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• W2084527777 abstract "Article17 January 2000free access New structural motifs on the chymotrypsin fold and their potential roles in complement factor B Hua Jing Hua Jing Center for Macromolecular Crystallography, School of Optometry, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Graduate Program in Biophysical Sciences and Pharmaceutical Design Program University of Alabama at Birmingham, Birmingham, AL, 35294 USA Present address: Center for Blood Research, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Yuanyuan Xu Yuanyuan Xu Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author Mike Carson Mike Carson Center for Macromolecular Crystallography, School of Optometry, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author Dwight Moore Dwight Moore Center for Macromolecular Crystallography, School of Optometry, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author Kevin J. Macon Kevin J. Macon Center for Macromolecular Crystallography, School of Optometry, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author John E. Volanakis John E. Volanakis Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Biomedical Sciences Research Center ‘A.Fleming’, Vari, 16672 Greece Search for more papers by this author Sthanam V.L. Narayana Corresponding Author Sthanam V.L. Narayana Center for Macromolecular Crystallography, School of Optometry, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author Hua Jing Hua Jing Center for Macromolecular Crystallography, School of Optometry, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Graduate Program in Biophysical Sciences and Pharmaceutical Design Program University of Alabama at Birmingham, Birmingham, AL, 35294 USA Present address: Center for Blood Research, Harvard Medical School, Boston, MA, 02115 USA Search for more papers by this author Yuanyuan Xu Yuanyuan Xu Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author Mike Carson Mike Carson Center for Macromolecular Crystallography, School of Optometry, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author Dwight Moore Dwight Moore Center for Macromolecular Crystallography, School of Optometry, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author Kevin J. Macon Kevin J. Macon Center for Macromolecular Crystallography, School of Optometry, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author John E. Volanakis John E. Volanakis Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Biomedical Sciences Research Center ‘A.Fleming’, Vari, 16672 Greece Search for more papers by this author Sthanam V.L. Narayana Corresponding Author Sthanam V.L. Narayana Center for Macromolecular Crystallography, School of Optometry, University of Alabama at Birmingham, Birmingham, AL, 35294 USA Search for more papers by this author Author Information Hua Jing1,2,3, Yuanyuan Xu4, Mike Carson1, Dwight Moore1, Kevin J. Macon1,4, John E. Volanakis4,5 and Sthanam V.L. Narayana 1 1Center for Macromolecular Crystallography, School of Optometry, University of Alabama at Birmingham, Birmingham, AL, 35294 USA 2Graduate Program in Biophysical Sciences and Pharmaceutical Design Program University of Alabama at Birmingham, Birmingham, AL, 35294 USA 3Present address: Center for Blood Research, Harvard Medical School, Boston, MA, 02115 USA 4Division of Clinical Immunology and Rheumatology, Department of Medicine, University of Alabama at Birmingham, Birmingham, AL, 35294 USA 5Biomedical Sciences Research Center ‘A.Fleming’, Vari, 16672 Greece *Corresponding author. E-mail: [email protected] The EMBO Journal (2000)19:164-173https://doi.org/10.1093/emboj/19.2.164 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Factor B and C2 are two central enzymes for complement activation. They are multidomain serine proteases and require cofactor binding for full expression of proteolytic activities. We present a 2.1 Å crystal structure of the serine protease domain of factor B. It shows a number of structural motifs novel to the chymotrypsin fold, which by sequence homology are probably present in C2 as well. These motifs distribute characteristically on the protein surface. Six loops surround the active site, four of which shape substrate-binding pockets. Three loops next to the oxyanion hole, which typically mediate zymogen activation, are much shorter or absent. Three insertions including the linker to the preceding domain bulge from the side opposite to the active site. The catalytic triad and non-specific substrate-binding site display active conformations, but the oxyanion hole displays a zymogen-like conformation. The bottom of the S1 pocket has a negative charge at residue 226 instead of the typical 189 position. These unique structural features may play different roles in domain–domain interaction, cofactor binding and substrate binding. Introduction Serine proteases with a chymotrypsin-like fold (SP) are ubiquitous and involved in a wide range of biological processes. Structural and functional studies on this family of proteases are directed to answering three major questions: what determines their catalytic efficiency, how do they recognize substrates and how is their activity regulated? Extensive pioneering studies on digestive SPs such as chymotrypsin, trypsin and elastase have provided fundamental answers to these questions. The catalytic efficiency is provided by a His57–Asp102–Ser195 triad, located near the oxyanion hole (reviewed in Kraut, 1977). The substrate-binding specificity and affinity are determined mostly by the geometrical and chemical nature of the substrate-binding pockets, especially the S1 pocket (reviewed in Perona and Craik, 1995; Czapinska and Otlewski, 1999). Blocking these pockets with synthetic peptide analogs or natural protein inhibitors provided insights into the mechanisms of SP inactivation (reviewed in Bode and Huber, 1992). The activation of an SP is attributed in most cases to the zymogen activation where a freely liberated N-terminus induces conformational changes in and around the active site (reviewed in Huber and Bode, 1978; Khan and James, 1998). In some cases, however, SP activation also requires specific protein–protein interactions such as enzyme–cofactor association (Banner et al., 1996; Wang et al., 1998), enzyme–substrate interactions (Volanakis and Narayana, 1996; Jing et al., 1998) and enzyme self-assembly (Pereira et al., 1998). Structural studies of these novel SPs have begun to reveal the diversity of their regulation mechanisms. The complement system, rich in such regulated SPs, contains eight proteolytic enzymes, all of which belong to the chymotrypsin fold group and cleave specific peptide bonds next to arginine residues (reviewed in Arlaud et al., 1998). Except for factor D, which has a single domain and interacts only with its natural substrate, all others have multiple domains and interact not only with the substrate, but also with certain cofactor proteins. Among these enzymes, factor B (FB) and C2 are homologous, and each supplies a catalytic subunit for the central C3/C5 convertases of complement. They also display structural and functional features distinct from those of the known SPs. FB and C2 have three domains each: an N-terminal Ba or C2b domain composed of three complement control protein (CCP) modules, followed by a von Willebrand factor type A (vWFA) domain and a C-terminal SP domain. In the presence of Mg2+, FB binds cofactor C3b to be activated by factor D, whereas C2 binds cofactor C4b and is activated by C1s (reviewed in Volanakis, 1988). The activation results in the release of their N-terminal domains and the generation of C3b-bound Bb or C4b-bound C2a, respectively, and the consequent protein complexes are named C3 convertases. Hence, the C-terminal SP domains of the catalytic subunits Bb and C2a lack the typical free N-terminus essential for the enzyme activation due to their linkage to the vWFA domains. The esterolytic activities of FB/Bb and C2/C2a towards their best synthetic substrates are ∼103-fold lower than that of trypsin (Kam et al., 1987), indicating a possible defect in the catalytic apparatus. No natural inhibitor has been identified for either enzyme. Instead, they are regulated by an association with the respective cofactor proteins. On the other hand, this bimolecular assembly is unstable and exhibits a half-life of <2 min under physiological conditions (Kerr, 1980; Pangburn and Müller-Eberhard, 1986). The dissociated Bb displays residual hemolytic and proteolytic activity (Fishelson and Müller-Eberhard, 1984), albeit with higher esterolytic activity than FB (Kam et al., 1987). The cofactor interacts with the Mg2+-binding motif on the vWFA domain in Bb/C2a (Horiuchi et al., 1991; Hourcade et al., 1995; Tuckwell et al., 1997) and possibly also with the SP domain (Lambris and Müller-Eberhard, 1984; Sanchez-Corral et al., 1990). Very little is known about the mode of cofactor binding and the position of the binding site(s) relative to the active site. To understand the regulation mechanism and to facilitate the design of complement inhibitors, we determined the crystal structure of the SP domain of FB (FBSP). The structure reveals many motifs novel to the chymotrypsin fold. Sequence homology with C2SP suggests that similar motifs are likely to be present in C2SP as well, although some surface features might be different. The structure is presented below with an emphasis on the novel motifs. Some suggestions on their functional correlates, such as domain–domain interaction and cofactor binding, are presented. Results Overall structure The FBSP structure was solved by a combination of multiple isomorphous replacement (MIR) and molecular replacement methods (Table I). All residues are in the most favored (85.6%) or additionally allowed (14.2%) regions of the Ramachandran plot except for one (K144) in the generously allowed region. The completeness of the two molecules in the asymmetric unit is slightly different for the N-terminal linker region and a surface loop (Table I). The rest of the two molecules superimpose well with a root-mean-square deviation of 0.34 Å for 277 matched (<3.8 Å) Cα atoms. Table 1. Statistics on diffraction data and refined structure Room temperature data Low temperature data Native TMLA Sm Native TMLA Cell dimensions 51.88, 75.94, 74.04, β = 96.65 51.64, 74.26, 73.26, β = 95.55 Resolution (Å) 2.3 2.8 2.8 2.1 2.3 Completeness (%) 97.7 96.0 91.0 99.2 97.5 Rsym (%) 7.3 6.0 4.1 6.9 8.1 Riso (%) – 12.2 8.9 – 16.4 Phasing power (acentric/centric) 1.05/0.88 1.33/1.00 tRCullis 0.84/0.88 0.78/0.70 Figure-of-merit 0.44/0.69 0.30/0.57 Refinement and structure statistics Resolution (Å) 20–2.1 No. of unique reflections 32 125 Rcryst 20.9 Rfree 24.3 No. of protein atoms 4508 No. of water atoms 517 A: A1k-V4, K9-K220a, Q220h-L250 B: S1b-K220a, V220i-L250 A: W5-E6-H7-R8, N220b-Q220c-K220d-R220e-Q220f-K220g B: A1k-D1j-P1i-D1h-E1g-S1f-Q1e-S1d-L1c, N220b-Q220c-K220d-R220e-Q220f-K220g-Q221h R.m.s.d. bond length (Å) 0.009 R.m.s.d. bond angle (°) 1.43 Luzzati error (work/test set) (Å) 0.26/0.30 No. of residues (A/B) 288/282 Average B-factor for protein (A/B) 31.99/24.04 Average B-factor for water 37.3 FBSP displays a two-domain β-barrel fold in which two β-sheets, composed of six β-strands each, are surrounded by surface helices and loops (Figure 1A and B). Compared with other SPs, the core β-sheets are similar, but many surface loops and helical regions are strikingly different. Long insertions and deletions are quite distinct in the structure-based sequence alignment with other SPs (Figure 1C). Interestingly, the insertions and deletions are clustered characteristically on the back, the right side and around the active site (refer to the orientation in Figure 1A). The active site also displays certain atypical conformations for some critical residues. Figure 1.Overall structure of FBSP and structure-based sequence alignment with 10 other SPs. The front view (A) and back view (B) of the overall structure in stereo ribbon diagrams are shown on the left, with corresponding surface charge potential shown on the right. The surface charge potential is plotted on a −10 to +10 scale using GRASP (Nicolls et al., 1991). The labeled surface regions are colored according to distribution. The active site is designated by S195. In the structure-based sequence alignment (C) with chymotrypsin (CHM), trypsin (TRP), factor D (FD), thrombin (THM), factor Xa (FXA), factor IXa (FIXA), protein C (PRC), urokinase-type plasminogen activator (UPA), tissue-type plasminogen activator (TPA) and C2SP, the structurally equivalent positions are shaded in yellow and the secondary structure elements are underlined (β-strands, green; α-helices, blue; 310-helices, cyan). The conserved residues are colored in green; a few FBSP- or C2SP-unique residues are highlighted in red; and the potential N-linked glycosylation sites in C2SP are in blue. The variable regions are outlined in magenta and their three-dimensional positions in FBSP are shown in (A) and (B). Download figure Download PowerPoint Three insertions on the back The N-terminal linker region (residues D1h–T11 in chymotrypsinogen numbering), the 125 insertion (T125a–D133) and the C-terminal insertion (Q243–L250) are clustered on the back of the structure (Figure 1B). The N-terminal linker associates with the main body of the SP domain through a disulfide bond between C1 and C122. The N-terminal end of this linker displays a helical conformation in one molecule but is disordered in the other molecule; the C-terminal end displays a flexible loop conformation. The 125 insertion folds into a helix–loop–helix motif where the N-terminal ends of the two helices are constrained by a unique disulfide bond (C125–C125p). The second helix is present at a similar position in a few other SPs, but the first helix and the middle loop regions are unique (Figure 1C). The C-terminal insertion is a loop whose conformation is stabilized by specific hydrophobic and electrostatic interactions involving its last four residues. These three regions are tightly associated with each other and with the main body by a network of disulfide bonds, hydrophobic interactions and hydrogen bonds. A nearby short 203 loop (K203–R206) enhances such an association by complementing with salt bridge partners. The combined motif is obviously rigid and displays three distinct positively charged, negatively charged and hydrophobic patches (Figure 1B). This motif may display a comparable conformation in C2SP, as indicated by similar residue compositions in the four involved regions (Figure 1C). Some differences, however, can be identified for a few exposed residues in the linker region (residues 3–9) and the C-terminal insertion (residues 245–246). Particularly, N5 and N9 in C2SP are potential N-linked glycosylation sites that are probably occupied (see Xu and Volanakis, 1999), suggesting that this segment might be surface exposed in C2/C2a. Mutation of a glycine at position 2 to an arginine was described as the cause of C2 secretion deficiency (Westel et al., 1996), probably due to the disruption of the nearby C1–C122 disulfide bond. Three deletions on the right side Three loops that are present in all other SPs, the 16 loop (16–23), the 71 loop (71–81) and the 142 loop (142–155), are nearly absent in FBSP (Figure 1C). The 16 loop is completely absent, while loops 71 and 142 have only eight residues in total as compared with 24–29 residues observed in other SPs. Notably, seven of the eight residues are charged, but most of them do not form salt bridges. Thus, these three deletions create a unique hydrophilic surface to the right of the active site. Similarly, C2SP has no 16 loop. Its 71 loop is longer than that in FBSP (seven residues versus three), its 142 loop is shorter (two residues versus five) and only two of the nine residues are charged. The 142 loop contains another potential N-linked glycosylation site at N142, which would make the deletion patch also hydrophilic in C2/C2a. These three loops typically mediate zymogen activation in other SPs (see Jing et al., 1999). Their consensus deletion in FBSP and C2SP may be related to their unique regulation mechanism. Six loops around the active site The active site is surrounded by loops 35, 60, 96, 172, 186 and 218, among which loops 96, 172 and 218 are exceptionally long (Figure 1). The 35 loop (V35–E40) and 60 loop (T60–S63) present on top of the substrate-binding cleft shape the upper wall of the S1′ and S2′ pockets with the side chains of T60, E40 and S41. The long 96 loop (I96–F98) and 172 loop (A172–V175) inter-digitate with each other on the left side of the active site. The side chain of Y99, by stacking with that of W215, divides the space between the S2 and S3 pockets (Figure 2B). The S2 pocket is small and the S3 is large, both being hydrophobic in nature, and correlate well with the specificity towards alanine or glycine at the P2 position and leucine at the P3 position of the natural substrates. These two pockets appear to be rigid because of the extensive interactions between loops 96 and 172. This may explain their strict substrate-binding specificity and imply that they are unlikely to undergo any substrate-induced conformational changes. Similar conformations may be expected for the corresponding loops in C2SP, as indicated by their high sequence homology (Figure 1C). Figure 2.Active site conformation and a model of Bb domain architecture. (A) Comparison of the essential active site structural elements in FBSP (gold) and in trypsin (silver) (catalytic triad, pink; oxyanion hole, red; non-specific substrate-binding site, green; specificity-determining residues, cyan). The unique salt bridge between D187 and K163 is in blue. A complementary ball-and-stick plot of the active site is shown in (B) together with the σ-weighted 2Fo − Fc electron density map (1.0 σ). Residues Lys, Ser and Pro (density in magenta) coming from an NCS-related 35 loop interact coincidentally with the S1, S2 and S3 pockets, respectively, in a substrate-like manner. (C) One of the possible positions of the vWFA domain relative to the SP domain as suggested by docking. Arrows point to the proposed cofactor-binding sites. This right-side view of the SP domain distinctly shows the distribution of the unique regions and their possible relationships with the vWFA domain and putative cofactor-binding sites. Download figure Download PowerPoint The 186 loop (V186–D187) is located underneath the S1 pocket (Figure 1A). It interacts with a short 163–164c loop through a buried salt bridge between D187 and K163 (Figure 2A) and an aromatic stacking between Y186c and Y161, both of which are unique to FBSP. The 186 loop in C2SP carries somewhat different residues, but the salt bridge between D187 and K163 might be conserved (Figure 1C). The 218 loop (V218–A224) is in front of the S1 pocket. Three exposed valine residues and a partially buried D218a in the beginning of this loop line the entrance to the S1 pocket (Figure 2B). C220 forms a disulfide bond with C191, which, due to insertions, is shifted by a few angstroms from its typical position (Figure 2A). Following C220, a stretch of approximately six unique charged or hydrophilic residues is invisible in the electron density maps (Table I). The corresponding region in C2SP is longer and has similar residue composition (Figure 1C), and thus may also display a highly flexible conformation. This atypically long and flexible loop, especially in proximity to the unique deletion patch and active site, probably contributes to the cofactor binding as discussed below. Within the active site The catalytic triad residues (H57, D102 and S195) and the non-specific substrate-binding site (W215–G216) display typical active conformations (Figure 2A). However, the oxyanion hole exhibits an atypical conformation due to an inward orientation of R192 carbonyl oxygen (Figure 2B). It forms a hydrogen bond with the amide group of S195, thus reducing the typical positive charge expected in the oxyanion hole. Residues 191–194 adopt a single turn 310-helix conformation, whose C-terminal end imposes additional negative potential to the oxyanion hole (Figure 2A). Such an inactive oxyanion hole conformation is similar to that observed in some SP zymogens (see Jing et al., 1999) and could possibly explain the low esterolytic activities of FB/Bb (Kam et al., 1987). A conformational change to an active oxyanion hole is obviously essential for efficient catalysis. In the absence of the positively charged N-terminus of the SP domain, D194 forms a hydrogen bond with the hydroxyl group of S140 (Figure 2A). In C2SP, S140 is absent, but a unique R43 residue might be close enough to form a salt bridge with E194. Other residues within the oxyanion hole, the catalytic triad and the non-specific substrate-binding site in C2SP are nearly identical to those in FBSP. At the bottom of the S1 pocket, residue D189, typically positioning the positively charged P1 residue of the substrate for the nucleophilic attack by S195, is replaced by N189 in FBSP and S189 in C2SP. However, a typical G226, located on the opposite side to D189 at the bottom of the S1 pocket, is replaced by D226 in both enzymes (Figure 1C). Compared with the typical position of the D189 carboxyl group, the D226 carboxyl group in FBSP is more elevated (Figure 2A). Its negative charge might be more dispersed due to hydrogen bonding with the atoms from N189, T190, R225 and a non-crystallographic symmetry (NCS)-related K37. This conformation is similar to that observed in a trypsin D189G/G226D mutant, which is also less active than the wild-type trypsin (Perona et al., 1993). However, relocating the negative charge from 226 to the typical 189 position in FB mutant D226N/N189D did not restore the catalytic efficiency closer to that of trypsin, as shown in our recent mutagenesis experiments (Xu et al., 2000). On the contrary, this mutant exhibits complete loss of hemolytic and C3 cleavage activity and ∼50% reduced activity in forming C3 convertase (Figure 3). The esterolytic activity of the mutant is also substantially reduced compared with the wild-type, mainly due to a reduction in the kcat value. These data indicate that residue D226 is the primary substrate determinant for P1-Arg binding, and a proper registration of the long P1-Arg side chain to D226 on one end and its scissile bond to the nucleophilic S195 on the other end is essential for efficient catalysis (Xu et al., 2000). Figure 3.Relative cobra venom factor (CoVF)-binding activity of FB mutants compared with the wild-type FB or Bb. The methods are described in Xu et al. (1999). Download figure Download PowerPoint The S1, S2 and S3 pockets are occupied coincidentally by the side chains of K37, S36c and P36b, respectively, from an NCS-related 35 loop in a substrate-like manner (Figure 2B). The amino group of K37 forms four hydrogen bonds with oxygen atoms from residues D226, T190 and V218. The carbonyl oxygen of P36b forms a hydrogen bond with the backbone amine of G216. This crystal packing artifact results in a shift of the loop 35 tip from its typical position, but it is unlikely to affect the S1 site conformation because each structural element in the active site region is engaged in a network of specific intramolecular interactions. The active site of FBSP displays an overall negative potential, which may provide the driving force to attract and position the natural substrates (Figure 1A). The presence of long insertion loops immediately around the active site may limit the accessibility of the FB active site to non-specific macromolecular substrates and natural SP inhibitors in blood. Discussion The crystal structure of FBSP reveals the presence of a unique bulky and rigid motif on the back, three distinct deletions on the right and six loop insertions around the active site. As described, four of the six loops (loops 35, 60, 96 and 172) dictate the specificity at the S1′, S2′, S2 and S3 pockets. The atypical D226 residue in the S1 pocket determines P1-Arg binding and catalysis (Xu et al., 2000). The remaining structural motifs, which have no corresponding regions in all other SPs with known structures, raise intriguing questions regarding their functional roles. Given that FB/Bb and C2/C2a are multidomain SPs and require cofactor binding to express proteolytic activity fully, it seems possible that these unique regions could be involved in the domain–domain interactions, cofactor binding and substrate binding. Domain architecture Several biophysical methods have shown that the three domains in FB constitute a compact and globular structure and that the two domains in Bb are in close contact (Smith et al., 1982, 1984; Ueda et al., 1987; Chamberlain et al., 1998; Hinshelwood and Perkins, 1998). The linker between the vWFA domain and the SP domain is inaccessible to limited proteolysis by elastase in the context of Bb, but is susceptible in free FB (Lambris and Müller-Eberhard, 1984), suggesting a possible rearrangement of the two domains from FB to Bb. To identify the possible domain arrangements, we performed a docking search using the FBSP crystal structure and an FB vWFA model built by Tuckwell et al. (1997), where the C-terminal region of the vWFA domain was restrained together with the N-terminal region of the SP domain. Although there were no clear-cut solutions, most of the best solutions placed the vWFA domain at the right of the back of the SP domain (see an example in Figure 2C). The Mg2+-binding site on top of the vWFA domain and the N-terminus of the vWFA domain are exposed from the domain interface. The residues in the contact regions come from bottom loop regions of the vWFA domain and from the unique insertion motif on the back of the SP domain, suggesting that the latter probably plays a role in the interactions with the vWFA domain. However, the exact orientation is hard to determine because the linker appears to be flexible in this structure. The N-terminal end of the SP domain could be traced to the end only for one molecule in the asymmetric unit (Table I). The C-terminal helix of the vWFA domain may also be flexible, because in isolated integrin Mac-1 I-domain (also a vWFA domain) this helix shifts in response to ligand binding (Lee et al., 1995; Liddington and Bankston, 1998). Such flexibility of the linker may reflect a structural basis for the domain rearrangement mentioned above. Alternatively, it could be simply a crystal packing artifact in the isolated SP domain. On the other hand, because of the presence of potential carbohydrates at N5, N9 and N142 in C2SP, the deletion patch is unlikely to be buried in the domain interfaces. The location of the Ba domain in FB is uncertain, but it should be in contact with both the vWFA and SP domains (Chamberlain et al., 1998; Hinshelwood and Perkins, 1998). In other multidomain SPs, the three most common domains preceding the SP domain are the EGF domain, the Kringle domain and the CCP module (Table II). However, only a few multidomain SP structures containing the EGF domains currently are available, e.g. factor Xa, factor IXa, factor VIIa and protein C (reviewed in Bode et al., 1997). Superposition of these structures onto FBSP showed that a similar C1–C122 disulfide bond is utilized to link the SP domain with its preceding domain. Similarly, the regions on the back of these SPs also mediate the domain interface. However, the FBSP- and C2SP-unique helix (E125b–A125g) in the motif on the back overlaps significantly with the core of the proximal EGF domain, suggesting that the vWFA–SP interface would be different from the EGF–SP interface. Table 2. Domain structures and cofactors of a few regulated SPsa SP Cofactor protein Domain structure Thrombin Thrombomodulin Gla-2×KR-SP Factor Xa Factor VIIIa/Va Gla-2×EGF-SP Factor IXa Factor VIIIa+Ca2+ Gla-2×EGF-SP Factor VIIa Tissue factor Gla-2×EGF-SP Protein C Protein S Gla-2×EGF-SP uPA uPA receptor EGF-KR-SP TPA Fibrin FD-EGF-2×KR-SP Plasmin Streptokinase/staphylokinase 5×KR-SP Tryptase Tetramerization SP C1r2 C1s2 As tetramer on C1q CUB-EGF-CUB-2× CP-SP MASP-1,-2 MBP CUB-EGF-CUB-2× CCP-SP FB/Bb C3b 3×CCP-vWFA-SP C2/C2a C4b 3×CCP-vWFA-SP Factor I Complement regulatory proteins FIMAC-SRCR-2×LDLRA-SP a Proteins: uPA, urokinase-type plasmin" @default.
• W2084527777 created "2016-06-24" @default.
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• W2084527777 date "2000-01-17" @default.
• W2084527777 modified "2023-10-16" @default.
• W2084527777 title "New structural motifs on the chymotrypsin fold and their potential roles in complement factor B" @default.
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