Crystallographic study of coenzyme, coenzyme analogue and substrate binding in 6-phosphogluconate dehydrogenase: implications for NADP specificity and the enzyme mechanism

mPGES-1 (microsomal prostaglandin E synthase-1) is a newly recognized target for the treatment of inflammatory diseases. As the terminal enzyme of the prostaglandin production pathway, mPGES-1 inhibition may have a low risk of side effects. Inhibitors of mPGES-1 have attracted considerable attention as next-generation anti-inflammatory drugs. However, as mPGES-1 is a membrane protein, its enzymatic mechanism remains to be disclosed fully. We used MD (molecular dynamics) simulations, mutation analysis, hybrid experiments and co-IP (co-immunoprecipitation) to investigate the conformation transitions of mPGES-1 during catalysis. mPGES-1 forms a homotrimer with three substrate-binding sites (pockets). In the MD simulation, only one substrate molecule could bind to one of the pockets and form the active complex, suggesting that the mPGES-1 trimer has only one pocket active at any given time. This one-third-of-the-sites reactivity enzyme mechanism was verified further by hybridization experiments and MD simulations. The results of the present study revealed for the first time a novel one-third-of-the-sites reactivity enzyme mechanism for mPGES-1, and the unique substrate-binding pocket in our model constituted an active conformation that was suitable for further enzymatic mechanism study and structural-based drug design against mPGES-1.

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Fibronexus-Like Adhesion Sites are Present at the Invasive Zones of Human Epidermoid Carcinomas

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100053280 ◽

1982 ◽

Vol 40 ◽

pp. 106-109

Author(s):

Irwin I. Singer

Keyword(s):

Cell Cycle ◽

Serum Concentration ◽

Substrate Binding ◽

High Serum ◽

Adhesion Sites ◽

Substrate Adhesion ◽

Focal Contacts ◽

Adhesion Complex ◽

Low Serum

Our previous results indicate that two types of fibronectin-cytoskeletal associations may be formed at the fibroblast surface: dorsal matrixbinding fibronexuses generated in high serum (5% FBS) cultures, and ventral substrate-adhering units formed in low serum (0.3% FBS) cultures. The substrate-adhering fibronexus consists of at least vinculin (VN) and actin in its cytoplasmic leg, and fibronectin (FN) as one of its major extracellular components. This substrate-adhesion complex is localized in focal contacts, the sites of closest substratum approach visualized with interference reflection microscopy, which appear to be the major points of cell-tosubstrate adhesion. In fibroblasts, the latter substrate-binding complex is characteristic of cultures that are arrested at the G1 phase of the cell cycle due to the low serum concentration in their medium. These arrested fibroblasts are very well spread, flattened, and immobile.

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Polymorphic phase transition of a membrane protein - analysis of continuous diffuse electron diffraction intensity

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142451 ◽

1986 ◽

Vol 44 ◽

pp. 166-167

Author(s):

Douglas L. Dorset ◽

Andrew K. Massalski

Keyword(s):

Molecular Sieve ◽

Three Dimensional ◽

Pore Geometry ◽

Two Dimensional ◽

Diffraction Intensity ◽

Polymorphic Phase Transition ◽

E Coli ◽

Crystallographic Study ◽

Hexagonal Form ◽

Polymorphic Forms

Matrix porin, the ompF gene product of E. coli, has been the object of a electron crystallographic study of its pore geometry in an attempt to understand its function as a membrane molecular sieve. Three polymorphic forms have been found for two-dimensional crystals reconstituted in phospholipid, two hexagonal forms with different lipid content and an orthorhombic form coexisting with and similar to the hexagonal form found after lipid loss. In projection these have been shown to retain the same three-fold pore triplet geometry and analyses of three-dimensional data reveal that the small hexagonal and orthorhombic polymorphs have similar structure as well as unit cell spacings.

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