Crystal structure of a complement factor D mutant expressing enhanced catalytic activity.

Complement factor D is a serine protease regulated by a novel mechanism that depends on conformational changes rather than cleavage of a zymogen for expression of proteolytic activity. The conformational changes are presumed to be induced by the single natural substrate, C3bB, and to result in reversible reorientation of the catalytic center and of the substrate binding site of factor D, both of which have atypical conformations. Here we report that replacement of Ser94, Thr214, and Ser215 of factor D (chymotrypsinogen numbering has been used for comparison purposes) with the corresponding residues of trypsin, Tyr, Ser, and Trp, is sufficient to induce substantially higher catalytic activity associated with a typical serine protease alignment of the catalytic triad residues His57, Asp102, and Ser195. These results provide a partial structural explanation for the low reactivity of “resting-state” factor D toward synthetic substrates.

Activation of complement leads to expression of important host defense functions and proceeds via pathways that consist of successive enzymatic amplification steps. In the alternative pathway, the first enzymatic reaction is catalyzed by factor D, a serine protease that cleaves factor B only in the context of a complex with C3b, a fragment of the third component of complement, C3 (1). Factor D is unique among serine proteases in that it requires neither enzymatic cleavage for expression of proteolytic activity nor inactivation by an inhibitor for its control. Regulation of factor D activity in blood is apparently attained by a novel mechanism that depends on conformational changes and allows for reversible expression of proteolytic activity (2,3). The putative conformational changes are believed to be induced by the natural substrate, C3bB, and to result in reorientation of the catalytic center and of the substrate binding site of factor D, both of which have atypical structures (4). Functional support for this hypothesis has been provided by the seemingly parodoxical observation that, while the reactivity of factor D with thioester substrates and active site inhibitors is 3-4 orders of magnitude lower than that of typical serine proteases (5,6), its proteolytic activity during complement activation is comparable to that of other complement enzymes. Obviously, esterolytic assays assess the resting-state inactive conformation of the enzyme, while complement activa-tion assays the substrate-induced proteolytically active conformation. Structural support for the inactive resting-state conformation hypothesis has been provided by the recently determined crystal structure of factor D (4).
The two non-crystallographically related molecules, A and B, present in the triclinic unit cell of factor D have typical serine protease structural folds. However, they display distinctive orientations of the side chains of the catalytic triad residues Asp 102 and His 57 (chymotrypsinogen numbering 1 has been used throughout this paper), while the orientation of the third member of the triad, Ser 195 , is similar to that of other serine proteases. In all serine proteases of known structure, the spatial relationships of these three residues are constant and essential for the formation of a functional unit responsible for catalytic activity (7)(8)(9)(10). By contrast, in molecule A of factor D, the carboxylate of Asp 102 is pointed away from His 57 and is freely accessible to the solvent. In molecule B, the imidazolium of His 57 is oriented away from Ser 195 , having assumed the energetically favored trans conformation (4). Neither of these orientations would allow expression of catalytic activity, indicating the need for a realignment of the catalytic triad residues, probably induced by the single natural substrate, C3b-complexed factor B. A possible structural explanation for the unusual disposition of Asp 102 /His 57 in factor D is provided by the substitution of a Ser for the bulky aromatic Tyr or Trp residue usually present at position 94 of serine proteases and also of a Thr for the invariant Ser 214 (Fig. 1). Both of these residues are among those identified by Blow et al. (7) as being important for shielding Asp 102 from solvent molecules in chymotrypsin. In addition, a Ser residue that substitutes for the conserved aromatic Trp or Phe at position 215 may also be a determinant of the unique conformation of resting-state factor D. Indeed, in molecule B of factor D, Ser 215 is positioned between Asp 102 and Ser 195 , preventing His 57 from assuming the active gauche conformation that is characteristic of serine proteases. Here we report that replacement of Ser 94 , Thr 214 , and Ser 215 of factor D with the corresponding residues of trypsin, Tyr, Ser, and Trp, is sufficient to induce typical serine protease alignment of the catalytic triad residues and substantially higher catalytic activity. These results provide a structural exegesis for the unique conformation of the active center of resting-state factor D and also for its low reactivity with synthetic esters. The data also demonstrate the previously unrecognized importance of residue 94 of serine proteases for catalysis. * This work was supported in part by National Institutes of Health Grants AI21067 and AI32949 and by Grant NAGW-813 from NASA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Division of Clinical Immunology and Rheumatology, UAB Station, THT 437, Birmingham, AL 35294-0006. Tel.: 205-934-5067; Fax: 205-934-2126. 1 For comparison purposes factor D residues are numbered similarly to chymotrypsin from 16 to 243. Nine residues present in chymotrypsin (37,116,117,203,204,205,206,244, and 245) but missing from factor D are absent from the numbering scheme. Conversely, 9 residues of factor D are not present in chymotrypsin (3 residues after position 61 and one each after positions 78, 124, 129, 170, 177, and 223). These are numbered as the preceding residue with an appropriate suffix: A, B, or C.

Construction of Wild-type (wt) and Mutant Factor D Recombinant
Plasmids-The human factor D cDNA hg31 (11) was cloned into the unique HindIII site of the eukaryotic expression vector pRc/CMV (Invitrogen, San Diego, CA) as described previously (12). The S94Y mutant was constructed from the wt 2 template by using the mutagenic oligonucleotide 5Ј-TGGTGTCGGGCTGGTAGTCCGGGTGG-3Ј (mutated nucleotides are boldface and underlined). The combination mutants S94Y/T214S, S94Y/S215W, and S94Y/T214S/S215W were constructed by using the S94Y mutant cDNA as template and the mutagenic oligonucleotides 5Ј-GCGAGCCCGAGGAGACCACGCCC-3Ј, 5Ј-CGAGC-CCCAGGTGACCACGC-3Ј, and 5Ј-AACGCGCGAGCCCCAGGAGAC-CACGCCC-3Ј, respectively. Mutants were constructed according to the method of Kunkel (13). All mutations were verified by nucleotide sequencing using the chain termination method (14). Oligonucleotides were synthesized by using a model 394 DNA/RNA synthesizer (Applied Biosystems, Foster City, CA).
Expression and Purification of wt and Mutant Recombinant Factor D-Thirty g of each recombinant pRc/CMV plasmid containing wt or mutant factor D cDNA were transfected into 4 ϫ 10 6 Chinese hamster ovary (CHO) cells by electroporation at 1500 V and 25 microfarads by using a Gene Pulser apparatus (Bio-Rad). The transfected CHO cells were grown in Dulbecco's modified Eagle's medium/Ham's F-12 (1:1) supplemented with 10% heat-inactivated fetal calf serum, 2 mM glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin in 8% CO 2 at 37°C. After 48 h of culture, stably transfected CHO cells were selected by growth for 2 weeks in the above medium supplemented with increasing concentrations (0.4 -1 mg/ml) of Geneticin-418 (Life Technologies, Inc.). Expression of factor D was assessed by a solid phase enzymelinked immunosorbent assay (ELISA) using the anti-factor D monoclonal antibody FD10 -1 in the solid-phase and the IgG fraction of a rabbit anti-factor D serum as detection reagent as described previously (12). For purification of factor D, transfected CHO cells were grown in serum-free CHO medium (Life Technologies, Inc.), supplemented with 10 mM HEPES in 1-liter spinner flasks at 37°C. Typically, after 5-7 days of culture, supernatants of pooled transfectants contained approximately 1 g/ml factor D, which was purified by successive chromatography on Bio-Rex 70 (Bio-Rad) and Mono S HR 5/5 (Pharmacia Biotech Inc.) as described (15). For crystallization of the triple mutant S94Y/ T214S/S215W, a single transfectant was isolated by a series of subcloning steps. The stable transfectant secreted approximately 5-10 g/ml factor D in 5 days.
Hemolytic Assay for Factor D-Sheep erythrocytes carrying human C3b (EC3b) were prepared as described previously (16). The complement proteins C3 (17), factor B (18), and properdin (19) were purified by previously described methods. Native factor D was purified from the urine of a patient with Fanconi's syndrome as described (20). Hemolytic titrations were performed by incubating 7.5 ϫ 10 6 EC3b with properdin (62 ng), factor B (250 ng), and appropriately diluted factor D samples in 200 l of half-strength Veronal-buffered saline, pH 7.3, containing 2.5% dextrose, 2.5 mM MgCl 2 , 10 mM EGTA, and 0.1% gelatin. The mixtures were incubated at 37°C for 30 min and then 250 l of guinea pig serum diluted 1/40 in Veronal-buffered saline (150 mM NaCl, 5 mM Veronal, pH 7.3) containing 10 mM EDTA and 0.1% gelatin (EDTA-GVB) was added. The mixtures were further incubated at 37°C for 30 min. The reaction was stopped by adding 500 l of ice-cold EDTA-GVB, and the percentage of lysis was calculated from the absorbance of the supernatants at 413 nm and used to calculate hemolytic units/ml.
Thioester Hydrolysis by Factor D-The assay measuring the rates of hydrolysis of the thioester substrate Z-Lys-SBzl by factor D was performed as described by Kam et al. (5). Z-Lys-SBzl was purchased from Calbiochem (San Diego, CA). Ellman's reagent 5,5Ј-dithiobis-(2-nitrobenzoic acid) (Aldrich) was used at a concentration of 20 mM as a chromogen of thioester hydrolysis (21). Assays were conducted in microtiter wells and the rate of hydrolysis was measured as absorbance at 405 nm by using a Vmax™ kinetic microplate reader (Molecular Devices, Menlo Park, CA). The kinetic constants were calculated from Lineweaver-Burk plots.
Crystallization and Data Collection-Diffraction quality crystals of the S94Y/T214S/S215W mutant were grown at 22°C by the vapor diffusion method by using 50 mM MES buffer and polyethylene glycol 6000 as precipitant. Crystals grew to a size of 0.3 mm ϫ 0.3 mm ϫ 0.5 mm in 3 days. Preliminary crystallographic analysis indicated that the crystals belonged to the hexagonal space group P3 1 21 with unit cell dimensions a ϭ b ϭ 45.4 Å, c ϭ 175.2 Å. The calculated solvent content (22) was 43%, indicating 1 molecule/asymmetric unit. Diffraction data were collected on a Xentronics (Siemens/Nicolet, Madison, WI) area detector, using Cu-K␣ radiation (40 mA, 100 kV) from a rotating anode generator. Under these conditions the crystals diffracted to a resolution of 2.0 Å. The XENGEN program package (23) was used to process the data.
Structure Determination-The structure of the S94Y/T214S/S215W mutant was solved by molecular replacement methods (24) using the atomic coordinates of native factor D (4) as search model. The starting coordinates were modified by the removal of the side chains of the three mutated residues and also of His 57 , Asp 102 , Asp 194 , Ser 195 , Ser 217 , and Arg 218 . Coordinates of solvent atoms were also excluded in molecular replacement studies. When molecule B of the native factor D triclinic cell was used for molecular replacement, the cross-rotation function calculations (8.0 to 4.0 Å resolution) yielded higher and cleaner peaks than when molecule A was used. Molecule B coordinates were therefore used for the molecular replacement studies. The search model was rotated and translated into the unit cell of the mutant by an R-factor search using XPLOR (25), which gave an R-factor of 38%. The positional and orientational parameters of the model were refined by using the rigid body refinement protocol in XPLOR (8.0 to 4.0 Å resolution), which gave a R-factor of 36%. This model was subjected to least square refinement of individual atoms with the PROLSQ program (26) using 8.0 to 3.0 Å data. A difference map constructed by using calculated phases to 3.0-Å resolution revealed the positions of the missing side chains (except for residues 216, 217, and 218). The model was then refined further by a combination of simulated annealing using XPLOR and restrained least-square refinement using PROLSQ. The current refined model includes residues 16 -243 and 59 water molecules. The model has been fit by visual inspection of electron density maps (2͉F o ͉ Ϫ ͉F c ͉) and (͉F o ͉ Ϫ ͉F c ͉) computed using calculated phases (27). Table I FIG. 1. Alignment of partial amino acid sequences of selected serine proteases. Residues participating in shielding the catalytic residue Asp 102 from the solvent (7) are shaded. Conserved residues are boxed. Asterisks indicate amino acid residues subjected to site-directed mutagenesis. Numbers at the top are for residues of the chymotrypsinogen sequence, while the numbers at the bottom are for the human factor D residues. HFD, human factor D; TRP, bovine pancreas trypsin; CHT, bovine pancreas chymotrypsin; ELA, porcine pancreas elastase. Sequence data are from Ref. 37, except for the human factor D sequence, which is taken from Ref. 11.
summarizes the crystal and x-ray diffraction data and the final refinement statistics.

RESULTS
Four mutant factor D cDNAs, S94Y, S94Y/T214S, S94Y/ S215W, and S94Y/T214S/S215W, were constructed for these studies. The mutant and wt factor D cDNAs were stably expressed in CHO cells, and the recombinant proteins were purified to homogeneity. The effects of the mutations on the catalytic efficiency of resting-state factor D were evaluated by an esterolytic assay using the thioester substrate Z-Lys-SBz1 (12). Results of multiple experiments are summarized in Table  II. Previously reported results for the T214S, S215W, and T214S/S215W mutants of factor D (12) have been included for comparison. As shown, mutation of Ser 94 to Tyr in the single S94Y mutant resulted in about 6-fold increase of the catalytic rate constant (k cat ). Larger increases in k cat were observed when the S94Y mutation was combined with mutations of Thr 214 to Ser and of Ser 215 to Trp. The triple mutant S94Y/ T214S/S215W had a k cat more than 16-fold higher than wt factor D and only 3.5-fold lower than trypsin (Table II). The enhanced k cat of all S94Y mutants and of the double T214S/ S215W mutant resulted in increased overall catalytic efficiency as assessed by the k cat /K m ratio, although the K m was affected only slightly if at all.
The proteolytic activity of mutant factor D was assessed by a sensitive hemolytic assay, and the results (Table III) were in general agreement with those obtained with the esterolytic assay. The S94Y mutant had higher hemolytic activity than wt factor D, and its activity was synergistically increased in combinations with the S215W and the double T214S/S215W mu-tations. As expected, all mutants cleaved factor B complexed with cobra venom factor, a C3b analogue of cobra venom, but none cleaved uncomplexed factor B (data not shown).
To investigate the structural correlates of the increased catalytic activity of mutant factor D, we determined the crystal structure of the triple mutant S94Y/T214S/S215W. The model has been refined to an R-factor of 19.8% (Table I) and is in agreement with the most favored region of a Ramachandran plot (28) (Fig. 2). Fig. 3 shows the electron density for the mutated residues 94, 214, and 215 in a difference map calculated by using molecular replacement phases (8.0 to 3.0 Å). Plots of mean temperature factors (B) per residue and of realspace density fit per residue (R) are shown in Fig. 4 (a and b,  respectively). The highest B-factors and worst real-space Rfactors are observed in the segment His 172 -Ile 177 . In contrast, in both molecules A and B of native factor D, the B-factors for this segment were lower than the average values (4). In typical serine proteases residue 172 is a highly conserved aromatic (Tyr or Trp), which is part of a type I ␤-turn and has been shown to be a determinant of substrate specificity (29). Thus, the apparent flexibility of His 172 in the mutant may have an  effect on substrate binding. The second highest B-factors were observed for the Asp 61 -Val 64 segment of mutant factor D. This region is flexible in molecule A, but quite rigid in molecule B of native factor D. Compared to chymotrypsin or trypsin, this segment of factor D has 3 or 4 additional residues, respectively.
Overall, the tertiary structure of the S94Y/T214S/S215W mutant is very similar to that of native factor D. The root mean square (r.m.s.) deviations between the coordinates of main chain and side chain atoms of the mutant and molecules A and B of factor D are shown in Fig. 5 (a and b, respectively). Substantial structural divergence between the mutant and both molecules of native factor D is observed in two segments. The first includes residues 169 -175, which in the mutant form a flexible loop thus making the observed r.m.s. shifts an unreliable indicator of actual structural differences. Furthermore, this segment partially overlaps the region (residues 172-177) with the highest B-factors and worst real-space R-factors (Fig.  4, a and b). Excluding residues 172-177, the r.m.s. shifts between the mutant and molecule A (Fig. 5a) are 0.95 Å for main chain atoms and 1.02 Å for side-chain atoms. The corresponding values for the comparison between the mutant and molecule B are 0.86 Å and 1.59 Å. By comparison, the r.m.s. shifts between molecules A and B of native factor D (4) are 0.96 Å for main chain atoms and 1.58 Å for side chain atoms. The second segment with significant r.m.s. deviations between the mutant and both molecules of factor D includes residues 214 -224. Residues 214 -220 form one of the three walls that line-up the primary specificity pocket of serine proteases (30). In addition, residues 214 -216 are part of the S 1 -S 3 subsite 3 that forms a ␤-pleated sheet with residues P 1 -P 3 of the substrate (31)(32)(33). Two of these residues, Thr 214 and Ser 215 , have been mutated, which probably accounts at least in part for the observed r.m.s. deviations. In addition, changes in the orientation of the side chains of Arg 218 and Arg 223 apparently contribute to the observed r.m.s. shifts. In native factor D Arg 218 forms a salt bridge with Asp 189 at the bottom of the primary specificity pocket (Fig. 6). This bridge probably contributes to the low reactivity of resting-state factor D with synthetic substrates, as it restricts access of the positively charged P 1 Arg residue to the negative charge of Asp 189 . In the mutant, Arg 218 seems to be flexible and its side chain points away from the carboxylate of Asp 189 and toward the solvent. Therefore, no salt bridge between this residue and Asp 189 can form. Instead, Arg 223 has been reoriented so that its guanidinium group is H-bonded to Asp 189 (Fig. 6). These changes are accompanied by a substantial narrowing of the primary specificity pocket of the mutant, apparently due to a movement of the segment formed by residues 214 -218 (Fig. 6).
Superposition of the mutant on molecule A results in substantial r.m.s. shifts around the Ser 94 3 Tyr substitution (Fig.  5a). Asp 102 of molecule A is pointed toward the solvent away from His 57 at the active center of factor D. In the mutant, Asp 102 is forced by the phenyl ring of Tyr 94 to assume a position similar to that observed in typical serine proteases (Fig. 6) and the entire loop joining residues Val 89 and Leu 103 has been rearranged in a way that resembles the corresponding region of trypsin. The movement of loop 89 -103 is mainly responsible for the observed average shift of 5.0 Å for main chain atoms and 8.2 Å for side chain atoms of this segment (Fig. 5a). In molecule B of factor D, the orientation of Asp 102 and of the entire loop 89 -103 is similar to that of trypsin, which accounts for the lack of large r.m.s. shifts between molecule B and the mutant (Fig.  5b). However, the imidazolium of His 57 , which has an atypical trans orientation in molecule B of native factor B, assumes the catalytically active gauche orientation in the mutant. This movement is probably responsible for the r.m.s. shifts observed in this region of the plot shown in Fig. 5b. Fig. 7 summarizes graphically the main structural differences between native and mutant factor D. The catalytic triad residues Asp 102 , His 57 , and Ser 195 of both molecules A and B present in the triclinic unit cell of native factor D have an atypical alignment inconsistent with catalysis. In molecule A, Asp 102 is turned away from His 57 , having gained access to the solvent, while in molecule B His 57 is pointed away from Ser 195 and Ser 215 is occupying the space usually occupied by His 57 . In contrast, in S94Y/T214S/S215W the catalytic triad residues display an orientation very similar to that of trypsin. This typical serine protease conformation of the catalytic triad probably accounts for the increased catalytic activity of the mutant compared to native factor D.

DISCUSSION
The present data clearly demonstrate that three residues, Ser 94 , Thr 214 , and Ser 215 , are responsible for the atypical orientation of the catalytic triad of native factor D. Replacement of these three residues with those present in the corresponding positions of trypsin, Tyr 94 , Ser 214 , and Trp 215 , resulted in a mutant enzyme with about 20-fold higher reactivity toward the synthetic thioester Z-Lys-SBzl than native factor D. The increased catalytic efficiency could be accounted for by an increase in k cat and could be attributed to structural changes of the catalytic center. Most importantly, in contrast to native factor D, the side chains of the catalytic residues Asp 102 and His 57 of the S94Y/T214S/S215W mutant have a typical serine protease orientation very similar to that seen in trypsin (Fig. 7).
Possible lattice effects were considered before concluding that the observed trypsin-like conformational changes could be attributed directly to the mutations. The triclinic unit cell of native factor D contains two non-crystallographically related molecules, whereas the triple mutant crystallizes in a different space group with one molecule in the asymmetric unit. Therefore, different lattice forces than those exercised on either molecule A or B of native factor D should be expected to im- 3 The nomenclature used for the individual amino acid residues (P 1 , P 2 , etc.) of a substrate and the corresponding subsites (S 1 , S 2 , etc.) of the enzyme is that of Schechter and Berger (39). pinge on the mutant, possibly contributing to the observed realignment of key catalytic residues. However, we have recently crystallized native factor D in space group P2 1 with one molecule in the asymmetric unit. The structure of this crystal form of native factor D has been determined by a combination of isomorphous replacement and molecular replacement meth-ods. The model has been refined to an R-factor of 18.8% by using 8.0 to 2.0 Å resolution data. The catalytic triad of this molecule has a conformation similar to that present in molecule B of the triclinic cell. Similar conformations also have been observed in inhibitor complexes of factor D. It thus seems unlikely that lattice forces are major contributors to the observed structural differences between native and triple mutant factor D.
The spatial relationships of the three catalytic residues are constant in all serine proteases of known structure and are stabilized by a network of H bonds (7,34). Specifically, H bonds between the N␦1 of His 57 and the O␦2 of Asp 102 , the N⑀2 of His 57 and the O␦ of Ser 195 , and the O␦2 of Asp 102 and O␥ of Ser 214 that are present in typical serine proteases are also present in the mutant factor D. In contrast the orientation of the side chains of these residues in native factor D precludes the formation of these H bonds. The integrity of the H bonds and the hydrophobic nature of the environment surrounding the buried Asp 102 are essential for the formation of a functional unit responsible for bond formation and cleavage during catalysis (7)(8)(9)(10). Hence, the proposal for a substrate-induced conformational change of the active center of factor D to explain the efficient activation of the alternative complement pathway (2,3,5).
All three mutated residues, Ser 94 , Thr 214 , and Ser 215 , apparently contribute to the unusual conformation of the catalytic triad of native factor D and the resulting low reactivity toward small synthetic thioester substrates. However, Ser 94 seems to be the principal determinant as indicated by the larger effect of the S94Y mutation on the k cat for Z-Lys-SBzl than those obtained for the single T214S and S215W mutants (Table II). The introduction of the bulky Tyr residue at position 94 is probably responsible for forcing Asp 102 and His 57 to assume an active conformation. This is suggested by the r.m.s. deviation plot (Fig. 5a), which shows substantial structural differences in the immediate vicinity of the S94Y substitution when the mutant is superposed on molecule A of factor D.
A contribution of Thr 214 is clearly indicated by the synergistic gain in k cat (Table II) observed when the T214S mutation is combined with the S94Y and S215W mutations. This effect is probably due to the formation of a H bond between the side chains of Ser 214 and Asp 102 , which helps stabilize Asp 102 . A role for the highly conserved aromatic at position 215 of serine proteases in catalysis has never been probed before. This residue is part of the S 1 -S 3 subsite in trypsin (33), which forms a ␤-pleated sheet with residues P 1 -P 3 of the substrate. In trypsin, Trp 215 is involved in the formation of a hydrophobic cluster The models were generated using the Ribbons program (38). Green is used for carbon, blue for nitrogen, and red for oxygen. The backbone is represented as a tube, and bonds joining the side-chain atoms C␤ onward are shown as cylinders. Each model shows the orientation of the catalytic triad and of residues in the immediate vicinity. Bovine trypsin coordinates were obtained from the Brookhaven Protein Data Bank. that also includes Tyr 172 , a structural determinant of substrate specificity (29). However, in the S94Y/T214S/S215W mutant loop 172-177 is flexible, precluding a hydrophobic interaction between His 172 and Trp 215 . Instead, Trp 215 is oriented in a way favoring hydrophobic interactions with His 57 (Fig. 7). Regardless of its precise structural contribution, the S215W mutation had a significant effect on catalytic efficiency against Z-Lys-SBzl (Table II).
An additional interesting structural difference between S94Y/T214S/S215W and native factor D involves Arg 218 . In both molecules of factor D, the guanidinium group of this residue forms a salt bridge with the carboxyl of Asp 189 (4). In trypsin and presumably also in all trypsin-like serine proteases, the carboxyl of Asp 189 forms a salt bridge with the side chain of the P 1 Arg or Lys residue of the substrate (35). This ionic interaction plays a major role in positioning the scissile bond of the substrate for hydrolysis. Thus, the Arg 218 -Asp 189 bridge of native D has been considered an important contributor to the low reactivity of resting-state factor D toward small synthetic substrates (4,12). Furthermore, it has been suggested that a reorientation of the side chain of Arg 218 is an important component of the putative substrate-induced conformational change that leads to efficient proteolysis of C3bbound factor B by factor D (4,12). Interestingly, mutation of Arg 218 to Gly, which is present in the corresponding position of trypsin, combined with deletion of Val 219 , which is absent from trypsin, resulted in a small increase of esterolytic activity and an almost complete loss of proteolytic activity (12). Indeed, even extensive mutations involving the substrate binding pocket and associated structural elements, which yielded a mutant factor D exhibiting 100-fold higher esterolytic activity than native factor D were associated with extremely low proteolytic activity attributable to the R218G and V219⌬ mutations (36). These findings were interpreted to indicate a direct interaction between Arg 218 and determinants on the C3bB complex, the natural substrate of factor D.
The present finding that the realignment of the catalytic triad residues induced by the S94Y/T214S/S215W mutations is associated with a reorientation of Arg 218 away from Asp 189 indicates a linkage among the conformations of these elements of the active center. It thus seems possible that the proposed interaction between Arg 218 and negatively charged residue(s) on C3bB not only makes Asp 189 available to the P 1 Arg 234 of factor B, but also contributes to the realignment of the catalytic triad residues. Obviously, additional conformational changes particularly of the substrate binding pocket are necessary for expression of efficient proteolytic activity. These changes were not induced by the mutation of the three residues investigated in the present study, as indicated by the geometry of the primary specificity pocket (Fig. 6) and the high K m of the triple mutant (Table II).