Apoptosis Signaling and 3D Structural Transitions. 

 

       Structural transitions involving proteins with well defined 3D structures are known to be important in apoptosis signaling (programmed cell death).  These proteins, which are classified as Bcl-2 apoptosis regulators, include BAX, BID, and Bcl-XL [12, 13].  While these three proteins have well defined 3D structures, as determined by NMR and x-ray crystallography, other regulators, including BIM, BAD, and BMF are intrinsically unstructured proteins [14] that bind various Bcl-2 family members to facilitate their function(s).  This project is focused on the transitional mechanism of Bcl-2 proteins (primarily BAX) to investigate their mechanism of 3D transition with the purpose of developing inhibitors (or activators) to control apoptosis in vivo.

 

 

 

 

 

Figure 1.  Helical interaction energy distribution in the Bcl-2 family of apoptotic regulators demonstrating variation in stabilizing regions, indicating variation in transitional mechanisms.  The energy values in this diagram are relative and do not represent actual values due to entropic factors not being in the calculations.    

 

 

Preliminary results from my lab (in collaboration with Dr. Khaled at UCF) have shown that the helical interaction energy of the carboxyl-terminal region of BAX (known as α9) is important in controlling membrane localization and apoptotic activity.  As a result of this finding, we determined that interaction of the α9 helix with an internal hydrophobic groove explained how BAX could transition from a soluble cytoplasmic protein to a membrane associated protein that appears to undergo homo-dimerization or oligomerization.  However, the initiating events that enabled interactions between the α9 helix and the hydrophobic groove of BAX, as well as interactions of BAX with other BCL-2 family members remain elusive.

To advance our understanding of this mechanism, we determined the helical interaction energies of all helices within the BAX protein to better understand the static packing arrangement of the helices across the entire 3D structure (Figure 1).  Our results demonstrated that BAX, and other Bcl-2 family members, have a significant variation in helical interaction energies (IEs) that may explain their ability to undergo structural transitions involved in apoptotic signaling events.  Three papers have been submitted based on these studies and I plan on continuing this work (in collaboration with Dr. Khaled) to fully characterize these transitional mechanisms.

The goals of determining the mechanism of transition of Bcl-2 proteins (beginning with BAX) and developing modulators for these transitions will be completed using a combination of x-ray crystallography, computational docking, NMR, mutagenesis, fluorescent probe labeling, and peptide binding studies.  These studies will be initiated by creating a “non-transitional” BAX protein that contains disulfide linkages that are engineered at specific locations in the protein.  This protein, which should maintain its 3D fold, will allow transitions that are dependent on the reducing environment and allow the transition to be initiated in a chemically dependent manner.  Various helices (in combinations of pairs) will be “locked” using disulfides at specific locations to inhibit mobility and apoptosis will be monitored using cellular assays and characterized using NMR and fluorescence spectroscopy (with fluorescent donor-acceptor pairs).  The hydrophobic groove of BAX (and other Bcl-2 family members) will be used in computational docking experiments (using Auto-DOCK and NCI library compounds) to find small molecules (or peptides) that promote transitions and force the terminal helices away from the structure.  X-ray crystallography will be used to find small molecule binding positions by soaking crystals of the disulfide locked proteins with analogs and peptides.

 

 

References.

 

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2.          Grundling, A., M.D. Gonzalez, and D.E. Higgins, Requirement of the Listeria monocytogenes broad-range phospholipase PC-PLC during infection of human epithelial cells. Journal of Bacteriology, 2003. 185(21): p. 6295-6307.

3.          Heffernan, B.J., et al., Bacillus anthracis phospholipases C facilitate macrophage-associated growth and contribute to virulence in a murine model of inhalation anthrax. Infection and Immunity, 2006. 74(7): p. 3756-3764.

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6.          Hondal, R.J., Zhao, Z., Kravchuk, A. V., Liao, H., Riddle, S. R., Yue, X., Bruzik, K. S., Tsai, M. D., Mechanism of phosphatidylinositol-specific phospholipase C: a unified view of the mechanism of catalysis. Biochemistry., 1998. 37(13): p. 4568-80.

7.          Heinz, D.W., Essen, L. O., Williams, R. L., Structural and mechanistic comparison of prokaryotic and eukaryotic phosphoinositide-specific phospholipases C. Journal of molecular biology., 1998. 275(4): p. 635-50.

8.          Kravchuk, A.V., Zhao, L., Bruzik, K. S., Tsai, M. D., Engineering a catalytic metal binding site into a calcium-independent phosphatidylinositol-specific phospholipase C leads to enhanced stereoselectivity. Biochemistry., 2003. 42(8): p. 2422-30.

9.          Apiyo, D., et al., X-ray structure of the R69D phosphatidylinositol-specific phospholipase C enzyme: Insight into the role of calcium and surrounding amino acids in active site geometry and catalysis. Biochemistry, 2005. 44(30): p. 9980-9989.

10.        Iwasaki, Y., Tsubouchi, Y., Ichihashi, A., Nakano, H., Kobayashi, T., Ikezawa, H., Yamane, T., Two distinct phosphatidylinositol-specific phospholipase Cs from Streptomyces antibioticus. Biochimica et biophysica acta., 1998. 1391(1): p. 52-66.

11.        Zhao, L., Liu, Y., Bruzik, K. S., Tsai, M. D., A novel calcium-dependent bacterial phosphatidylinositol-specific phospholipase C displaying unprecedented magnitudes of thio effect, inverse thio effect, and stereoselectivity. Journal of the American Chemical Society., 2003. 125(1): p. 22-3.

12.        Chan, S.L. and V.C. Yu, Proteins of the Bcl-2 family in apoptosis signalling: From mechanistic insights to therapeutic opportunities. Clinical and Experimental Pharmacology and Physiology, 2004. 31(3): p. 119-128.

13.        Petros, A., E. Olejniczak, and S. Fesik, Structural biology of the Bcl-2 family of proteins. Biochim Biophys Acta, 2004. 1644(2-3): p. 83-94.

14.        Hinds, M.G., et al., Bim, Bad and Bmf: intrinsically unstructured BH3-only proteins that undergo a localized conformational change upon binding to prosurvival Bcl-2 targets. Cell Death and Differentiation, 2007. 14(1): p. 128-136.

15.        Southan, C., A genomic perspective on human proteases as drug targets. Drug Discovery Today, 2001. 6(13): p. 681-688.

16.        Borgono, C.A. and E.P. Diamandis, The emerging roles of human tissue kallikreins in cancer. Nature Reviews Cancer, 2004. 4(11): p. 876-890.