The role of Asn14 in the stability and conformation of the reactive-site loop of winged bean chymotrypsin inhibitor: crystal structures of two point mutants Asn14-->Lys and Asn14-->Asp.

A double-headed chymotrypsin inhibitor, WCI, from winged bean seeds was cloned for structural and biochemical studies. The inhibitor was subjected to two point mutations at a conserved position, Asn14. This residue, known to have a pivotal role in stabilizing the first reactive-site loop (Gln63-Phe68) of the inhibitor, is highly conserved in the sequences of the other members of Kunitz (STI) family as well as in the sequences of Kazal family of serine protease inhibitors. The mutants, N14K and N14D, were subjected to biochemical assay and their characteristics were compared with those of the recombinant inhibitor (rWCI). Crystallographic studies of the recombinant and the mutant proteins are discussed. These studies were primarily aimed at understanding the importance of the protein scaffolding towards the conformational rigidity of the reactive-site loop. Our analysis reveals that, as the Lys14 side chain takes an unusual fold in N14K and the Asp14 side chain in N14D interacts with the loop residues by water-mediated hydrogen bonds, the canonical conformation of the loop has remained effectively intact in both the mutant structures. However, minor alterations such as a 2-fold increase in the inhibitory affinity towards the cognate enzyme were observed.     

Potential antiradiation drugs containing no nitrogen, and related compounds

Capabilities are reported of di- and higher sulfides (RSnR') terminated by sulfinate functions [-S(O)O-] for protecting mice against otherwise lethal effects of ionizing radiation. With the use of congeners, structure-activity correlations are developed for the effects of esterification of the sulfinate function, of changing the length of the chain of sulfur atoms, of reduction to a mercapto sulfinate, and of changing the substituents R and R' to chiral and other types of groups. Neither a trisulfide nor a sulfinate by itself was significantly radioprotective. The key requirement for radio-protection in the series appears to be the presence of a sulfur function (-Sn-) from which a thiol can be engendered by a neighboring-group effect of an electron-donating group; sulfoxide functions may afford alternatives to sulfinate functions as such neighboring groups. The relevance of structure-activity relations to the chemical and biological mechanisms involved in the radioprotective activities is discussed.     

Three-dimensional structure of Gln25-ribonuclease T1 at 1.84-A resolution: structural variations at the base recognition and catalytic sites.

The structure of the Gln25 variant of ribonuclease T1 (RNase T1) crystallized at pH 7 and at high ionic strength has been solved by molecular replacement using the coordinates of the Lys25-RNase T1/2'-guanylic acid (2'GMP) complex at pH 5 [Arni et al. (1988) J. Biol. Chem. 263, 15358-15368] and refined by energy minimization and stereochemically restrained least-squares minimization to a crystallographic R-factor of 14.4% at 1.84-A resolution. The asymmetric unit contains three molecules, and the final model consists of 2302 protein atoms, 3 sulfates (at the catalytic sites), and 179 solvent water molecules. The estimated root mean square (rms) error in the coordinates is 0.15 A, and the rms deviation from ideality is 0.018 A for bond lengths and 1.8 degrees for bond angles. Significant differences are observed between the three molecules in the asymmetric unit at the base recognition and catalytic sites.     

Lck-dependent tyrosyl phosphorylation of the phosphotyrosine phosphatase SH-PTP1 in murine T cells.

The phosphorylation and dephosphorylation of proteins on tyrosyl residues are key regulatory mechanisms in T-cell signal transduction and are controlled by the opposing activities of protein tyrosine kinases and phosphotyrosyl phosphatases (PTPs). In T cells, several nontransmembrane protein tyrosine kinases are associated with receptors; for example, Lck is bound to the coreceptors CD4 and CD8 and becomes activated upon their stimulation. In comparison, little is known about the role of nontransmembrane PTPs in early T-cell signaling. SH-PTP1 (PTP1C, HCP, SHP) is a nontransmembrane PTP expressed primarily in hematopoietic cells, including T cells. We have found that SH-PTP1 is basally phosphorylated on serine in resting T cells. Upon stimulation of CD4 or CD8 either in a T-cell hybridoma cell line or in primary thymocytes, SH-PTP1 becomes tyrosyl phosphorylated. Moreover, SH-PTP1 is constitutively phosphorylated on tyrosine in the Lck-overexpressing lymphoma cell line LSTRA. SH-PTP1 is also a good substrate for recombinant Lck in vitro. Comparisons of the tryptic phosphopeptide maps of wild-type SH-PTP1 and deletion and point mutations establish that the two sites (Y-536 and Y-564) which are directly phosphorylated by Lck in vitro are also phosphorylated in vivo in LSTRA cells. One of these sites (Y-564) is phosphorylated in T cells in response to Lck activation. We conclude that SH-PTP1 undergoes Lck-dependent tyrosyl phosphorylation in T cells and likely plays a role in early T-cell signaling.     

Crystal and molecular structure of human plasminogen kringle 4 refined at 1.9-A resolution.

The crystal structure of human plasminogen kringle 4 (PGK4) has been solved by molecular replacement using the bovine prothrombin kringle 1 (PTK1) structure as a model and refined by restrained least-squares methods to an R factor of 14.2% at 1.9-A resolution. The K4 structure is similar to that of PTK1, and an insertion of one residue at position 59 of the latter has minimal effect on the protein folding. The PGK4 structure is highly stabilized by an internal hydrophobic core and an extensive hydrogen-bonding network. Features new to this kringle include a cis peptide bond at Pro30 and the presence of two alternate, perpendicular, and equally occupied orientations for the Cys75 side chain. The K4 lysine-binding site consists of a hydrophobic trough formed by the Trp62 and Trp72 indole rings, with anionic (Asp55/Asp57) and cationic (Lys35/Arg71) charge pairs at either end. With the adjacent Asp5 and Arg32 residues, these result in triply charged anionic and cationic clusters (pH of crystals at 6.0), which, in addition to the unusually high accessibility of the Trp72 side chain, serve as an obvious marker of the binding site on the K4 surface. A complex intermolecular interaction occurs between the binding sites of symmetry-related molecules involving a highly ordered sulfate anion of solvation in which the Arg32 side chain of a neighboring kringle occupies the binding site.     

Ensuring the adaptive potential of Coastal wetlands of India- the need of the hour for sustainable management

India is endowed with a variety of coastal wetlands viz., mangroves, seagrasses, saltmarshes, coral reefs, lagoons and tidal flats, and the country is also a signatory to the Ramsar Convention on Wetlands and the Convention of Biological Diversity, besides having a robust framework of laws and policies, governing the wetland conservation. However, the conservation strategies can better be improved in the context of increasing pressures and threats and limited success of restoration/rehabilitation. Land conversion and ecological degradation of coastal wetlands are the stressors, associated with rapid coastal developmental activities and climate change. The coastal wetlands require desired habitat niche and hence, the conversion of coastal wetlands to other land uses (including agricultural and urban lands) may lead to permanent loss, whereas ecologically degraded coastal wetlands may be resilient if supported by effective protection measures. Preventing the habitat conversion and maximizing the adaptive potential (viz., the ability of populations or species to adapt to rapid environmental change with minimal disruption) by preserving the ecological health are the need of the hour to safeguard the existing coastal wetlands and sustain the provisional ecosystem services offered by them rather than short-term increase in area by unproductive restoration/rehabilitation efforts. Since coastal wetlands are flow through ecosystems, preserving the hydrological connectivity, facilitating the connectivity between adjacent ecosystems and protection of natural corridors are potential strategies that are required to enhance the adaptive potential of coastal wetlands. This analysis calls for site-specific, long-term and integrated ecosystem-based protection, management and rehabilitation strategies based on scientific principles and enforcing the effective legislative measures to regularize the coastal developmental activities in India.