Complement factor D, a novel serine protease

Abstract

Factor D is unique among serine proteases in that it requires neither enzymatic cleavage for expression of proteolytic activity nor inactivation by a serpin for its control. Regulation of factor D activity is instead attained by a novel mechanism that depends on reversible conformational changes for expression and control of catalytic activity. These conformational changes are believed to be induced by the single natural substrate, C3bB, and to result in realignment of the catalytic triad, the specificity pocket, and the nonspecific substrate binding site, all of which have atypical conformations. Mutational studies have defined structural determinants responsible for these unique structural features of factor D and for the resultant low reactivity with synthetic esters.Keywords: alternative complement pathway; catalytic triad; complement; enzyme kinetics; mutagenesis; serine proteases Complement is an important effector system of host defense. It consists of multiple, functionally linked proteins that mediate acute inflammatory reactions, clearance of foreign cells and molecules, and killing of susceptible cells. Activation of the complement system is necessary for expression of these activities and proceeds via pathways consisting of successive enzymatic amplification steps (Campbell et al., 1988; Muller-Eberhard, 1988). The key event in complement activation is the cleavage of a single peptide bond on the a-chain of the third component of complement, C3. The reaction is catalyzed by either of two endopeptidases, termed C3-convertases. In the alternative pathway of complement activation, the enzymatic reaction leading to the formation of the C3-convertase is catalyzed by a serine protease, termed factor D. This enzyme exhibits unique functional properties that are ideally suited to its role as the initiating enzyme of an activation cascade. Unlike other mammalian serine proteases in blood, factor D requires neither enzymatic cleavage for expression of proteolytic activity nor inactivation by an inhibitor for its control. Instead, transition of factor D from the catalytically inactive to the active state seems t o be effected by fully reversible conformational changes. A description of the structural correlates of these unusual functional features has begun to emerge from recent crystallographic and mutational studies. In this paper, we will review these studies in the context of " ..

Manduca sexta Hemolymph Proteinase 21 Activates Prophenoloxidase-activating Proteinase 3 in an Insect Innate Immune Response Proteinase Cascade

“…Two other well known examples of this type of serine proteinase are mannose-binding lectin-associated serine proteinase-2 and horseshoe crab clotting factor C (35)(36). Complement factor D, although cleaved at a putative zymogen activation site, is not active until it binds to its natural protein substrate C3bB, which induces a realignment of the catalytic triad to a functional conformation (37). Factor D and HP21 have in common the properties of being relatively insensitive to inhibition by DFP (requiring concentrations of 10 -20 mM) and that they do not efficiently hydrolyze small peptide substrates 4 (25,(37)(38)(39).…”

“…Complement factor D, although cleaved at a putative zymogen activation site, is not active until it binds to its natural protein substrate C3bB, which induces a realignment of the catalytic triad to a functional conformation (37). Factor D and HP21 have in common the properties of being relatively insensitive to inhibition by DFP (requiring concentrations of 10 -20 mM) and that they do not efficiently hydrolyze small peptide substrates 4 (25,(37)(38)(39). 3 We propose that proHP21 is activated by a conformational change that occurs when proHP21 binds to HP14 and proPAP3.…”

Deciphering complement mechanisms: The contributions of structural biology

“…Factor D is the smallest in size of the complement proteases. It consists solely of a typical chymotrypsin-like serine protease domain (Volanakis and Narayana, 1996), while the other proteases are all endowed with additional structural modules. These modules are attached to the N-terminal ends of the respective serine protease domains.…”

“…Unlike most of the serine proteases, no zymogen form of factor D exists and no known serpin-like inhibitor for this enzyme has been identified in the circulation (Volanakis and Narayana, 1996). Activation and regulation of this unique enzyme remained a mystery for enzymologists until structural biologists were able to apply a combination of X-ray crystallography, sitedirected mutagenesis and measurements of enzyme kinetics to the problem.…”

“…Then factor D reverts back to the 'resting enzyme' status in the circulation. This unique method of proteolysis has been termed 'substrate-induced' catalysis (Volanakis and Narayana, 1996), in which the essential activation and regulation of factor D are carried out by subtle and reversible conformational changes that are possible mainly due to three amino acid substitutions at critical positions.…”