Lectins, multivalent carbohydrate-binding proteins of non-immune origin, have the unique ability to decode the information contained in complex carbohydrate structures of glycoproteins and glycolipids by stereo-specifically recognizing and binding to carbohydrates and carbohydrate linkages. The ubiquitous distribution of lectins in all forms of life and viruses along with their involvement in various biological processes such as cell-cell communication, host-pathogen interaction, cancer metastasis, embryogenesis, tissue development and mitogenic stimulation further emphasizes the importance of lectins in biological systems. Although not much is known about the endogenous roles of plant lectins, they constitute the most thoroughly studied class of lectins. On the basis of their subunit folds plant lectins have been divided in six major classes. They include jelly roll fold lectins (or legume lectins), hevein domain lectins (or cereal lectins), β-trefoil fold lectins, β-prism II fold lectins (or bulb lectins), β-prism I fold lectins and the most recently discovered lectin homologous to cyanovirin-N (http://www.cermav.cnrs.fr/lectines). Interestingly, of these, lectin subunits harbor an approximate three-fold symmetry in three cases and each subunit is believed to have evolved through successive gene duplication, fusion and divergent evolution. One of the major research activities in this laboratory involves structural studies on plant lectins. Decades of extensive studies in the laboratory have shed light on various structural and functional aspects of lectins such as variability in quaternary association, lectin-carbohydrate interactions, strategies for generating ligand specificity and multivalency. Furthermore, the β-prism I fold was first identified as a lectin fold in this laboratory through the X-ray analysis of the methyl-α-galactose complex of jacalin, one of the two lectins from the seeds of Artocarpus integrifolia. Subsequently, many other lectins with the same fold have been structurally characterized here and else where (http://www.cermav.cnrs.fr/lectines). They include mannose specific tetrameric artocarpin and dimeric banana lectin studied in this laboratory. Also investigated here is the structure of first dimeric β-prism II fold lectin, namely, garlic lectin. The subsequent work, carried out by the author, on the structure and dynamics of three-fold symmetric lectins form the subject matter of this thesis. Different web-servers available at NCBI and EXPASY web sites were used for sequence annotation studies. MRBAYES and MEGA were used for phylogenetic analysis. Molecular dynamics (MD) simulations were carried out using the simulation package GROMACS v.3.3.1. OPLS-AA/L and GLYCAM-06 force fields were used for proteins and carbohydrates respectively. Simulations were performed in explicit water system with TIP4P water model under NPT conditions with unit dielectric constant. The hanging drop method was used for crystallizing banana lectin and its complexes. Intensity data were collected on a MAR 345 image plate mounted on a Rigaku RU200 rotating-anode X-ray generator. The Oxford cryosystem was used when collecting data at low temperature. The data were processed using DENZO and SCALEPACK of HKL suite of programs. The structure factors from the processed data were calculated using TRUNCATE of CCP4 suite of programs. The molecular replacement program MOLREP was used for structure solution. Structure refinements were carried out using the CNS software package and REFMAC of CCP4. Model building was done using the molecular graphics program COOT. INSIGHT II, ALIGN, CONTACT, MUSTANG and SC of CCP4 were used for analysis of structural features. PROCHECK and web-server MOLPROBITY were used for the validation of the refined structures. The β-prism II fold lectins of known structure, all from monocots, invariably have three carbohydrate-binding sites in each subunit / domain. Until recently, β-prism I fold lectins of known structure were all from dicots and they exhibited one carbohydrate-binding site per subunit / domain. However, the recently determined structure of the β-prism I fold lectin from banana, a monocot, has two very similar carbohydrate-binding sites. This prompted a detailed analysis of all the sequences appropriate for the two lectin folds and which carry one or more relevant carbohydrate-binding motifs. The recent observation of a β-prism I fold lectin, griffithsin, with three binding sites in each domain further confirmed the need for such an analysis. The detailed sequence and phylogenetic analysis of all the β-prism I fold lectin or lectin-like sequences, available then, with particular attention to their carbohydrate-binding sites in them, in conjunction with the analysis of available three-dimensional structures demonstrate substantial diversity in the number of binding sites, unrelated to the taxonomical position of the plant source. However, the number of binding sites and the symmetry within the sequence exhibit reasonable correlation. The distribution of the two families of β-prism fold lectins among plants and the number of binding sites in them, appear to suggest that both of them arose through successive gene duplication, fusion and divergent evolution of the same primitive carbohydrate-binding motif involving a Greek key. Analysis with sequences in individual Greek keys as independent units lends further support to this conclusion. It would seem that the prepondence of three carbohydrate-binding sites per domain in monocot lectins, particularly those with the β-prism II fold, is related to the role of plant lectins in defence. Jacalin is the most thoroughly studied β-prism I fold lectin. A wealth of structural and thermodynamic data, mostly from this laboratory, led to a thorough characterization of carbohydrate-recognition in the case of jacalin. One aspect of jacalin that has not been investigated so far was its dynamics. The issue was addressed through reasonably long MD simulations, in explicit solvent system using all atom force field, of all the jacalin-carbohydrate complexes of known structure, models of unliganded molecules derived from the complexes and also models of relevant complexes where X-ray structures are not available. Results of the simulations and the available crystal structures involving jacalin permit delineation of the relatively rigid and flexible regions of the molecule and the dynamical variability of the hydrogen bonds involved in stabilizing the structure. Local flexibility appears to be related to solvent accessibility. Hydrogen bonds involving side chains and water bridges involving buried water molecules appear to be important in the stabilization of loop structures. The lectin-carbohydrate interactions observed in crystal structures, the average parameters pertaining to them derived from simulations, energetic contribution of the stacking residue estimated from quantum mechanical calculations and the scatter of the locations of carbohydrate and carbohydrate-binding residues, are consistent with the known thermodynamic parameters of jacalin-carbohydrate interactions. The simulations, along with X-ray results, provide a fuller picture of carbohydrate binding by jacalin than provided by crystallographic analysis alone. The simulations confirm that in the unliganded structures water molecules tend to occupy the positions occupied by carbohydrate oxygens in the lectin-carbohydrate complexes. Population distributions in simulations of the free lectin, the ligands and the complexes indicate a combination of conformational selection and induced fit. Mannose-specific β-prism I fold lectins, like lectins belonging to other plant families, exhibit interesting variability in their quaternary association. Mannose specific artocarpin and MornigaM are tetrameric, heltuba is octameric in the crystal structure and banana lectin and calsepa are dimeric. The modes of the dimerization in the last two are however, entirely different. This variability was explored through modelling and molecular dynamics simulations based on the known three-dimensional structures. This study, which combines computational approaches and results of X-ray analyses, provides valuable insights into the origin of the variability in quaternary association. MD simulations on individual subunits and the oligomers provide insights into the changes in the structure brought about in the protomers on oligomerization, including swapping of the N-terminal stretch in one instance. The regions which undergo changes also tend to exhibit dynamic flexibility during MD simulations. The internal symmetries of individual oligomers are substantially retained during the calculations. Simulations were also carried out on models using all possible oligomers employing the four different protomers. The unique dimerization pattern observed in calsepa could be traced to unique substitutions in a peptide stretch involved in dimerization. The impossibility of a specific mode of oligomerization involving a particular protomer is often expressed in terms of unacceptable steric contacts or dissociation of the oligomer during simulations. The calculations also lead to a rationale for the observation of a heltuba tetramer in solution although the lectin exists as an octamer in the crystal, in addition to providing insights into relations among evolution, oligomerization and ligand binding. The known crystal structures of banana lectin in its native and ligand bound forms revealed interesting features including the presence of two functional carbohydrate-binding sites per subunit. However, some confusion remained on the role of glycosidic linkage in carbohydrate-binding. The three crystal structures reported in this thesis provide information on details of the interactions of mannose and mannosylα-1,3-mannose with banana lectin and evidence for the binding of glucosyl-α-1,2glucose to the lectin. The known structures involving the lectin include a complex with glucosyl-β-1,3-glucose. Modelling studies on the three disaccharide complexes with the reducing end and the non-reducing end at the primary binding site are also presented here. The results of the X-ray and modelling studies show that the disaccharides with an α-1,3 linkage prefers to have the non-reducing end at the primary binding site while the reducing end is preferred at the site when the linkage is β-1,3 in mannose/glucose specific β-prism I fold lectins. In the corresponding galactose-specific lectins, however, α-1,3 linked disaccharides cannot bind the lectin with the non-reducing end at the primary binding site on account of steric clashes with an aromatic residue which occurs only when the lectin is galactose-specific. MD simulations based on the known structures involving banana lectin enrich the information on lectin-carbohydrate interactions obtained from crystal structures. They demonstrate that conformational selection as well as induced fit operate when carbohydrates bind to banana lectin. Snake gourd seed lectin (SGSL) isolated from Trichosanthes anguina is a glycosylated, galactose-specific, non-toxic lectin similar to type II ribosome inactivating proteins (RIPs) with a molecular weight of ~53kDa. It was established through preliminary X-ray studies that chain A with molecular weight of ~23kDa adopts the same fold as that of type I RIPs and the toxic chain of type II RIPs. Chain B with molecular weight ~32kDa has two β-trefoil fold domains and is responsible for the lectin activity of the protein. The two chains are connected with a disulphide bond. The sequence of the protein could not be determined using conventional methods despite extensive effort. It was derived from X-ray data at 2.4 Å resolution, which was used for structure analysis. The non-toxicity of SGSL appears to result from a combination of changes in the catalytic site in chain A and sugar-binding site in chain B. Detailed analysis of the sequences of type II RIPs of known structure and their homologues with unknown structure, provide valuable insights into the evolution of this class of proteins. It also indicates some variability in carbohydrate-binding sites, which appears to contribute to different levels of toxicity exhibited by lectins from various sources. In addition to the work on plant lectins, the author was also involved in studies on the crystal structures of the adipic acid complexes of L- and DL-Lysine. This investigation is presented in an appendix. A part of the work presented in the thesis has been reported in the following publications. Sharma, A., Thamotharan, S., Roy, S., & Vijayan, M. (2006). X-ray studies of crystalline complexes involving amino acids and peptides. XLIII. Adipic acid complexes of L- and DL-lysine. Acta Cryst, C62, o148-o152. Sharma, A., Chandran, D., Singh, D.D., & Vijayan, M. (2007). Multiplicity of carbohydrate-binding sites in beta-prism fold lectins: occurrence and possible evolutionary implications. J Biosci, 32, 1089-1110. Sharma, A., Sekar, K., & Vijayan, M. (2009). Structure, dynamics, and interactions of jacalin. Insights from molecular dynamics simulations examined in conjunction with results of X-ray studies. Proteins, 77, 760-777.

For a long time, the scientific community underestimated the value of carbohydrates and the approach of most scientists to the complex world of glycans was apprehensive. The scenario, however, has changed today. With the development of new research tools and methodologies the study of carbohydrates and glycoconjugates has progressed rapidly, increasing our understanding of these molecules. Carbohydrates are most abundant amongst biological polymers in nature and vital for life processes. In their simplest form, they serve as a primary source of energy to most living organisms. In generalis, they exist as complex structures (glycans), and as conjugates of protein (glycoproteins, proteoglycans), lipids (glycolipids) and nucleosides (UDP-Glucose). Defined in the broadest sense, the study of glycans in all their forms and their interacting partners is termed “Glycobiology”. Glycans are ubiquitously found in nature decorating cells of almost all types with a “sugar coat”. They are also present within the cytoplasm, as well as in the extra-cellular matrix. They have key roles in a broad range of biological processes, including signal transduction, cell development and immune responses. All living organisms have evolved to express proteins that recognize discrete glycans and mediate specific physiological or pathological processes. One major class of such proteins is “Lectins”. Found in all forms of life, they are characterized by their ability to recognize carbohydrates. They are proteins of non-immune origin that bind glycans reversibly with a high degree of stereo-specificity in a non-catalytic manner. It must be emphasized that they are a different class from glycan-specific antibodies. Lectins were first discovered in plants and a large amount of work has been carried out on plant lectins to decipher their structural organization, mode of interaction with substrate and as models to study protein stability and folding. Study on animal and microbial lectins, on the other hand, gathered momentum only recently. In spite of this, more is known about their function in animals and micro-organisms rather than in plants. Lectin-glycan binding is implicated in several important biological processes such as protein folding, trafficking, host-pathogen interactions, immune cell responses and in malignancy and metastasis. Most lectins have one or more carbohydrate recognition domains (CRDs) which often share either 3-D structural features or amino acid sequence. New members of a family can be identified using either sequence or structural homology. Interestingly, it turns out that several plant and microbial lectins have structural or sequential similarity with animal lectins , revealing that these CRDs are evolutionarily related. This thesis, entitled “Structural, Biophysical and Biochemical Studies on Mannose-specific Lectins”, focuses on three lectins, Banana lectin (Banlec), Calreticulin (CRT) and Peptide-N-Glycanase (PNGase). Although all three lectins have distinct biological functions, they share a common ligand specificity at the monosaccharide level i.e. mannose. This thesis, besides characterizing these lectins, studies in detail, the difference in the mode of interaction with their ligands. Chapter 1 is a general introduction on lectins, glycan-lectin interactions and the various techniques that are employed to characterize these interactions. Several principles have emerged about the nature of glycan–lectin interactions. It has been observed that the binding sites for low molecular weight glycans are of relatively low affinity (Kd values in the high micromolar to low millimolar range). Selectivity is mostly achieved via a combination of hydrogen bonds and by van der Waals packing of the hydrophobic faces of monosaccharide rings against aromatic amino acid side chains. Further selectivity and enhanced affinity can be achieved by additional contacts between the glycan and the protein. It is notable that the actual region of contact between the saccharide and the polypeptide typically involves only one to three monosaccharide residues. As a consequence of all of the above, these lectin-binding sites tend to be of relatively low affinity, although they can exhibit high specificity. It is intriguing to observe that such low-affinity sites have the ability to mediate biologically relevant interactions. There are many different ways to study binding of glycans to proteins, and each approach has its advantages and disadvantages in terms of thermodynamic rigor, amounts of protein and glycan needed, and the speed of analysis. In examining these interactions, two broad categories of techniques are applied: (1) kinetic and near-equilibrium methods, such as titration calorimetry; and (2) non-equilibrium methods such as glycan microarray screening and ELISA-based approaches. Two of the most widely used biophysical approaches for examining glycan-lectin interactions at the molecular level are X-ray crystallography and nuclear magnetic resonance (NMR). However, as small molecules often co-crystallize with a lectin better than large molecules, a lot of our knowledge about glycan–lectin interactions at the atomic level is based on co-crystals of lectins with unnatural ligands. Thus, a great challenge exists in attempting to understand glycan–lectin interactions in the context of natural glycans present as glycoproteins, glycolipids, or proteoglycans. Chapter 2 introduces Banana lectin and describes the stability studies carried out. The unfolding pathway of Banlec was determined using GdnCl induced denaturation. Analysis of isothermal denaturation provided information on its conformational stability and the high values of ΔG of unfolding at various temperatures indicated the strength of inter-subunit interactions. It was found that Banlec is a very stable protein and denatures only at high chaotrope concentrations. The basis of the stability may be attributed to strong hydrogen bonds at the dimeric interface along with the presence of water bridges. This is a very unique example in proteins where subunit association is not a consequence of the predominance of hydrophobic interactions. High temperature molecular dynamics simulations have been utilized to monitor and understand early stages of thermally induced unfolding of Banlec. The present study investigates the behavior of the dimeric protein at four different temperatures. The process of unfolding was monitored by monitoring the radius of gyration, the rms deviation of each residue, change in relative solvent accessibility and the pattern of inter- and intra-subunit interactions. The overall study demonstrates that the Banlec dimer is a highly stable structure, the stability in most part contributed by interfacial interactions. The pattern of hydrogen bonding within the subunits and at the interface across different stages has been analyzed and has provided the rationale for its intrinsic high stability. In Chapter 3 the conformational and dynamic behaviour of three mannose containing oligosaccharides, a tetrasaccharide with α1→2, and α1→3, and a penta- and a heptasaccharide with α1→2, α1→3, and α1→6 linkages has been evaluated. Molecular mechanics, molecular dynamics simulations and NMR spectroscopy methods were used for evaluation. It is found that they display a fair amount of conformational freedom, with one major and one minor conformation per glycosidic linkage. The evaluation of their recognition by Banlec has been performed by STD NMR methods and a preliminary view of their putative interaction mode has been carried out by means of docking procedures. In Chapter 4 the conformational behaviour of three mannose containing oligosaccharides, namely, the α1→3[α1→6] trisaccharide, the heptasaccharide with α1→2, α1→3, and α1→6 linkages and the tetrasaccharide consisting of α1→3 and α1→2 linkages, when bound to Banlec has been evaluated by trNOE NMR methods and docking calculations. It is found that the molecular recognition event involves a conformational selection process, with only one of the conformations, among those available to the sugar in free state, being recognised at the lectin binding site. It is known that many proteins, including members of the Jacalin-related lectin family (of which Banlec is a member), bind the high-mannose saccharides found on the surface of the HIV-associated envelope glycoprotein, gp120, thus interfering with the viral life cycle, potentially providing a manner of controlling a variety of infections, including HIV. These proteins are thought to recognize the high-mannose type glycans with subtly different structures, although the precise specificities are yet to be clarified. This study was carried out to gain a better understanding of these protein-carbohydrate recognition events. Chapter 5 reports interactions of Calreticulin (CRT) with the trisaccharide Glcα1-3Manα1-2Man. Previously in our laboratory it was established using modeling studies the residues in CRT important for sugar binding. Here, the relative roles of Trp-319, Asp-317 and Asp-160 for sugar binding have been explored by using site-directed mutagenesis and isothermal titration calorimetry (ITC). Residues corresponding to Asp-160 and Asp-317 in calnexin (CNX) are known to play important roles in sugar binding. The present study demonstrates that Asp-160 is not involved in sugar binding, while Asp-317 plays a crucial role. Further, it is also validated that hydroxyl-pi interactions of the sugar with Trp-319 dictate sugar binding in CRT. This study defines further the binding site of CRT and also highlights its subtle differences with that of CNX. Additionally, mono-deoxy analogues of the trisaccharide unit Glcα1-3Manα1-2Man have been used to determine the role of various hydroxyl groups of the sugar substrate in sugar-CRT interactions. Using the thermodynamic data obtained by carrying out ITC of CRT with these analogues, it is demonstrated that the 3-OH group of Glc1 plays an important role in sugar-CRT binding, whereas the 6-OH group does not. Also, the 4-OH, 6-OH of Man2 and 3-OH, 4-OH of Man3 in the trisaccharide are involved in binding, of which 6-OH of Man2 and 4-OH of Man3 have a more significant role to play. Therefore, the interactions between the substrate sugar of glycoproteins and the lectin chaperone CRT are further delineated. Chapter 6 introduces Peptide-N-Glycanase (PNGase) and delineates the various interactions involved in the binding of oligomannose structures of glycoproteins to the C-terminal domain (the carbohydrate recognition module) of PNGase. ITC is used to characterize the interaction to oligosaccharides in terms of affinity, stoichiometry, enthalpy, entropy and heat capacity changes with the mouse PNGase C-terminal domain. Using the thermodynamic data obtained, it was determined that PNGase requires the tri-mannoside moiety of the native glycan on glycoproteins as the basic minimum entity for recognition and binding. Additional mannose moieties on the glycan do not significantly interact with PNGase and therefore no enhancement in binding affinity is observed (unlike CRT) which is in concordance with its role of stripping glycans from misfolded glycoproteins targeted for degradation via the ERAD (Endoplasmic reticulum assisted degradation) pathway. Chapter 7 briefly summarizes all the findings of the research carried out and presents a comparative analysis of the three lectins studied. Appendix A: Protein folding in the ER is assisted by molecular chaperones. Lectin chaperones such as CRT and CNX assist the folding of glycoproteins by their N-linked oligosaccharide chains. Dynamic processing of the original glycan chain of (GlcNAc)2(Man)9(Glc)3 to remove the terminal glucose moieties is essential for accurate folding. Proteins that attain their native conformation are then transported to the Golgi complex for further glycan modifications. In case of aberrant folding the proteins are retrotranslocated into the cytosol, ubiquitinated, deglycosylated and degraded by the proteasome. Peptide-N-glycanase is a cytosolic enzyme that releases N-glycans from glycoproteins and glycopeptides. PNGase is now widely recognized as a major participant in protein quality control machinery for ERAD or the proteasomal degradation of retrotranslocated glycoproteins. It is therefore desirable to synthesize fluorescently labeled glycoprotein substrates which will provide direct understanding of how, when and where, the interaction between the substrate and the enzyme occurs. Towards this goal, cloning of GFP and RFP tagged full length mouse and human PNGase and CRT was carried out which is described in this section.