source at the Argonne National Laboratory. Prior to data collection, crystals were soaked for several seconds in a reservoir solution supplemented with 20%–25% ethylene glycol as a cryoprotectant and then flash-frozen in a nitrogen stream at 100 K using nylon loops. The data sets for inhibitor complexes were collected on a Rigaku R-Axis IV imaging-plate detector using CuK␣ radiation from an in-house Rigaku rotating-anode X-ray generator operating at 50 kV and 100 mA. All data sets were processed and scaled using programs DENZO and SCALEPACK (Otwinowski and Minor, 1997). X-ray diffraction statistics are summarized in Table 1.

Structure Determination, Model Building, and Refinement Initial phases for the BbC428-C435 data were obtained by molecular replacement using the factor B SP structure (1DLE) as a search model with the AMoRe program (Navaza, 1994). A clear solution was obtained in the cross rotation function and subsequent translation function which was subjected to rigid body refinement using the program package CNS (Brunger et al., 1998) and the 20–2 A˚ diffraction data. Examination of the resultant electron density map revealed extra density corresponding to the vWFA domain. Further phase improvement was performed using density modification by solvent flipping and density truncation without phase extension in CNS. The initial Fourier map calculated with the improved phases clearly revealed electron density for more than 80% of the vWFA domain structure. Chain tracing and model building were carried out with the program QUANTA2000 (Molecular Simulations, Inc). Several rounds of model building and refinement with the addition of water molecules at the final stage gave a final Rcryst of 0.22 and an Rfree of 0.245. The final model contains four disordered regions two each in vWFA (345-350; 390) and SP domains (479-480; 701-708). The structures of BbC428-C435-BCX583 and BbC428-C435-DIP complexes were solved by molecular replacement using the structure of BbC428-C435. No extensive model building and refinement were required for the complexes structures since both were very similar to BbC428-C435. Refinement statistics for all three structures are listed in Table 1. Structural superpositions were analyzed using the program TOP (Lu, 2000), and figures were generated using Ribbons (Carson, 1997).

Binding of Recombinant Bb to Cobra Venom Factor Binding of purified recombinant wild-type and BbC428-C435 was determined by enzyme-linked immunosorbent assay as described (Tuckwell et al., 1997). Microplate wells were precoated with CVF (Quidel) at 10 ␮g/ml overnight at 4⬚C. Serial dilutions of Bb starting at 10 ␮g/ ml in half-strength veronal-buffered saline (pH 7.4) containing 1% BSA, 1 mM MgCl2, were added to the CVF-coated wells and incubated at 37⬚C for 2 hr. Bound Bb was detected with rabbit antiBb IgG and goat anti-rabbit IgG alkaline phosphatase conjugate (Jackson Laboratories). Two additional controls, factor B and factor B plus factor D, were also tested under the same conditions. Results represent the mean ⫾ SD values of four separate experiments.

Structure of Engineered Bb Segment of Factor B 27

Esterolytic Activity of Recombinant Bb Kinetic constants for the hydrolysis of the thioester Boc-Gly-LeuAla-Arg-SBzl (Richman Chemical Inc.) were measured by the method described previously (Xu et al., 2000). Wt Bb or BbC428-C435 (0.11–0.38 ␮M) was incubated with 0.08 to 0.8 mM synthetic substrate in the presence of 1.6 mM 5,5-dithiobis-(2-nitrobenzonic acid) (Sigma). Rates of hydrolysis were measured kinetically for 15 min on a Vmax Microplate reader (Molecular Devices). Results represent the average values of two separate assays. Acknowledgments The project is funded by NIH grant (AI39818) and NASA Cooperative Agreement NCC8-246 to S.V.L.N. High-resolution diffraction data for this study were collected at Argonne National Laboratory, Structural Biology Center beamline 19-BM. We thank Dr. Y.S. Babu (BioCryst Pharmaceuticals, Inc) for the kind donation of the BCX583 compound. Thanks to Dr. Holly Jing (AstraZeneca, Boston) for useful discussions during the initial stages of this work. We thank MeeKyung Ahn-Oh and Zhihong Yu for technical assistance. Received: October 21, 2003 Revised: February 4, 2004 Accepted: February 13, 2004 Published: April 8, 2004 References Acevedo, F., and Vesterberg, O. (2003). Nickel and cobalt activate complement factor C3 faster than magnesium. Toxicology 185, 9–16. Brunger, A.T., Adams, P.D., Clore, G.M., DeLano, W.L., Gros, P., Grosse-Kunstleve, R.W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N.S., et al. (1998). Crystallography & NMR system: a new software suite for macromolecular structure determination. Acta Crystallogr. D Biol. Crystallogr. 54, 905–921. Carson, M. (1997). RIBBONS. Methods Enzymol. 277, 493–505. Emsley, J., Knight, C.G., Farndale, R.W., Barnes, M.J., and Liddington, R.C. (2000). Structural basis of collagen recognition by integrin ␣2␤1. Cell 101, 47–56. Fishelson, Z., and Muller-Eberhard, H.J. (1984). Residual hemolytic and proteolytic activity expressed by Bb after decay-dissociation of C3b,Bb. J. Immunol. 132, 1425–1429. Fishelson, Z., and Muller-Eberhard, H.J. (1982). C3 convertase of human complement: enhanced formation and stability of the enzyme generated with nickel instead of magnesium. J. Immunol. 129, 2603– 2607. Fishelson, Z., Pangburn, M.K., and Muller-Eberhard, H.J. (1983). C3-convertase of the alternative complement pathway. Demonstration of an active stable C3b.B(Ni) complex. J. Biol. Chem. 258, 7411– 7415. Fujii, S., and Hitomi, Y. (1981). New synthetic inhibitors of C1r, C1 esterase, thrombin, plasmin, kallikrein and trypsin. Biochim. Biophys. Acta 661, 342–345. Hinshelwood, J., and Perkins, S.J. (2000). Metal-dependent conformational changes in a recombinant vWF-A domain from human factor B: a solution study by circular dichrosim, fourier transform infrared and 1H NMR spectroscopy. J. Mol. Biol. 298, 135–147. Holm, L., and Sander, C. (1994). Searching protein structure database has come of age. Proteins 19, 165–173. Horiuchi, T., Kim, S., Matsumota, M., Watanabe, I., Fujita, S., and Volanakis, J.E. (1993). Human complement factor B: cDNA cloning, nucleotide sequencing, phenotypic conversion by site-directed mutagenesis and expression. Mol. Immunol. 30, 1587–1592. Ikari, N., Hitomi, Y., Niinobe, M., and Fujii, S. (1983). Studies on esterolytic activity of alternative complement component factor B. Biochim. Biophys. Acta 742, 318–323. Jing, H., Babu, Y.S., Moore, D., Kilpatrick, J.M., Liu, X.-Y., Volanakis, J.E., and Narayana, S.V.L. (1998). Structure of native and complexed complement factor D: implications of the atypical His57 conforma-

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Structure of the ␣L I domain and its complex with ICAM-1 reveal a shape-shifting pathway for integrin regulation. Cell 112, 99–111. Smith, C.A., Vogel, C.-W., and Muller-Eberhard, H.J. (1984). MHC class III products: an electron microscopic study of the C3 convertases of human complement. J. Exp. Med. 159, 324–329. Takagi, J., Erickson, H., and Springer, T.A. (2001). C-terminal opening mimics ‘inside-out’ activation of integrin ␣5␤1. Nat. Struct. Biol. 5, 412–416. Taniguchi-Sidle, A., and Isenman, D.E. (1994). Interactions of human complement component C3 with factor B and with complement receptors type 1 (CR3,CD35) and type 3 (CR3, CD11b/CD18) involve an acidic sequence at the N-terminus of the C3 ␣⬘-chain. J. Immunol. 153, 5285–5302. Taylor, F.R., Bixler, S.A., Budman, J.I., Wen, D., Karpusas, M., Ryan, S.T., Jaworski, G.J., Safari-Fard, A., Pollard, S., and Whitty, A. (1999). Induced fit activation mechanism of the exceptionally specific serine protease, complement factor D. Biochemistry 38, 2849–2859. Tuckwell, D.S., Xu, Y., Newham, P., Humphries, M.J., and Volanakis, J.E. (1997). Surface loops adjacent to the cation-binding site of the complement factor B von Willebrand factor type A module determine the C3b binding specificity. Biochemistry 36, 6605–6613. Xu, Y., Circolo, A., Jing, H., Wang, Y., Narayana, S.V.L., and Volanakis, J.E. (2000). Mutational analysis of the primary substrate specificity pocket of complement factor B. J. Biol. Chem. 275, 378–385. Xu, Y., Narayana, S.V.L., and Volanakis, J.E. (2001). Structural biology of the alternative pathway convertase. Immunol. Rev. 180, 123–135. Accession Numbers The sequences have been deposited in the Protein Data Bank under ID codes 1RRK for BbC428-C435, 1RTK for BbC428-C435-BCX583, and 1RS0 for BbC428-C435-DIP.