M. Vijayan

A novel mode of carbohydrate recognition in jacalin, a Moraceae plant lectin with a β-prism fold

Jacalin, a tetrameric two-chain lectin (66,000 Mr) from jackfruit seeds, is highly specific for the tumour associated T-antigenic disaccharide. The crystal structure of jacalin with methyl-α-D-galactose reveals that each subunit has a three-fold symmetric β-prism fold made up of three four-stranded β-sheets. The lectin exhibits a novel carbohydrate-binding site involving the N terminus of the α-chain which is generated through a post-translational modification involving proteolysis, the first known instance where such a modification has been used to confer carbohydrate specificity. This new lectin fold may be characteristic of the Moraceae plant family. The structure provides an explanation for the relative affinities of the lectin for galactose derivatives and provides insights into the structural basis of its T-antigen specificity.

Role of water in plasticity, stability, and action of proteins: the crystal structures of lysozyme at very low levels of hydration.

Earlier studies involving water‐mediated transformations in lysozyme and ribonuclease A have shown that the overall movements in the protein molecule consequent to the reduction in the amount of surrounding water are similar to those that occur during enzyme action, thus highlighting the relationship among hydration, plasticity, and action of these enzymes. Monoclinic lysozyme retains its crystallinity even when the level of hydration is reduced further below that necessary for activity (about 0.2 gram of water per gram of protein). In order to gain insights into the role of water in the stability and the plasticity of the protein molecule and the geometrical basis for the loss of activity that accompanies dehydration, the crystal structures of monoclinic lysozyme with solvent contents of 17.6%, 16.9%, and 9.4% were determined and refined. A detailed comparison of these forms with the normally hydrated forms show that the C‐terminal segment (residues 88–129) of domain I and the main loop (residues 65–73) in domain II exhibit large deviations in atomic positions when the solvent content is reduced, although the three‐dimensional structure is essentially preserved. Many crucial water bridges between different regions of the molecule are conserved in spite of differences in detail, even when the level of hydration is reduced well below that required for activity. The loss of activity that accompany dehydration appears to be caused by the removal of functionally important water molecules from the active‐site region and the reduction in the size of the substrate binding cleft. Proteins 32:229–240, 1998.

Isomorphous replacement and anomalous scattering

When two or more crystals have the same structure except for the replacement or addition of one or a few (usually heavy) atoms, phase determination using isomorphous replacement can be resorted to. In most practical instances, the atomic form factor is a real number. When the absorption edge of an atom is close to the wavelength of the incident radiation, the form factor becomes complex and the atom becomes an anomalous scatterer. Anomalous scattering can be used for phase determination and also for determining the absolute configuration. The effects of isomorphous replacement and anomalous scattering are often complementary. Although they have been in use from the 1930s, the most useful applications of the two approaches have been in macromolecular crystallography from the 1950s. Robust methods have been developed for their use in the determination and refinement of heavy-atom positions in protein heavy-atom derivatives and in the calculation of phase angles. With the advent of tunable synchrotron radiation, methods based only on anomalous scattering have become prominent. This chapter describes the development in the area up to the mid-1980s. The subsequent developments in isomorphous replacement and anomalous scattering have been almost exclusively concerned with macromolecular crystallography. They are discussed in International Tables for Crystallography Volume F .

Crystal structure of the jacalin-T-antigen complex and a comparative study of lectin-T-antigen complexes

Thomsen-Friedenreich antigen (Galbeta1-3GalNAc), generally known as T-antigen, is expressed in more than 85% of human carcinomas. Therefore, proteins which specifically bind T-antigen have potential diagnostic value. Jacalin, a lectin from jack fruit (Artocarpus integrifolia) seeds, is a tetramer of molecular mass 66kDa. It is one of the very few proteins which are known to bind T-antigen. The crystal structure of the jacalin-T-antigen complex has been determined at 1.62A resolution. The interactions of the disaccharide at the binding site are predominantly through the GalNAc moiety, with Gal interacting only through water molecules. They include a hydrogen bond between the anomeric oxygen of GalNAc and the pi electrons of an aromatic side-chain. Several intermolecular interactions involving the bound carbohydrate contribute to the stability of the crystal structure. The present structure, along with that of the Me-alpha-Gal complex, provides a reasonable qualitative explanation for the known affinities of jacalin to different carbohydrate ligands and a plausible model of the binding of the lectin to T-antigen O-linked to seryl or threonyl residues. Including the present one, the structures of five lectin-T-antigen complexes are available. GalNAc occupies the primary binding site in three of them, while Gal occupies the site in two. The choice appears to be related to the ability of the lectin to bind sialylated sugars. In either case, most of the lectin-disaccharide interactions are at the primary binding site. The conformation of T-antigen in the five complexes is nearly the same.

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. Proteins 1999;35:58–69.

Structure of Mycobacterium tuberculosis single-stranded DNA-binding protein. Variability in quaternary structure and its implications

Single-stranded DNA-binding protein (SSB) is an essential protein necessary for the functioning of the DNA replication, repair and recombination machineries. Here we report the structure of the DNA-binding domain of Mycobacterium tuberculosis SSB (MtuSSB) in four different crystals distributed in two forms. The structure of one of the forms was solved by a combination of isomorphous replacement and anomalous scattering. This structure was used to determine the structure of the other form by molecular replacement. The polypeptide chain in the structure exhibits the oligonucleotide binding fold. The globular core of the molecule in different subunits in the two forms and those in Escherichia coli SSB (EcoSSB) and human mitochondrial SSB (HMtSSB) have similar structure, although the three loops exhibit considerable structural variation. However, the tetrameric MtuSSB has an as yet unobserved quaternary association. This quaternary structure with a unique dimeric interface lends the oligomeric protein greater stability, which may be of significance to the functioning of the protein under conditions of stress. Also, as a result of the variation in the quaternary structure the path adopted by the DNA to wrap around MtuSSB is expected to be different from that of EcoSSB.

Crystal Structures of Mycobacterium smegmatis RecA and Its Nucleotide Complexes

The crystal structures of Mycobacterium smegmatis RecA (RecA(Ms)) and its complexes with ADP, ATPgammaS, and dATP show that RecA(Ms) has an expanded binding site like that in Mycobacterium tuberculosis RecA, although there are small differences between the proteins in their modes of nucleotide binding. Nucleotide binding is invariably accompanied by the movement of Gln 196, which appears to provide the trigger for transmitting the effect of nucleotide binding to the DNA-binding loops. These observations provide a framework for exploring the known properties of the RecA proteins.

Molecular interactions and aggregation involving amino acids and peptides and their role in chemical evolution

The earth is believed to have originated some 4.5 billion years ago. The surface of the nascent earth was devoid of any trace of organic matter, let alone life. It is at present characterized by a rich variety of different forms of life. How did this come about? How did life originate and evolve into innumerable forms? The answer to this question is generally attempted in two phases. First, how did the molecules of life come into being and organize themselves into a primitive self-replicating system or primitive cell? Secondly, how did the present-day organisms evolve from the primitive cell or cells? The first question is concerned with chemical evolution and the subsequent origin of life, and the second with biological evolution. It may be noted that chemical evolution must have taken place in a comparatively short span of time on the geological time scale. There is evidence to suggest that life existed as early as 3.6 to 3.8 billion years ago (Nisbet, 1985). The earth, after its formation some 4.5 billion years ago, must have taken several hundred million years to cool down sufficiently for the formation of the crust and to be able to support water and organic matter on its surface. Allowing for this time lag and bearing in mind that life probably originated some 3.6 to 3.8 billion years ago, chemical evolution appears to have been completed in a few hundred million years in the early phase of earths existence. No traces of the processes of chemical evolution remain. Therefore, direct answers to questions concerning chemical evolution cannot be obtained. Results of informed speculation, simulation experiments, observations of meteorites etc., need to be put together even to get a blurred picture of chemical evolution. Indeed such a picture, partly clear and partly hopelessly confusing, has emerged during the last few decades.

Structural studies on MtRecA-nucleotide complexes: Insights into DNA and nucleotide binding and the structural signature of NTP recognition

RecA protein plays a crucial role in homologous recombination and repair of DNA. Central to all activities of RecA is its binding to Mg+2‐ATP. The active form of the protein is a helical nucleoprotein filament containing the nucleotide cofactor and single‐stranded DNA. The stability and structure of the helical nucleoprotein filament formed by RecA are modulated by nucleotide cofactors. Here we report crystal structures of a MtRecA‐ADP complex, complexes with ATPγS in the presence and absence of magnesium as well as a complex with dATP and Mg+2. Comparison with the recently solved crystal structures of the apo form as well as a complex with ADP‐AlF4 confirms an expansion of the P‐loop region in MtRecA, compared to its homologue in Escherichia coli, correlating with the reduced affinity of MtRecA for ATP. The ligand bound structures reveal subtle variations in nucleotide conformations among different nucleotides that serve in maintaining the network of interactions crucial for nucleotide binding. The nucleotide binding site itself, however, remains relatively unchanged. The analysis also reveals that ATPγS rather than ADP‐AlF4 is structurally a better mimic of ATP. From among the complexed structures, a definition for the two DNA‐binding loops L1 and L2 has clearly emerged for the first time and provides a basis to understand DNA binding by RecA. The structural information obtained from these complexes correlates well with the extensive biochemical data on mutants available in the literature, contributing to an understanding of the role of individual residues in the nucleotide binding pocket, at the molecular level. Modeling studies on the mutants again point to the relative rigidity of the nucleotide binding site. Comparison with other NTP binding proteins reveals many commonalties in modes of binding by diverse members in the structural family, contributing to our understanding of the structural signature of NTP recognition. Proteins 2003;50:474–485.

Structural Basis of the Carbohydrate Specificities of Jacalin: An X-ray and Modeling Study

The structures of the complexes of tetrameric jacalin with Gal, Me-alpha-GalNAc, Me-alpha-T-antigen, GalNAcbeta1-3Gal-alpha-O-Me and Galalpha1-6Glc (mellibiose) show that the sugar-binding site of jacalin has three components: the primary site, secondary site A, and secondary site B. In these structures and in the two structures reported earlier, Gal or GalNAc occupy the primary site with the anomeric carbon pointing towards secondary site A. The alpha-substituents, when present, interact, primarily hydrophobically, with secondary site A which has variable geometry. O-H..., centered pi and C-H...pi hydrogen bonds involving this site also exist. On the other hand, beta-substitution leads to severe steric clashes. Therefore, in complexes involving beta-linked disaccharides, the reducing sugar binds at the primary site with the non-reducing end located at secondary site B. The interactions at secondary site B are primarily through water bridges. Thus, the nature of the linkage determines the mode of the association of the sugar with jacalin. The interactions observed in the crystal structures and modeling based on them provide a satisfactory qualitative explanation of the available thermodynamic data on jacalin-carbohydrate interactions. They also lead to fresh insights into the nature of the binding of glycoproteins by jacalin.