The opportunistic bacterial pathogen Pseudomonas aeruginosa uses type IV pili (T4P) for adherence to, and rapid colonization of, surfaces via twitching motility. T4P are formed from thousands of pilin (PilA) subunits. Two groups of P. aeruginosa pilins were described previously (I and II), distinguished by protein length and sequence. PilA_I was glycosylated with an O-antigen subunit through the action of PilO/TfpO, encoded downstream of pilA_I. To determine if additional pilin variants existed, analysis of the pilin locus of >300 P. aeruginosa strains from a variety of environments was conducted. Three additional pilin alleles were discovered, each of which was invariantly associated with a unique, previously unidentified, downstream gene(s): pilA_III+tfpY, pilAIV+tfpW+tfpX, pilA_V+tfpZ. This survey also revealed that strains with group I T4P were more commonly associated with respiratory infections than strains with other pilins, suggesting that glycosylated T4P may confer a colonization advantage in this environment. The newly identified group IV pilin, represented by strain Pa5196, migrated aberrantly through SDS-PA gels, suggesting it was also glycosylated, a hypothesis confirmed by periodic acid-Schiff staining and mass spectrometry (MS) analyses. Disruption of Pa5196 O-antigen biosynthesis did not prevent the production of glycosylated pilins, demonstrating that these pilins were modified in a novel manner, unlike group I pilins. Using MS, nuclear magnetic resonance spectroscopy and site-directed mutagenesis, the Pa5196 pilins were shown to be uniquely modified with homo-oligosaccharides of mycobacterial-like α-1,5-D-arabinofuranose at multiple locations. Residues Thr64 and Thr66, located on the αβ-loop region of the protein, appear to be the preferred, but not exclusive sites of modification, each being modified with up to four D-Araf sugars. This region of the pilin is partially surface-exposed in the pilus, therefore modification of these sites may influence the surface chemistry of the fibre. Residues Ser81, Ser82, Ser85 and Ser89, located in the β-strand region, were also modified, mainly with mono- and disaccharides. Bioinformatic analyses and mutagenesis of TfpW suggest that this novel protein is an arabinosyltransferase necessary for PilA_IV modification. This research has increased our understanding of the complexity of this virulence factor, and may aid in development of new therapeutics for P. aeruginosa and mycobacterial infections.

abstract: Conformational changes in biomolecules often take place on longer timescales than are easily accessible with unbiased molecular dynamics simulations, necessitating the use of enhanced sampling techniques, such as adaptive umbrella sampling. In this technique, the conformational free energy is calculated in terms of a designated set of reaction coordinates. At the same time, estimates of this free energy are subtracted from the potential energy in order to remove free energy barriers and cause conformational changes to take place more rapidly. This dissertation presents applications of adaptive umbrella sampling to a variety of biomolecular systems. The first study investigated the effects of glycosylation in GalNAc2-MM1, an analog of glycosylated macrophage activating factor. It was found that glycosylation destabilizes the protein by increasing the solvent exposure of hydrophobic residues. The second study examined the role of bound calcium ions in promoting the isomerization of a cis peptide bond in the collagen-binding domain of Clostridium histolyticum collagenase. This study determined that the bound calcium ions reduced the barrier to the isomerization of this peptide bond as well as stabilizing the cis conformation thermodynamically, and identified some of the reasons for this. The third study represents the application of GAMUS (Gaussian mixture adaptive umbrella sampling) to on the conformational dynamics of the fluorescent dye Cy3 attached to the 5' end of DNA, and made predictions concerning the affinity of Cy3 for different base pairs, which were subsequently verified experimentally. Finally, the adaptive umbrella sampling method is extended to make use of the roll angle between adjacent base pairs as a reaction coordinate in order to examine the bending both of free DNA and of DNA bound to the archaeal protein Sac7d. It is found that when DNA bends significantly, cations from the surrounding solution congregate on the concave side, which increases the flexibility of the DNA by screening the repulsion between phosphate backbones. The flexibility of DNA on short length scales is compared to the worm-like chain model, and the contribution of cooperativity in DNA bending to protein-DNA binding is assessed. / Dissertation/Thesis / Ph.D. Chemistry 2011

The journey of structural studies on lectins, starting with ConA in the 70s, has crossed many milestones. Lectins, multivalent carbohydrate-binding proteins of non-immune origin, specifically bind diverse sugar structures. They have received considerable attention in recent times on account of the realization of the importance of protein-sugar interactions, especially at the cell surface, in biological recognition. They occur in plants, animals, fungi, bacteria and viruses. Plant lectins constitute about 40% of the lectins of known structure. They can be classified into five structural groups, each characterized by a specific fold. Among them, legume lectins constitute the most extensively investigated group. Basic Winged bean lectin (WBAI) is a glycosylated, homodimeric, legume lectin with Mr 58000. The structure of WBAI complexed with methyl-a-galactose, determined earlier in this laboratory, provided information about the oligomeric state and the carbohydrate specificity of the lectin in terms of lectin-monosaccharide interactions. The present work was initiated to understand the carbohydrate specificity of the lectin, especially at the oligosaccharide level, with special reference to its blood group specificity. The hanging drop method was used for crystallizing WBAI and its complexes. Intensity data were collected on Mar Research imaging plates mounted on Rigaku RU-200 or ULTRAX-18 X-ray generators. 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 AMoRe was used for structure solution. Structure refinement was carried out using the CNS software package. Model building was done using the molecular graphics program O. INSIGHT II, ALIGN, CONTACT and PROCHECK of CCP4 were used for the analysis and validation of the refined structures. WBAI exhibits differential affinity for different monosaccharide derivatives of galactose. In order to elucidate the structural basis for this differential affinity, the crystal structures of the complexes of basic winged bean lectin with galactose, 2-methoxygalactose, N-acetylgalactosamine and methyl-a-N-acetylgalactosamine have been determined. Lectin-sugar interactions involve four hydrogen bonds and a stacking interaction in all of them. In addition, a N-H O hydrogen bond involving the hydroxyl group substituted at C2 exists in the galactose and 2-methoxygalactose complexes. The additional hydrophobic interaction, involving the methyl group, in the latter leads to the higher affinity of the methyl derivative. In the lectin - N- acetylgalactosamine complex the N-H O hydrogen bond is lost, but a compensatory hydrogen bond involving the oxygen atom of the acetamido group is formed. In addition, the CH3 moiety of the acetamido group is involved in hydrophobic interactions. Consequently, the 2-methyl and the acetamido derivatives of galactose have nearly the same affinity for the lectin. The methyl group, a-linked to the galactose, takes part in additional hydrophobic interactions. Therefore, methyl-a- N-acetylgalactosamine has higher affinity than N-acetylgalactosamine to the lectin. The structures of basic winged bean lectin-sugar complexes provide a framework for examining the relative affinity of galactose and galactosamine for the lectins that bind to them. The complexes also lead to a structural explanation for the blood group specificity of basic winged bean lectin, in terms of its monosaccharide specificity. The Tn-determinant (GalNAc-a-O-Ser/Thr) is a human specific tumor associated carbohydrate antigen. Having epithelial origin, it is expressed in many carcinogenic tumors including breast, prostate, lung and pancreatic cancers. The crystal structure of WBAI in complex with GalNAc-a-O-Ser (Tn-antigen) has been elucidated, in view of its relevance to diagnosis and prognosis of various human cancers. The Gal moiety occupies the primary binding site and makes interactions similar to those found in other Gal/GalNAc specific legume lectins. The nitrogen and oxygen atoms of the acetamido group of the sugar make two hydrogen bonds with the protein atoms whereas its methyl group is stabilized by hydrophobic interactions. A water bridge formed between the terminal oxygen atoms of the serine residue of the Tn-antigen and the side chain oxygen atom of Asn128 of the lectin increase the affinity of the lectin for Tn-antigen compared to that for GalNAc. A comparison with the available structures reveals that while the interactions of the glyconic part of the antigen are conserved, the mode of stabilization of the serine residue differs and depends on the nature of the protein residues in its vicinity. The structure provides a qualitative explanation for the thermodynamic parameters of the formation of the complex of the lectin with Tn-antigen. Modelling studies indicate the possibility of an additional hydrogen bond with the lectin when the antigen is part of a glycoprotein. WBAI binds A-blood group substance with higher affinity and B-blood group substance with lesser affinity. It does not bind the O substance. The crystal structures of the lectin, complexed with A -reactive and B - reactive di and tri saccharides, have been determined. In addition, the complexes of the lectin with fucosylated A- and B-trisaccharides and with a variant of the A-trisaccharide have been modelled. These structures and models provide valuable insights into the structural basis of blood group specificities. All the four carbohydrate binding loops of the lectin contribute to the primary combining site while the loop of variable length contributes to the secondary binding site. In a significant advance to the current understanding, the interactions at the secondary binding site also contribute substantially, albeit in a subtle manner, to determine the blood group specificity. Compared to the interactions of the B- trisaccharide with the lectin, the third sugar residue of the A -reactive trisaccharide forms an additional hydrogen bond with a lysine residue in the variable loop. In the former, the formation of such a hydrogen bond is prevented by a shift in the orientation of the third sugar resulting from an internal hydrogen bond in it. The formation of this bond is also facilitated by an interaction dependent change in the rotamer conformation of the lysyl residue of the variable loop. Thus, the difference in the interactions at the secondary site is generated by coordinated movements in the ligand as well as the protein. A comparison of the crystal structure and the model of the complex involving the variant of the A-trisaccharide results in the delineation of the relative contributions of the interactions at the primary and the secondary sites in determining blood group specificity. At the disaccharide level, WBAI exhibits higher affinity for á1-3 linked Gal/GalNAc containing oligosaccharides, compared to that of other á linked oligosaccharides. With an objective of understanding the preferential binding of WBAI for á 1-3 linked Gal/GalNAc containing oligosaccharides, crystal structure of the complexes of the lectin with Galá1-4Gal, Galá1-4GalâEt and Galá1-6Gal have been determined. The reducing sugar of the disaccharides with linkages other than á1-3 binds to the lectin through a water bridge whereas the same sugar moiety with á 1-3 linkage makes direct interactions with the loop L4 of the protein. The modelling study on the complex of the lectin with Galá1-2Gal further upholds this observation. Different structures involving WBAI, reported earlier and presented here, were used to investigate the plasticity of the lectin. The front curved â-sheet, which nestles the metal binding region and on which the carbohydrate binding loops are perched, is relatively rigid. On the contrary, the flat back â-sheet, involved in the quaternary association in legume lectins, is flexible. This flexibility is probably necessary to account for the variation in quaternary structure. With the results presented in this thesis, 14 crystal structures of WBAI, in the free form and in complex with different sugars, have been reported, all from this laboratory. It is now, perhaps, appropriate to examine the new information and insights gained from these investigations, on the structure and function of the lectin. Earlier X-ray studies of WBAI contributed substantially in establishing that legume lectins are a family of proteins in which small alterations in essentially the same tertiary structure lead to large alterations in quaternary association. Structural studies on WBAI, particularly those reported here, also contributed to the elucidation of the nuances of carbohydrate recognition by lectins. A comparative study of the available structures also revealed the flexible and rigid regions of the protein. The study of the influence of covalently linked sugars on the structure of Erythrina corallodendron lectin (ECorL), a homolog of WBAI, is the content of appendix of the thesis. The three-dimensional structure of the recombinant form of Erythrina Corallodendron lectin(rECorL) complexed with lactose, has been elucidated by X-ray crystallography. Comparison of this non-glycosylated structure with that of the native glycosylated lectin reveals that the tertiary and quaternary structures are identical in the two forms, with local changes observed at one of the glycosylation sites(Asn17). These changes take place in such a way that hydrogen bonds with the neighbouring protein molecules in rECorL compensate those made by the glycan with the protein in ECorl. contrary to an earlier report, this study demonstrates that the glycan attached to the lectin does not influence the oligomeric state of the lectin. Identical interactions between the lectin and the non-covalently bound lactose in the two forms indicate, in line with earlier reports, that glycosylation does not affect the carbohydrate specificity of the lectin. The present study, the first of its kind involving a glycosylated protein with a well defined glycan and the corresponding deglycosylated form, provides insights into the structural aspects of protein glycosylation.

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Proteins, participating in a myriad of biological function, are at the core of all cellular activities occurring within living organisms. Therapeutic proteins, hence constitute a major part of the pharmaceutical industry. The glycoprotein hormones follicle stimulating hormone (FSH), luteinizing hormone (LH), thyroid stimulating hormone (TSH) and human chorionic gonadotropin (CG) regulate various reproductive and metabolic functions in humans and hence have high therapeutic potentials. The increasing demand of recombinant proteins for therapeutic uses drives the development of better expression systems. The methylotrophic yeast Pichia pastoris, has been termed as an industrial workhorse for heterologous protein expression. However, the N-glycosylation in yeast is of the high mannose type, resulting in a reduced serum half-life of the recombinant proteins. In the current work, we have re-engineered the Pichia N-glycosylation pathway to mimic the human type of N-glycosylation. Towards this end, we abolished the yeast native N-glycosylation and introduced enzymes from various eukaryotic sources into the system. These modifications resulted in the conversion of the yeast Man9-20GlcNAc2 glycan structure to a more human like GlcNAc2Man3GlcNAc2 form on over 70 % of the heterologous expressed proteins. In order to demonstrate the application of these strains as efficient protein expression hosts, the glycoengineerd Pichia was used for large scale expression of the glycoprotein hormones, hCG and FSH. The purified recombinant hormones were found to have binding affinities and structure similar to that of the natural hormones. These recombinant hormones were also able to elicit over two fold responses in animal models compared to buffer controls and the activity was comparable to the natural counterparts. Thus, we report the generation of a glycoengineered Pichia pastoris, which can be considered as a serious contender for the expression of glycosylated proteins of therapeutic importance.