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Further Structural Studies on Jacalin and Genomics Search for Mycobacterial and Archeal LectinsAbhinav, K V January 2016 (has links) (PDF)
This thesis consists of two parts. The first part is concerned with further structural and related studies of jacalin, one of the two lectins found in jack fruit seeds. The second part deals with the search of mycobacterial and archeal genomes for lectins.
The β-prism I fold was identified as a lectin fold through the X-ray analysis of jacalin way back in 1996. Subsequent structural studies on jacalin are described in the first chapter in context of the overall efforts on lectins with particular reference to those on lectins with β-prism I fold. The structure of jacalin has been thoroughly characterized through the analysis of several crystals. The extended binding site of the lectin, made up of the primary binding site and secondary sites A and B, has also been characterized through studies on different jacalin-sugar complexes. However, nuances of jacalin-carbohydrate interactions remain underexplored with respect to two specific issues. The first issue is concerned with the structural basis for the lower affinity of jacalin for β-substituted sugars. The second has to do with the influence of the anomeric nature of the glycosidic linkage on the location of the reducing and non-reducing sugars in disaccharides when interacting with jacalin. Part of the work described in the thesis addresses these two issues.
It was surmised that the lower affinity of β-galactosides to jacalin as compared to α-galactosides, is caused by steric interactions of the substituents in the former with the protein. This issue is explored both energetically and structurally in Chapter 2 using appropriately derivatized monosaccharide complexes of jacalin. It turns out that the earlier surmise is not correct. The interactions of the substituent with the binding site remain essentially the same irrespective of the anomeric nature of the substitution. This is achieved through a distortion of the sugar ring in β-galactosides. The difference in energy, and therefore affinity, is caused by the distortion of the sugar ring in β-galactosides. The elucidation of this unprecedented distortion of the ligand as a strategy for modulating affinity is of general interest. The crystal structures also provide a rationale for the relative affinities of the different carbohydrate ligands to jacalin.
The crystal structures of jacalin complexed with α-linked oligosaccharides Gal α-(1,4) Gal and Gal α-(1,3) Gal β-(1,4) Gal, as described in Chapter 3, have been determined with the primary objective of exploring the effect of linkage on the location of reducing and non-reducing sugars in the extended binding site of the lectin, an issue which has not been studied thoroughly. Contrary to the earlier surmise based on simple steric considerations, the two structures demonstrate that α-linked sugars can bind to jacalin with non-reducing sugar at the
primary binding site. This is made possible substantially on account of the hitherto underestimated plasticity of a non-polar region of the extended binding site. Modelling studies involving conformational search and energy minimization, along with available crystallographic and thermodynamic data, indicate a strong preference for complexation with Gal β-(1,3) Gal with the reducing Gal at the primary site, followed by that with Gal α-(1,3) Gal, with the reducing or non-reducing Gal located at the primary binding site. This observation is in consonance with the facility of jacalin to bind mucin type O-glycans containing T-antigen core.
Crystal structures of jacalin in complex with GlcNAc β-(1,3) Gal-β-OMe and Gal β-(1,3) Gal-β-OMe have also been described in Chapter 4. The binding of the ligands to jacalin is similar to that of analogous α-substituted disaccharides. However, the β-substituted β-(1,3) linked disaccharides get distorted at the anomeric centre and the glycosidic linkage. The distortion results in higher internal energies of the ligands leading to lower affinity to the lectin. This confirms the possibility of using ligand distortion as a strategy for modulating binding affinity. Unlike in the case of β-substituted monosaccharides bound to jacalin, where a larger distortion at the anomeric centre was observed, smaller distortions are distributed among two centres in the structures of the two β-substituted β-(1,3) linked disaccharides presented here. These disaccharides, like the unsubstituted and α-substituted counterparts, bind jacalin with the reducing Gal at the primary binding site, indicating that the lower binding affinity of β-substituted disaccharides is not enough to overcome the intrinsic propensity of Gal β-(1,3) Gal based disaccharides to bind jacalin with the reducing sugar at the primary site.
Although originally isolated from plants, lectins were also found subsequently in all forms of life, including bacteria. Studies on microbial lectins have not been as extensive as on those from plants and animals, although there have been some outstanding individual investigations on bacterial toxins like ADP-ribosylating toxins and neurotoxins. In addition to bacterial toxins, adhesins, β-trefoil lectins and cyanobacterial lectins form other important subgroups which have been explored using crystallography. Features pertaining to their three dimensional folds, carbohydrate specificity and biological properties are described in Chapter 5, to set the stage for the work discussed in the second part of the thesis. Studies on mycobacterial lectins were unexplored until work was initiated in the area in this laboratory some years ago. One of the lectins, identified on the basis of a bioinformatics search of M. tuberculosis H37Rv genome was cloned, expressed and crystallized. Also cloned, expressed and crystallized is another lectin from M. smegmatis. Biophysical and modelling studies were carried out on the full length protein containing this lectin. However, systematic efforts on mycobacterial lectins were conspicuous by their absence. The first chapter (Chapter 6) in the second part of the thesis is concerned with a genomic search for lectins in mycobacterial genomes. It was also realized that hardly anything is known about archeal lectins. Therefore, as discussed in the final chapter, a genomic search for archeal lectins was undertaken.
Sixty-four sequences containing lectin domains with homologs of known three-dimensional structure were identified through a search of mycobacterial genomes and are described in detail in Chapter 6. They appear to belong to the β-prism II, the C-type, the Microcystis virdis (MV), and the β-trefoil lectin folds. The first three always occur in conjunction with the LysM, the PI-PLC, and the β-grasp domains, respectively while mycobacterial β-trefoil lectins are unaccompanied by any other domain. Thirty heparin binding hemagglutinins (HBHA), already annotated, have also been included in the study although they have no homologs of known three-dimensional structure. The biological role of HBHA has been well characterized. A comparison between the sequences of the lectin from pathogenic and non-pathogenic mycobacteria provides insights into the carbohydrate binding region of the molecule, but the structure of the molecule is yet to be determined. A reasonable picture of the structural features of other mycobacterial proteins containing one of the four lectin domains can be gleaned through the examination of homologous proteins, although the structure of none of them is available. Their biological role is yet to be elucidated. The work presented here is among the first steps towards exploring the almost unexplored area of the structural biology of mycobacterial lectins.
As mentioned in Chapter 7, forty six lectin domains, which have homologues among well established eukaryotic and bacterial lectins of known three dimensional structure, have been identified through a search of 165 archeal genomes using a multi-pronged approach involving domain recognition, sequence search and analysis of binding sites. Twenty one of them have the 7-bladed β-propeller lectin fold while 16 have the β-trefoil fold and 7 the legume lectin fold. The remainder assumes the C-type lectin, the β-prism I and the tachylectin folds. Acceptable models for almost all of them could be generated using the appropriate lectins of known three dimensional structure as templates, with binding sites at one or more expected locations. The work represents the first comprehensive bioinformatics study of archeal lectins. The presence of lectins with the same fold in all domains of life indicates their ancient origin well before the divergence of the three branches. Further work is necessary to identify archeal lectins which have no homologues among eukaryotic and bacterial species.
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Structural and Related Studies on Mycobacterial LectinsPatra, Dhabaleswar January 2014 (has links) (PDF)
This thesis is concerned with the first ever X-ray crystallographic and complimentary solution studies on mycobacterial lectins. Lectins, described as multivalent carbohydrate binding proteins of non-immune origin, are found in all kingdoms of life. As explained in the introductory chapter, those from plants and animals are the best characterized in terms of structure and function. Although not that extensive, important studies have been carried out on viral, fungal and parasite lectins as well. Bacterial lectins studied so far can be classified in to fimbrial, surface and secretory (or toxic). Applications of lectins include blood typing, cell separation and purification of glycoconjugates, mitogenic stimulation of lymphocytes, mapping of neuronal pathways and drug targeting and delivery. The work reported in the thesis lies at the intersection of two major long range programs in this laboratory, one on lectins and the other on mycobacterial proteins. Three putative lectins Rv1419 and Rv2813 from M. tuberculosis and MSMEG_3662 from M. smegmatis were chosen for exploratory studies on the basis of preliminary genomic searches.
Exploratory studies on Rv1419, Rv2813 and MSMEG_3662 are described in the second chapter. MSMEG_3662 contains two domains, a LysM domain and a lectin domain (MSL) connected by a long polypeptide chain. The two M. tuberculosis proteins, full length MSMEG_3662 and MSL were cloned, expressed, purified and characterized. Rv2813 did not show any appreciable agglutination activity. It showed ATPase activity. Clearly the protein was not a lectin. Rv1419, full length MSMEG_3662 and MSL exhibited lectin characteristics. Among them, Rv1419 and MSL could be crystallized. Preliminary X-ray diffraction studies on them were carried out.
Rv1419 could be successfully expressed only once. However, that was enough for the determination of crystal structure and the glycan array analysis of the lectin (Chapter 3). The monomeric lectin has a β-trefoil fold. It has high affinity for LacNAc and its Neu5Ac derivatives. Modeling studies using complexes of homologous structures, led to the identification of two carbohydrate binding sites on the lectins. Sequence comparisons of Rv1419 with homologous proteins with known structures and phylogenetic analysis involving them provide interesting insights into the relationship among trefoil lectins from different sources.
X-ray crystal structure analysis of MSL and its complexes with mannose and methyl-α-mannose, the first comprehensive effort of its kind on a mycobacterial lectin, reveals a structure very similar to β-prism II fold lectins from plant sources, but with extensive unprecedented domain swapping in dimer formation (Chapter 4). The two subunits in a dimer often show small differences in structure, but the two domains, not always related by 2-fold symmetry, have the same structure. Each domain carries three sugar-binding sites, similar to those in plant lectins, one on each Greek key motif. The occurrence of β-prism II fold lectins in bacteria, with characteristics similar to those from plants, indicates that this family of lectins is of ancient origin and had evolved into a mature system before bacteria and plants diverged. In plants, the number of binding sites per domain varies between one and three, whereas the number is two in the recently reported lectin domains from Pseudomonas putida and Pseudomonas aeruginosa. An analysis of the sequences of the lectins and the lectin domains shows that the level of sequence similarity among the three Greek keys in each domain has a correlation with the number of binding sites in it. Furthermore, sequence conservation among the lectins from different species is the highest for that Greek key which carries a binding site in all of them. Thus, it would appear that carbohydrate binding influences the course of the evolution of the lectin.
LysM domains have been recognized in bacteria and eukaryotes as carbohydrate-binding protein modules, but the mechanism of their binding to chitooligosaccharides is underexplored. Binding of a full length MSMEG_3662 containing LysM and lectin (MSL) domains to chitooligosaccharides has been studied using isothermal titration calorimetry and fluorescence titration (Chapter 5). This investigation demonstrates the presence of two binding sites of non-identical affinities per dimeric MSL-LysM molecule. Affinity of the molecule for chitooligosaccharides correlates with the length of the carbohydrate chain. Its binding to chitooligosaccharides is characterized by negative cooperativity in the interactions of the two domains. Apparently, the flexibility of the long linker that connects the LysM and MSL domains plays a facilitating role in this recognition. The LysM domain in MSL-LysM, like other bacterial domains but unlike plant LysM domains, recognizes equally well peptidoglycan fragments as well as chitin polymers. Interestingly, in the present case two LysM domains are enough for binding to peptidoglycan in contrast to the three reportedly required by the LysM domains of Bacillus subtilis and Lactococcus lactis. Also, the affinity of MSL-LysM for chitooligosaccharides is higher than that of LysM-chitooligosaccharide interactions reported so far.
A part of the work presented in this thesis has been reported in the following publications:
• Patra D, Mishra P, Surolia A, Vijayan M. 2014. Structure, interactions and evolutionary implications of a domain-swapped lectin dimer from Mycobacterium smegmatis. Glycobiology, 24:956-965.
• Patra D, Sharma A, Chandran D, Vijayan M. 2011. Cloning, expression, purification, crystallization and preliminary X-ray studies of the mannose-binding lectin domain of MSMEG_3662 from Mycobacterium smegmatis. Acta Crystallogr Sect F Struct Biol Cryst Commun, 67:596-599.
• Patra D, Srikalaivani R, Misra A, Singh DD, Selvaraj M, Vijayan M. 2010. Cloning, expression, purification, crystallization and preliminary X-ray studies of a secreted lectin (Rv1419) from Mycobacterium tuberculosis. Acta Crystallogr Sect F Struct Biol Cryst Commun, 66:1662-1665.
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