Calpains (calcium-dependent cytoplasmic cysteine proteinases) are implicated in processes such as cytoskeleton remodeling and signal transduction. The 2.3-Å crystal structure of full-length heterodimeric [80-kDa (dI-dIV) ؉ 30-kDa (dV؉dVI)] human m-calpain crystallized in the absence of calcium reveals an oval disc-like shape, with the papain-like catalytic domain dII and the two calmodulin-like domains dIV؉dVI occupying opposite poles, and the tumor necrosis factor ␣-like ␤-sandwich domain dIII and the N-terminal segments dI؉dV located between. Compared with papain, the two subdomains dIIa؉dIIb of the catalytic unit are rotated against one another by 50°, disrupting the active site and the substrate binding site, explaining the inactivity of calpains in the absence of calcium. Calcium binding to an extremely negatively charged loop of domain dIII (an electrostatic switch) could release the adjacent barrel-like subdomain dIIb to move toward the helical subdomain dIIa, allowing formation of a functional catalytic center. This switch loop could also mediate membrane binding, thereby explaining calpains' strongly reduced calcium requirements in vivo. The activity status at the catalytic center might be further modulated by calcium binding to the calmodulin domains via the Nterminal linkers.T he calpains (EC 3.4.22.17; Clan CA, family C02) are a family of calcium-dependent cytosolic cysteine proteinases. They seem to catalyze limited proteolysis of proteins involved in cytoskeletal remodeling and signal transduction but are also implicated in other physiological and pathophysiological processes, such as cell cycle regulation, apoptosis, muscular dystrophies, cataractogenesis, and Alzheimer's or Parkinson's diseases (1-5). In mammals, the calpain family comprises several ''tissuespecific'' isoforms (n-calpains) besides two ''ubiquitous'' isoenzymes (-and m-calpains). In lower organisms such as insects, nematodes, fungi, and yeast, a number of ''atypical'' calpain homologues have been found.The ubiquitous -and m-calpains (calpains I and II), by far the best characterized calpains, are heterodimers comprising distinct but quite homologous 80-kDa ''large'' L-subunits and a common 30-kDa ''small'' S-subunit. On the basis of amino acid homologies, the L-and S-subunits have been described as consisting of four domains, dI to dIV, and of two domains, dV and dVI, respectively, with domain dII somewhat resembling papain and the calmodulin-like domains dIV and dVI containing EF-hands (6, 7). On exposure to calcium at concentrations of 5-50 M (-calpain) and 200-1,000 M (m-calpain), both calpains are activated and partially autolyzed. In vivo, both calpains seem to be active at physiological calcium concentrations of 100-300 nM, however, suggesting that other factors such as phospholipids might play a role in activation in addition.The crystal structures of rat and porcine domain dVI in the absence and presence of calcium have been determined (8, 9). For a full understanding of the activation mechanism and the functioning of calp...

We determined the temporal dynamics of cambial activity and xylem cell differentiation of Scots pine (Pinus sylvestris L.) within a dry inner Alpine valley (750 m a.s.l., Tyrol, Austria), where radial growth is strongly limited by drought in spring. Repeated micro-sampling of the developing tree ring of mature trees was carried out during two contrasting years at two study plots that differ in soil water availability (xeric and dry-mesic sites). In 2007, when air temperature at the beginning of the growing season in April exceeded the long-term mean by 6.4 degrees C, cambial cell division started in early April at both study plots. A delayed onset of cambial activity of c. 2 weeks was found in 2008, when average climate conditions prevailed in spring, indicating that resumption of cambial cell division after winter dormancy is temperature controlled. Cambial cell division consistently ended about the end of June/early July in both study years. Radial enlargement of tracheids started almost 3 weeks earlier in 2007 compared with 2008 at both study plots. At the xeric site, the maximum rate of tracheid production in 2007 and 2008 was reached in early and mid-May, respectively, and c. 2 weeks later at the dry-mesic site. Since in both study years more favorable growing conditions (i.e., an increase in soil water content) were recorded during summer, we suggest a strong sink competition for carbohydrates to mycorrhizal root and shoot growth. Wood formation stopped c. 4 weeks earlier at the xeric compared with the dry-mesic site in both years, indicating a strong influence of drought stress on cell differentiation. This is supported by radial widths of earlywood cells, which were found to be significantly narrower at the xeric than at the dry-mesic site (P < 0.05). Repeated cellular analyses during the two growing seasons revealed that, although spatial variability in the dynamics and duration of cell differentiation processes in P. sylvestris exposed to drought is strongly influenced by water availability, the onset of cambial activity and cell differentiation is controlled by temperature.

The binding of RBI to TMA constitutes a new inhibition mechanism for alpha-amylases and should be general for all alpha-amylase inhibitors of the cereal inhibitor superfamily. Because RBI inhibits two important digestive enzymes of animals, it constitutes an efficient plant defense protein and may be used to protect crop plants from predatory insects.

The three-dimensional structure of the bifunctional alpha-amylase/trypsin inhibitor (RBI) from seeds of ragi (Eleusine coracana Gaertneri) has been determined in solution using multidimensional 1H and 15N NMR spectroscopy. The inhibitor consists of 122 amino acids, with 5 disulfide bridges, and belongs to the plant alpha-amylase/trypsin inhibitor family for which no three-dimensional structures have yet been available. The structure of the inhibitor was determined on the basis of 1131 interresidue interproton distance constraints derived from nuclear Overhauser enhancement measurements and 52 phi angles, supplemented by 9 psi and 51 chi 1 angles. RBI consists of a globular four-helix motif with a simple "up-and-down" topology. The helices are between residues 18-29, 37-51, 58-65, and 87-94. A fragment from Val 67 to Ser 69 and Gln 73 to Glu 75 forms an antiparallel beta-sheet. The fold of RBI represents a new motif among the serine proteinase inhibitors. The trypsin binding loop of RBI adopts the "canonical", substrate-like conformation which is highly conserved among serine proteinase inhibitors. The binding loop is stabilized by the two adjacent alpha-helices 1 and 2. This motif is also novel and not found in known structures of serine proteinase inhibitors. The three-dimensional structure of RBI together with biochemical data suggests the location of the alpha-amylase binding site on the face of the molecule opposite to the site of the trypsin binding loop. The RBI fold should be general for all members of the RBI family because conserved residues among the members of the family from the core of the structure.