Understanding substrate binding and product release in cytochrome P450 (CYP) enzymes is important for explaining their key role in drug metabolism, toxicity, xenobiotic degradation and biosynthesis. Here, molecular simulations of substrate and product exit from the buried active site of a mammalian P450, the microsomal CYP2C5, identified a dominant exit channel, termed pathway (pw) 2c. Previous simulations with soluble bacterial P450s showed a different dominant egress channel, pw2a. Combining these, we propose two mechanisms in CYP2C5: (i) a one-way route by which lipophilic substrates access the enzyme from the membrane by pw2a and hydroxylated products egress along pw2c; and (ii) a two-way route for access and egress, along pw2c, for soluble compounds. The proposed differences in substrate access and product egress routes between membranebound mammalian P450s and soluble bacterial P450s highlight the adaptability of the P450 fold to the requirements of differing cellular locations and substrate specificity profiles.

Matrix metalloproteinases (MMPs) are involved in the remodeling processes of the extracellular matrix and the basement membrane. Most MMPs are composed of a regulatory, a catalytic, and a hemopexin subunit. In many tumors the expression of MMP-9 correlates with local tumor growth, invasion, and metastasis. To analyze the role of the hemopexin domain in these processes, the MMP-9 hemopexin domain (MMP-9-PEX) was expressed as a glutathione S-transferase fusion protein in Escherichia coli. After proteolytic cleavage, the isolated PEX domain was purified by size exclusion chromatography. In a zymography assay, MMP-9-PEX was able to inhibit MMP-9 activity. The association and dissociation rates for the interaction of MMP-9-PEX with gelatin were determined by plasmon resonance. From the measured rate constants, the dissociation constant was calculated to be K d ‫؍‬ 2,4 ؋ 10 ؊8 M, demonstrating a high affinity between MMP-9-PEX and gelatin. In Boyden chamber experiments the recombinant MMP-9-PEX was able to inhibit the invasion of melanoma cells secreting high amounts of MMP-9 in a dose-dependent manner. These data demonstrate for the first time that the hemopexin domain of MMP-9 has a high affinity binding site for gelatin, and the particular recombinant domain is able to block MMP-9 activity and tumor cell invasion. Because MMP-9 plays an important role in metastasis, this antagonistic effect may be utilized to design MMP inhibition-based cancer therapy.Matrix metalloproteinases (MMPs) 1 are a family of zinc metallo-endopeptidases secreted by cells. They are responsible for most of the turnover of matrix components. The MMPs are produced as zymogens with a signal sequence and propeptide segment that has to be removed during activation. The propeptide domain contains a conserved cysteine that chelates the zinc in the active site. The gelatinases MMP-2 and MMP-9 contain fibronectin type II domains that are inserted in the middle of the catalytic domain, presumably to enhance substrate binding (1). MMP-9 also has a collagen type V-like domain located between the catalytic and the C-terminal hemopexin domain (Fig. 1). All but two MMPs (MMP-7 and MMP-26) contain a regulatory subunit, the hemopexin domain, separated from the catalytic domain by a variable hinge region (2). This domain is thought to confer much of the substrate specificity to the MMPs (3). It is involved in activation as well as inhibition of MMPs (3,4) and may enhance substrate binding and specificity (5). The hinge region also confers specificity to the MMPs either by direct binding of the substrate or by setting the orientation of the hemopexin domain and the catalytic domain (6). The hemopexin domain of MMP-2 is known to bind heparin (7). Heparin has been shown to potentiate the activities of some MMPs, and MMPs are often found associated with heparin sulfate glycosaminoglycans on the cell surface (8). The overall three-dimensional structure of the hemopexin domain is a four-bladed propeller with a calcium binding site nestled in the folds (3). A fragme...

Amoebapore A is a 77-residue protein from the protozoan parasite and human pathogen Entamoeba histolytica. Amoebapores lyse both bacteria and eukaryotic cells by pore formation and play a pivotal role in the destruction of host tissues during amoebiasis, one of the most life-threatening parasitic diseases. Amoebapore A belongs to the superfamily of saposin-like proteins that are characterized by a conserved disulfide bond pattern and a fold consisting of five helices. Membrane-permeabilizing effector molecules of mammalian lymphocytes such as porcine NK-lysin and the human granulysin share these structural attributes. Several mechanisms have been proposed to explain how saposin-like proteins form membrane pores. All mechanisms indicate that the surface charge distribution of these proteins is the basis of their membrane binding capacity and pore formation. Here, we have solved the structure of amoebapore A by NMR spectroscopy. We demonstrate that the specific activation step of amoebapore A depends on a pH-dependent dimerization event and is modulated by a surface-exposed histidine residue. Thus, histidine-mediated dimerization is the molecular switch for pore formation and reveals a novel activation mechanism of pore-forming toxins.The protozoan parasite, Entamoeba histolytica, inhabits the colon of infected humans. It is the causative agent of human amoebiasis that often leads to tissue damage, colitis, and extraintestinal abscesses (1). Amoebiasis is the second leading cause of death from parasitic diseases worldwide (2). About 50 million people suffer from invasive amoebiasis, of whom up to 100,000 die annually (3).In the amoebic trophozoite, several factors have been identified that are involved in pathogenesis. In addition to a galactose-/N-acetylgalactosamine-specific lectin on the amoebic surface that mediates adhesion to colonic mucus and host cells (4) and secreted cysteine proteinases that disintegrate tissues by cleaving extracellular matrix proteins (5), a family of membrane-active polypeptides have been discovered (6). These polypeptides exist as three isoforms and are named amoebapore A, B, and C, respectively. They are capable of lysing a broad spectrum of target cells, including human host cells and bacteria. It has been recently shown that trophozoites of E. histolytica lacking amoebapore A, due to transcriptional silencing of the encoding gene, became avirulent (7), demonstrating that this protein is a key pathogenicity factor of the parasite. All three amoebapore isoforms have been isolated and biochemically characterized, and their primary structure has been elucidated by molecular cloning of the genes of their precursors (8 -10). The mature proteins consist of 77 amino acid residues each and are localized within cytoplasmic granules. The overall sequence identity between the three amoebapores is between 35 and 57% (10). Despite the substantial sequence divergence, they possess a characteristic disulfide bond pattern and a single conserved C-terminal histidine residue. The secondary structur...