STRUCTURES OF NATIVE AND COMPLEXED COMPLEMENT FACTOR-D - IMPLICATIONSOF THE ATYPICAL HIS57 CONFORMATION AND SELF-INHIBITORY LOOP IN THE REGULATION OF SPECIFIC SERINE-PROTEASE ACTIVITY

Factor D is a serine protease essential for the activation of the alte rnative pathway of complement; The structures of native factor D and a complex formed with isatoic anhydride inhibitor were determined at re solution of 2.3 and 1.5 Angstrom, respectively, in an isomorphous mono clinic crystal form containing one molecule per asymmetric unit. The n ative structure was compared with structures determined previously in a triclinic cell containing two molecules with different active site c onformations. The current structure shows greater similarity with mole cule B in the triclinic cell, suggesting that this may be the dominant factor D conformation in solution. The major conformational differenc es with molecule A in the triclinic cell are located in four regions, three of which are close to the active site and include some of the re sidues shown to be critical for factor D catalytic activity. The confo rmational flexibility associated with these regions is proposed to pro vide a structural basis for the previously proposed substrate-induced reversible conformational changes in factor D. The high-resolution str ucture of the factor D/isatoic anhydride complex reveals the binding m ode of the mechanism-based inhibitor. The higher specificity towards f actor D over trypsin and thrombin is based on hydrophobic interactions between the inhibitor benzyl ring and the aliphatic side-chain of Arg 218 that is salt bridged with Asp189 at the bottom of the primary spec ificity (S1) pocket. Comparison of factor D structural variants with o ther serine protease structures revealed the presence of a unique ''se lf-inhibitory loop''. This loop (214-218) dictates the resting-state c onformation of factor D by (1) preventing His57 from adopting active t automer conformation, (2) preventing the P1 to P3 residues of the subs trate from forming anti-parallel beta-sheets with the non-specific sub strate binding loop, and (3) blocking the accessibility of Asp189 to t he positively charged P1 residue of the substrate. The conformational switch from resting-state to active-state can only be induced by the s ingle macromolecular substrate, C3b-bound factor B. This self-inhibito ry mechanism is highly correlated with the unique functional propertie s of factor D, which include high specificity toward factor B, low est erolytic activity toward synthetic substrates, and absence of regulati on by zymogen and serpin-like or other natural inhibitors in blood. (C ) 1998 Academic Press.