NMR structure of CmPI-II, a non-classical Kazal protease inhibitor: Understanding its conformational dynamics and subtilisin A inhibition.

Subtilisin-like proteases play crucial roles in host-pathogen interactions. Thus, protease inhibitors constitute important tools in the regulation of this interaction. CmPI-II is a Kazal proteinase inhibitor isolated from Cenchritis muricatus that inhibits subtilisin A, trypsin and elastases. Based on sequence analysis it defines a new group of non-classical Kazal inhibitors. Lacking solved 3D structures from this group prevents the straightforward structural comparison with other Kazal inhibitors. The 3D structure of CmPI-II, solved in this work using NMR techniques, shows the typical fold of Kazal inhibitors, but has significant differences in its N-terminal moiety, the disposition of the CysI-CysV disulfide bond and the reactive site loop (RSL) conformation. The high flexibility of its N-terminal region, the RSL, and the α-helix observed in NMR experiments and molecular dynamics simulations, suggest a coupled motion of these regions that could explain CmPI-II broad specificity. The 3D structure of the CmPI-II/subtilisin A complex, obtained by modeling, allows understanding of the energetic basis of the subtilisin A inhibition. The residues at the P2 and P2' positions of the inhibitor RSL were predicted to be major contributors to the binding free energy of the complex, rather than those at the P1 position. Site directed mutagenesis experiments confirmed the Trp14 (P2') contribution to CmPI-II/subtilisin A complex formation. Overall, this work provides the structural determinants for the subtilisin A inhibition by CmPI-II and allows the designing of more specific and potent molecules. In addition, the 3D structure obtained supports the existence of a new group in non-classical Kazal inhibitors.

Crystal structure of the complex of porcine pancreatic elastase with TEI-8362.

The crystal structure of porcine pancreatic elastase (PPE) complexed with a new benzoxazinone inhibitor, TEI-8362, of human neutrophil elastase (HNE) was determined at 1.8 A resolution. The hydroxyl oxygen of Ser195 opened the benzoxazinone by nucleophilic attack and formed a covalent bond with the carbonyl carbon. Hydrophobic interaction between the terminal benzene of TEI-8362 and the S4 pocket is reinforced by the side chain of Arg217 and has an impact on the ligand binding conformation. Two additional interactions with the oxyanion hole and His57 are introduced to the benzoxazinone structure of TEI-8362. These combinatorial interactions will also exist in HNE and cause high preference of TEI-8362 for HNE.

The effect of caloric restriction on the aortic tissue of aging rats.

Connective tissue shows peculiar and complex age-related modifications, which can be, at least in part, responsible for altered functions and increased susceptibility to diseases. Food restriction has long been known to prolong life in rodents, having antiaging effects on a variety of physiologic and pathologic processes. Therefore, the aorta has been investigated in rats fed normal or hypocaloric diet, from weaning to senescence. Compared with controls, caloric-restricted animals showed less pronounced age-dependent alterations such as elastic fiber degradation, collagen accumulation and cellular modifications. Immunocytochemical analyses revealed that elastic fibers were positively labelled for biglycan, decorin, ApoB100 (LDL), ApoA1 (HDL) and elastase and that the intensity of the reactions was time- and diet-dependent. With age, the major changes affecting aortic elastic fibers were increased positivity for decorin, LDL and elastase. Compared with age-matched normal fed rats, caloric restricted animals revealed lower content of LDL, decorin and elastase and higher positivity for HDL. These data suggest that a caloric restricted diet might influence the aging process of the arterial wall in rats, delaying the appearance of age-related degenerative features, such as structural alterations of cells and matrix and modified interactions of elastin with cells and with other extracellular matrix molecules.

Crystallization of a complex between an elastase-specific inhibitor elafin and porcine pancreatic elastase.

A complex between synthetic elafin, an elastase-specific inhibitor, and porcine pancreatic elastase was crystallized using 2-methyl-2,4-pentanediol as precipitant. The crystals belong to the monoclinic space group P2(1) with cell parameters a = 37.91 A, b = 73.32 A, c = 48.92 A, beta = 105.4 degrees, and one complex molecule in the asymmetric unit. The crystals diffract X-rays to 1.9 A resolution and are suitable for crystallographic structure analysis.

Low-resolution real-space envelopes: an approach to the ab initio macromolecular phase problem.

An ab initio approach to the phase problem in macromolecular x-ray crystallography is described. A random gas of hard-sphere point scatterers is allowed to condense under the constraint of the solvent fraction and the restraint of the observed Fourier amplitude data. Two applications to real macromolecular examples are discussed. This method produces an approximate outline of the bulk solvent regions and thus yields a low-resolution picture of the unit cell that can be extended to higher resolutions in special cases, such as through the use of molecular replacement or of noncrystallographic symmetry-based phase extension.