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.     

Graphical representation of the salient conformational features of protein residues.

A composite plot for depicting in two dimensions the conformation and the secondary structural features of protein residues has been developed. Instead of presenting the exact values of the main- and side-chain torsion angles (φ, psi and chi(1)), it indicates the region in the three-dimensional conformational space to which a residue belongs. Other structural aspects, like the presence of a cis peptide bond and disulfide linkages, are also displayed. The plot may be used to recognize patterns in the backbone and side-chain conformation along a polypeptide chain and to compare protein structures derived from X-ray crystallography, NMR spectroscopy or molecular modelling studies and also to highlight the effect of mutation on structure.     

Complex carbohydrate-lectin interaction at the interface: a model for cellular adhesion. I. Effect of vesicle size on the kinetics of aggregation between a fatty acid conjugate of lectin and a liposomal asialoganglioside

Two types of phospholipid vesicles capable of mutual recognition have been tailor-made to serve as a model system for the study of carbohydrate-mediated cellular adhesion. One of the vesicles contained a fatty acid conjugate of a galactose specific lectin (lectin vesicle) and the other an asialoganglioside with a reactive terminal galactose residue (galactose vesicle). The kinetics of aggregation of these two types of vesicles was followed by monitoring time-dependent change in turbidity. A 10-100-fold enhancement in the forward rate constant (kf ranging from 7.1 x 10(5) to 4.5 x 10(7) M-1.s-1 at 27 degrees C) was observed when compared with that for the lectin-galactose system in solution (kf being 4.5 x 10(5) M-1.s-1), reported in the literature. A study of the influence of vesicle size on the rate of aggregation showed that enhancement depended on the curvature of the galactose vesicle rather than the density of asialoganglioside suggesting a possible diffusion in the plane of the membrane. The ratio, kf/kd is found to be approx. 10(10) M-1 indicating that the formation of multiple bonds plays a role for stable adhesion.     

Chemical modification studies on a lectin from Saccharomyces cerevisiae (baker''s yeast).

The effect of chemical modification on a galactose-specific lectin isolated from a fatty acid auxotroph of Saccharomyces cerevisiae was investigated in order to identify the type of amino acids involved in its agglutinating activity. Modification of 50 free amino groups with succinic anhydride or citraconic anhydride led to an almost complete loss of activity. This could not be protected by the inhibitory sugar methyl alpha-D-galactopyranoside. Treatment with N-bromosuccinimide and N-acetylimidazole, for the modification of tryptophan and tyrosine residues, did not affect lectin activity. Modification of carboxy groups with glycine ethyl ester greatly affected lectin activity, although sugars afford partial protection. Modification of four thiol groups with N-ethylmaleimide was accompanied by a loss of 85% of the agglutinating activity, and two thiol groups were found to be present at the sugar-binding site of the lectin. Modification of 18 arginine residues with cyclohexane-1,2-dione and 26 histidine residues with ethoxyformic anhydride led to a loss of lectin activity. However, in these cases, modification was not protected by the abovementioned inhibitory sugar, suggesting the absence of these groups at the sugar-binding site. In all the cases, immunodiffusion studies with modified lectin showed no gross structural changes which could disrupt antigenic sites of the lectin.     

Structural investigation of Klebsiella serotype K10 capsular polysaccharide

The primary structure of Klebsiella serotype K10 capsular polysaccharide has been investigated using mainly the techniques of methylation, partial hydrolysis, and 1H and 13C NMR spectroscopy. The polysaccharide was found to consist of hexasaccharide repeating units having the following structure: (formula; see text)     

Geometry of interaction of metal ions with sulfur-containing ligands in protein structures

An analysis of the geometry of binding of metal ions by cysteine and methionine residues in protein structures has been made by using the Protein Data Bank. Metal ions have a distinct model of binding to each of these residues, and this is independent of the nature of the metal center or the type of protein. Metal ions tend to approach the sulfur of Met roughly 38 degrees from the perpendicular to the plane through atoms C gamma-S delta-C epsilon. For Cys, the approach direction is such that the M...S gamma-C beta-C alpha torsional angle is about +/- 90 or 180 degrees. The side-chain conformation of the cysteine residue is affected by the presence of the metal ion; there is a shift from the g+ conformation toward g- and mainly t conformations. When two Cys residues at positions i-3 and i bind to the same metal center, there appears to be some restriction on the geometry of metal binding by the residue i; for such a residue chi 1 and M...S gamma-C beta-C alpha angles are likely to be around 60 degrees and 270 degrees, respectively. Met and Cys residues coordinating to a metal ion are usually from coil or turn regions of the protein structure.     

Abnormalities of the erythrocyte membrane in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is a hematologic malignancy characterized by excessive growth of myeloid cells and their progenitors. The proportion of spectrin dimers compared to tetramers extracted from membranes at 4 degrees C, under low ionic strength conditions, increased in CML erythrocytes. These also displayed abnormal thermal sensitivity (between 45 and 46 instead of 49 degrees C). Crosslinking with the bifunctional reagent, dimethyl adipimidate (8.6 A) showed significant organizational modification of not only spectrin, but other cytoskeletal components such as ankyrin, bands 4.2 and 5. Enhanced concanavalin A (Con-A) agglutinability of CML erythrocytes also suggests altered topographic distribution of a functionally important membrane protein, band 3. The anion transport activities of erythrocytes from patients with CML and normal donors were comparable. In CML erythrocytes, significant reduction in the number of ankyrin-binding sites, present in the cytoplasmic domain of band 3, may lead to partial loss of cytoskeletal anchorage to the bilayer and account for their increased Con-A agglutinability and heat sensitivity and may lead to their premature removal from the circulation.