Presentation on theme: "Abstract: MUC1 mucin protein is naturally expressed on the surface of epithelial cells in humans; it is found on the apical surface of ductal epithelia."— Presentation transcript:
Abstract: MUC1 mucin protein is naturally expressed on the surface of epithelial cells in humans; it is found on the apical surface of ductal epithelia tissues of the breast, pancreas, airways, and the gastrointestinal tract. MUC1 mucin is also expressed in cancer cells in the breast, pancreas, and ovaries. Tumor mucin is immogenic and different in the glycogen structure. The antibody against the MUC1 tumor mucin, known as monoclonal antibody SM3 is made up of two large subunits (211 and 212 residues); its crystal structure complexed with the antigenic peptide ligand TSAPDTRPAPGST is known. Dynamic studies of the SM3 complexed with the antigenic peptide antigen, using VMD and NAMD, have shown hydrophobic interactions between the ligand and the aromatic rings of the protein. These attractions are observed between Pro4 of the ligand and the aromatic rings of Trp33, Tyr35, Trp94 of the SM3. There are also strong hydrophobic attractions between Pro4 and Pro8 of the ligand and Tyr32 of the protein. There is intra hydrogen-bond stabilization within the peptide antigen between Pro4 and Thr6 as well as between Asp5 side chain and Arg7. The mapping of this binding site could allow for the docking of alternate antigen in the future. Objective: Tyrosine 32 fluctuations with respect to Proline 4, 8, 10 and Alanine 3: Matt Moris, Sudeep Bhattacharyya, Thao Yang Chemistry Department University of Wisconsin-Eau Claire Chemistry Department University of Wisconsin-Eau Claire Matt Moris, Sudeep Bhattacharyya, Thao Yang Chemistry Department University of Wisconsin-Eau Claire Chemistry Department University of Wisconsin-Eau Claire To determine which residues of the antigen and antibody SM3 are responsible for binding and to predict what types of attractive forces are responsible for binding in order to determine the type of alternate ligands to place in the binding pocket. Molecular Dynamics Studies of Monoclonal Antibody SM3 with a Peptide Antigen Intramolecular stabilization: Hydrophobic rings: To prepare the monoclonal antibody SM3 and its peptide antigen for dynamic studies, first download the.pdb file from http://www.pdb.org, then run a script (.tcl file) to add hydrogen and generate a.psf file via the TkConsole in VMD 1.8.7. Next, run another script (.tcl file) to generate a new.pdb file and.psf file placing the molecule in a water sphere with a radius of 34 Å from the molecule’s center of mass. Then use NAMD graphical interface, a feature of VMD 1.8.7, to generate a dynamics (.dcd) file. Generate the dynamics file using 5,000 time steps of minimization, and 50,000 time steps (50 ps) for the molecular dynamics, under the conditions of 310 K and 1.01325 bar. This results in 1003 different dynamic conformations which can be used to conduct research.http://www.pdb.org Preparation: Figure 3a was rendered using VMD1.8.7 to show the relationship in space between the -carbon of Tyrosine-32 and the -carbons of Alanine-3, Proline-4, proline-8 and proline-10. Figure 3b shows that the proline rings of the ligand move in harmony with tyrosine-32, it also shows that the -carbon of alanine-3 moves in harmony with tyrosine-32 but it fluctuates more than the proline residues. This suggests that the prolines are critical residues in the binding pocket. 1. Dokurno, P., Lally, J M., Bates, P. A., Taylor-Papadimitriou, J., BandH., A. Snary, D. & Freemont, P. S. (1997). Crystallisation of an antitumour antibody SM3 complexed with a peptide epitope. Acta Crystallog. Sect. D, 53, 780-781. 2. Hull, S.R., Bright, A., Carraway, K. L., Abe, M., Hayes, D.F. & Kufe, D. W. (1989). Oligosaccharise differences in the DF3 sialomucin antigen from normal human milk and BT-20 human breast carcinomas cell line. Cancer Commun. 1, 261-267 3. Girling, A., Bartkova, J., Burchell, J., Gendler, S., Gillett, C. & Taylor-Papadimitriou, J. (1989). A core protein epitote of polymorphic Epithelial Mucin detected by the monoclonal-antibody SM-3 is selectively exposed in a range of primary carcinomas. Int. J. Cancer, 43, 1072-1076. 4.Granowska, M., Biassoni, L., Carrol, M. J., Howell, R., Mather, S. J., Ellison, D., Granowski, A. & Britton, K. E. (1996). Breast-cancer TC-99 M SM3 radioimmunoscintigraphy. Acta Oncologica, 35, 319-321. 5. Rees, A. R., Staunton, D., Webster, D. M., Searle, S. J., Henry, A. H. & Pedersen, J. T. (1994). Antibody design – beyond the natural limits. Trends Biotechnol. 12, 199-206. Background: The crystal structure for a fragment of SM3 with a 13-residue MUC1 peptide antigen is known . Tumour Mucin antigen is overly expressed in breast cancer . The SM3 antibody reacts with a cryptic epitote present in cancerous mucin in more than 90% of breast cancer carcinomas . The highly specified interaction between the SM3 antibody and carcinoma-associated mucin presents a potentially useful tool in the diagnosis and treatment of breast cancer . Understanding this interation at the molecular level may allow for the engineering of alternative reactive antigens . References: Results: Conclusions: In addition to the intermolecular attractions between the peptide antigen and the mucin antibody. There is also intramolecular hydrogen bonding between the carbonyl oxygen of proline-4 and the nitrogen of threonine-6, and between the -oxgens of aspartic acid-5 and the nitrogen of arginine-7 as indicated by the red dashed lines located on Fig. 2a. Figure 2b shows that there is very little fluctuation between the distances of the atoms involved in the intramolecular stabilizations. The blue curve represents the hydrogen bond length variations between Pro4 and Thr6, and the red curve, the hydrogen bond length variations between Asp5 and Arg7. Neither curve varies more than 2 Å. This data was gathered using the bond-label tool located in VMD 1.8.7. Figure 2a Figure 2b Figure 4 shows the attractions between Proline- 4 of the peptide ligand to trytophan-91, tryptophan- 96, tyrosine-35 of chain-L and Tryptophan-33 of chain-H. The proline residues of the peptide ligand are stabilized by hydrophobic rings. Figure 4 Figure 3a Figure 3b Ca SM3 RMSD: Figure 5a Figure 5b Figure 5a shows the RMSD of every fifth residue on both peptide chains of the antigen. In both Fig. 5a and 5b Red represents chain-L, blue represents chain-H, and yellow represents chain-P, the peptide antigen This data was generated using the NAMD RMSD trajectory tool located in VMD 1.8.7. Figure 5b was rendered using VMD 1.8.7, The Orange VDW represents the relative minima of chain-H indicated by Fig. 5a and the Green VDW represents the relative minima of chain-L. This indicates that both chains are critical for binding. Figure 6 Figure 6 is a contour surface map of the peptide antigen generated by Origin 8. It shows the RMSD of the peptide backbone as it evolves during the dynamics. Residues 5, 6 and 7 have the most motion as indicated by the red, orange and yellow colors. This indicates that this region does not bind to the SM3 as tightly as the other part. Hydrophobic interactions are crucial to the mucin peptide antigen binding to the monoclonal SM3 antibody. The proline rings of the peptide enhance the SM3’s ability to bind to the peptide. The terminal residues of the peptide antigen are more tightly bound to the SM3 than the central residues. Residues 5, 6, and 7 of the peptide have more motion indicating they are not bound as tightly. Figure 1 In the future, we will try placing alternate ligands in the binding pocket of the SM3 to see if they are capable of binding to the antibody and how binding affects the mucin peptide antigen conformations. Future Work: Figure 1 Shows the SM3 antibody and its peptide antigen in a water sphere, the image was rendered using VMD 1.8.7.