Mutational Analysis of the Primary Substrate Specificity Pocket of Complement Factor B

Abstract

Factor B is a serine protease, which despite its trypsin-like specificity has Asn instead of the typical Asp at the bottom of the S 1 pocket (position 189, chymotrypsinogen numbering). Asp residues are present at positions 187 and 226 and either one could conceivably provide the negative charge for binding the P 1 -Arg of the substrate. Determination of the crystal structure of the factor B serine protease domain has revealed that the side chain of Asp 226 is within the S 1 pocket, whereas Asp 187 is located outside the pocket. To investigate the possible role of these atypical structural features in substrate binding and catalysis, we constructed a panel of mutants of these residues. Replacement of Asp 187 caused moderate (50 -60%) decrease in hemolytic activity, compared with wild type factor B, whereas replacement of Asn 189 resulted in more profound reductions (71-95%). Substitutions at these two positions did not significantly affect assembly of the alternative pathway C3 convertase. In contrast, elimination of the negative charge from Asp 226 completely abrogated hemolytic activity and also affected formation of the C3 convertase. Kinetic analyses of the hydrolysis of a P 1 -Arg containing thioester by selected mutants confirmed that residue Asp 226 is a primary structural determinant for P 1 -Arg binding and catalysis.Complement is a major effector system of host defense. Activation of complement leads to the generation of protein fragments and protein-protein complexes that mediate acute inflammatory responses, phagocytosis and killing of pathogens, and regulation of adaptive immune responses. Activation-associated production of biologically active protein fragments is catalyzed by a group of eight atypical complement serine proteases (SPs) 1 of the chymotrypsin superfamily (1). Understanding the structural basis for the highly restricted proteolytic activity of these SPs is an important first step toward pharmacologic control of complement activation (2). Members of the chymotrypsin family have very similar three-dimensional structures but distinct substrate specificities. To a great extent specificity is determined by the side chains of the amino acid residues that line up the primary substrate specificity pocket (S 1 site). The pocket has three walls formed by residues 189 -195, 214 -220, and 225-228 (chymotrypsinogen numbering has been used for all SPs or SP domains throughout this paper) (3). The presence at the bottom of the pocket of Asp 189 endows trypsin with preference for positively charged Arg and Lys residues (4, 5), whereas in chymotrypsin the specificity for bulky aromatics is largely determined by Ser 189 (6). Residues at position 216 and 226 also contribute to substrate specificity (7). All complement SPs exhibit trypsinlike specificity for positively charged Arg residues and all have an Asp at position 189, except for factor B and C2 (Fig. 1).Factor B and C2 are structurally similar modular proteins that play a central role in complement activation by providing the catalytic subunits of...

New structural motifs on the chymotrypsin fold and their potential roles in complement factor B

“…The esterolytic activity of the mutant is also substantially reduced compared with the wild‐type, mainly due to a reduction in the k cat 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).…”

“…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.…”

Origin and Expansion of the Serine Protease Repertoire in the Myelomonocyte Lineage

“…In the human genome, the only examples are CFD, CFB, and a C1r-like serine protease, called C1r-LP. The aspartate residue at position 189 is replaced by serine residues in factor C2 and C1r-LP and by asparagine in CFB [43][44][45]. The C1r-related serine protease is synthesized as a complex-type zymogen and is able to cleave the haptoglobin precursor chain already very early in the ER after an arginine residue [44,46].…”

Deciphering complement mechanisms: The contributions of structural biology

“…However, the crystal structures of factor B (Milder et al, 2007;Ponnuraj et al, 2004), C2 (Krishnan et al, 2007;Milder et al, 2006) and their individual domains (Bhattacharya et al, 2004;Xu et al, 2000), in combination with site-directed mutagenesis and enzyme kinetic studies, revealed some interesting structural details that correlate with both the narrow substrate specificity and the unique activating mechanisms of these enzymes. These findings revealed that the four essential structural features (Fig.…”