Structural similarity and functional diversity in proteins containing the legume lectin fold.

Knowledge of structural relationships in proteins is increasingly proving very useful for in silico characterizations and is also being exploited as a prelude to almost every investigation in functional and structural genomics. A thorough understanding of the crucial features of a fold becomes necessary to realize the full potential of such relationships. To illustrate this, structures containing the legume lectin-like fold were chosen for a detailed analysis since they exhibit a total lack of sequence similarity among themselves and also belong to diverse functional families. A comparative analysis of 15 different families containing this fold was therefore carried out, which led to the determination of the minimal structural principles or the determining region of the fold. A critical evaluation of the structural features, such as the curvature of the front sheet, the presence of the hydrophobic cores and the binding site loops, suggests that none of them are crucial for either the formation or the stability of the fold, but are required to generate diversity and specificity to particular carbohydrates. In contrast, the presence of the three sheets in a particular geometry and also their topological connectivities seem to be important. The fold has been shown to tolerate different types of protein-protein associations, most of them exhibiting different types of quaternary associations and some even existing as complexes with other folds. The function of every family in this study is discussed with respect to its fold, leading to the suggestion that this fold can be linked to carbohydrate recognition in general.

Crystal structures of Mycobacterium tuberculosis RecA and its complex with ADP-AlF(4): implications for decreased ATPase activity and molecular aggregation.

Sequencing of the complete genome of Mycobacterium tuberculosis, combined with the rapidly increasing need to improve tuberculosis management through better drugs and vaccines, has initiated extensive research on several key proteins from the pathogen. RecA, a ubiquitous multifunctional protein, is a key component of the processes of homologous genetic recombination and DNA repair. Structural knowledge of MtRecA is imperative for a full understanding of both these activities and any ensuing application. The crystal structure of MtRecA, presented here, has six molecules in the unit cell forming a 6(1) helical filament with a deep groove capable of binding DNA. The observed weakening in the higher order aggregation of filaments into bundles may have implications for recombination in mycobacteria. The structure of the complex reveals the atomic interactions of ADP-AlF(4), an ATP analogue, with the P-loop-containing binding pocket. The structures explain reduced levels of interactions of MtRecA with ATP, despite sharing the same fold, topology and high sequence similarity with EcRecA. The formation of a helical filament with a deep groove appears to be an inherent property of MtRecA. The histidine in loop L1 appears to be positioned appropriately for DNA interaction.

Variability in quaternary association of proteins with the same tertiary fold: a case study and rationalization involving legume lectins.

Legume lectins constitute a family of proteins in which small alterations arising from sequence variations in essentially the same tertiary structure lead to large changes in quaternary association. All of them are dimers or tetramers made up of dimers. Dimerization involves side-by-side or back-to-back association of the flat six-membered beta-sheets in the protomers. Variations within these modes of dimerization can be satisfactorily described in terms of angles defining the mutual disposition of the two subunits. In all tetrameric lectins, except peanut lectin, oligomerization involves the back-to-back association of side-by-side dimers. An attempt has been made to rationalize the observed modes of oligomerization in terms of hydrophobic surface area buried on association, interaction energy and shape complementarity, by constructing energy minimised models in each of which the subunit of one legume lectin is fitted in the quaternary structure of another. The results indicate that all the three indices favor and, thus, provide a rationale for the observed arrangements. However, the discrimination provided by buried hydrophobic surface area is marginal in a few instances. The same is true, to a lesser extent, about that provided by shape complementarity. The relative values of interaction energy turns out to be a still better discriminator than the other two indices. Variability in the quaternary association of homologous proteins is a widely observed phenomenon and the present study is relevant to the general problem of protein folding.

Carbohydrate specificity and quaternary association in basic winged bean lectin: X-ray analysis of the lectin at 2.5 A resolution.

The structure of basic Winged Bean Agglutinin (WBAI) with two dimeric molecules complexed with methyl-alpha-D-galactopyranoside in the asymmetric unit, has been determined by the molecular replacement method and refined with 2.5 A X-ray intensity data. The polypeptide chain of each monomer has the characteristic legume lectin tertiary fold. The structure clearly defines the lectin-carbohydrate interactions. It reveals how the unusually long variable loop in the binding region endows the lectin with its characteristic sugar specificity. The lectin forms non-canonical dimers of the type found in Erythrina corallodendron lectin (EcorL) even though glycosylation, unlike in EcorL, does not prevent the formation of canonical dimers. The structure thus further demonstrates that the mode of dimerisation of legume lectins is not necessarily determined by the covalently bound carbohydrate but is governed by features intrinsic to the protein. The present analysis and our earlier work on peanut lectin (PNA), show that legume lectins are a family of proteins in which small alterations in essentially the same tertiary structure lead to wide variations in quaternary association. A relationship among the non-canonical modes of dimeric association in legume lectins is presented.

X-ray studies on crystalline complexes involving amino acids and peptides. XXXII. Effect of chirality on ionisation state, stoichiometry and aggregation in the complexes of oxalic acid with DL- and L-lysine.

Crystals of the oxalic acid complex of DL-lysine (triclinic P1; a = 5.540(1), b = 10.764(2), c = 12.056(2) A, alpha = 77.8(1), beta = 80.6(1), gamma = 75.6(1).; R = 4.7% for 2023 observed reflections) contain lysine and semioxalate ions in the 1:1 ratio, whereas the ratio of lysine and semioxalate/oxalate ions is 2:3 in the crystals of the L-lysine complex (monoclinic P2(1); alpha = 4.906(1), b = 20.145(4), c = 12.455(1) A, beta = 92.5(1).; R = 4.4% for 1494 observed reflections). The amino acid molecule in the L-lysine complex has an unusual ionisation state with positively charged alpha- and side-chain amino groups and a neutral carboxyl group. The unlike molecules aggregate into separate alternating layers in the DL-lysine complex in a manner similar to that observed in several of the amino acid complexes. The L-lysine complex exhibits a new aggregation pattern which cannot be easily explained in terms of planar features, thus emphasizing the fundamental dependence of aggregation on molecular characteristics. Despite the differences in stoichiometry, ionisation state and long-range aggregation patterns, the basic element of aggregation in the two complexes exhibits considerable similarity.

X-ray studies on crystalline complexes involving amino acids and peptides. XXXI. Effect of chirality on ionization state, stoichiometry and aggregation in the complexes of oxalic acid with L- and DL-histidine.

Crystals of the oxalic acid complex of L-histidine (orthorhombic P2(1)2(1)2(1); a = 5.535(4), b = 6.809(4), c = 26.878(3) A; R = 3.6% for 1188 observed reflections) contain histidine molecules and semi-oxalate ions in the 1:1 ratio, while the ratio is 1:2 in the crystals of the DL-histidine complex (monoclinic P2(1)lc; a = 6.750(7), b = 10.139(2), c = 19.352(2) A, beta = 90.8 degrees; R = 3.7% for 3176 observed reflections). The histidine molecule in the latter has an unusual ionization state with positively charged amino and imidazole groups and a neutral carboxyl group. The molecule has the sterically least favourable allowed conformation with the side chain imidazole ring staggered between the alpha-amino and the alpha- carboxyl (carboxylate) groups, in both the structures. The unlike molecules aggregate into separate alternating layers in both of them. There are elements of similarity in the aggregation patterns in the semi-oxalate layers in the two complexes, but the patterns in the amino acid layers are entirely different. Interestingly, the crystal structure of L-histidine semi-oxalate has broad similarities with that of DL-histidine = glycolate, demonstrating how broad features of aggregation could be retained inspite of changes in chirality and composition. The unusual ionization state of the amino acid molecule in the DL-histidine complex is reflected in a hitherto unobserved aggregation pattern in its crystal structure.