X-ray crystallographic analysis of protease/inhibitor complexes and fibrinogen binding proteins

• Main Author: Bice, William Todd
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2003
• Subjects: Biochemistry; Molecular biology
• ISBN: 9780496795291; 0496795295

In this work, the structures of seven protein and protein-inhibitor complexes have been determined using X-ray protein crystallography techniques. The proteins are divided into three main groups: calcium (Ca2+)-activated proteolytic system, trypsin (TRY)/factor D (FD) bound to the small molecule inhibitor, 4-[2-aminoethyl] benzenesulfonyl fluoride (PEFA) and fibrinogen-binding adhesions from Staphylococcus aureus. The Ca2+-activated proteolytic system is composed of a cysteine protease, calpain, along with its endogenous inhibitor calpastatin. Presented are the crystal structure of a complex between a calpastatin peptide and the calcium-binding domain DVI of calpain. Two crystal structures of a nineteen residue peptide corresponding to the C region of the first inhibitory domain (DIC19) bound to DVI of calpain were determined by molecular replacement (MR) methods to 2.5 Å and 2.2 Å resolution. The crystal structures of the native DVI and its inhibitor 3-(4-iodophenyl)-2-mercapto-(Z)-2-propenoic acid complex were determined with the help of MR methods to 2.0 Å and 2.3 Å resolutions, respectively. FD is a TRY-like serine proteases which is critical for the activation of the alternative pathway of complement through cleavage of factor B bound to C3b. Presented are the crystal structures of a complex between the serine proteases, bovine pancreatic β-TRY and human FD, with PEFA. The two structures were determined with the help of MR methods to 1.9 Å and 2.4 Å, respectively. In both of the inhibitor complexes, PEFA covalently binds the catalytic triad Ser195 and extends away from the S1 pocket. The β-TRY complex also contains the additional non-specific serine protease inhibitor benzamidine, which is in the S1 pocket salt bridged to Asp189. Clumping factor B (CLFB) of S. aureus is a surface protein that binds to fibrinogen. We previously localized the fibrinogen binding to the C-terminal portion of the clumping factor A region. Presented here is the crystal structure of the minimum ligand binding region of CLFB determined by multiple isomorphous replacement methods to 2.5 Å resolution. The polypeptide consists of two independently folded domains that have a composition dominated by a DE-variant of the immunoglobulin-like β-sheet secondary structure. Mutagenesis studies revealed that the N and C-terminal residues, which interact extensively with neighbouring molecules in the crystal structure, are critical for the fibrinogen binding.