Structure-Function Studies of membrane associated Inositol Signaling Enzymes. 

 

         Phosphatidylinositol (PI) specific phospholipase C (PI-PLC) enzymes are involved in the production of PI (or its derivatives) and diacylglycerol (DAG) in eukaryotic signaling cascades (Scheme 1) [1].  The bacterial enzymes are known to function as virulence factors during the development of diseases such as meningitis, sepsis, and anthrax [2-5].  While the bacterial and eukaryotic enzymes both use general acid/base (GA/GB) mechanisms to catalyze the cleavage of P-O bonds during catalysis [6], larger eukaryotic enzymes utilize Ca⁺² to regulate catalysis by stabilizing the transition state of the reaction [7].  In smaller bacterial enzymes this stabilization is typically achieved by arginine that can be replaced by Ca⁺² upon mutation to aspartate [8].  The x-ray structure of this enzyme was solved in my lab in 2005 and demonstrated an active site similar to the mammalian enzyme [9]. 

 

 

 Scheme 1.  Mechanism of hydrolysis for PI-PLC enzymes to liberate cyclic inositol (cIP) and DAG.   

 

To further our understanding of this class of signaling enzymes, I will continue to investigate the structural and mechanistic changes that occur upon Ca⁺² binding in PI-PLCs as well as the structural and functional changes that have taken place during evolution of Ca⁺² independent enzymes to Ca⁺² dependent ones.  This study was initiated using the Ca⁺² dependent PI-PLC from Streptomyces antibioticus (saPLC) [10].  This enzyme is the only reported Ca⁺² dependent bacterial PI-PLC and has been characterized by kinetic methods demonstrating a mechanism similar to the mammalian enzyme with unusual stereoselectivity and an efficient kinetic rate [11].  As with other bacterial enzymes, saPLC shares moderate sequence similarity with mammalian PI-PLCs, including the requisite GA/GB histidines and amino acids proposed to bind Ca⁺².  SaPLC also shows lower sequence homology with known bacterial enzymes supporting the possibility that it is evolutionarily related to both types of PI-PLCs, yet structurally and mechanistically distinct.

The goals of these studies will be to; a) determine the structure of saPLC alone and in the presence of substrate analogs and inhibitors, b) perform site-specific mutagenesis experiments to investigate the roles of tryptophan(s) in membrane association and/or catalysis, c) investigate the dynamics of this enzyme (and mutants) using ¹H-¹⁵N HSQC experiments to following protein conformational changes during lipid and/or inhibitor binding, and d) develop lead compounds for drug development based on the PI-PLCs active site structure and membrane association properties.

 

 

 

Figure 1.  Crystals of saPLC grown in my lab and the resulting diffraction pattern.  Selenomethionine and heavy atom derivatives are currently being prepared to obtain phases and complete the structure in the free and bound forms. 

 

 

These goals will be accomplished using x-ray crystallography for structure determination combined with fluorescence spectroscopy and NMR to monitor dynamic structural changes.  Diffraction quality (2.1 Å) crystals have been grown in my lab (Figure 1) and phase determination is currently underway.  Additionally, studies demonstrating dynamic changes in tryptophan fluorescence upon lipid and inositol binding have been completed and tryptophan-to-alanine mutants are currently being characterized.  These structure-function studies will be combined with additional biophysical, mutagenesis, and kinetic data to fully characterize this enzyme as a Ca⁺² dependent PI-PLC model system while pursuing crystals and biophysical characterization of the larger mammalian enzymes and their complexes.

 

 

References.

 

1.          Rong, R., et al., Phospholipase activity of phospholipase C-gamma 1 is required for nerve growth factor-regulated MAP kinase signaling cascade in PC12 cells. Journal of Biological Chemistry, 2003. 278(52): p. 52497-52503.

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.

4.          Szitasova, I., H. Drahovska, and J. Turna, Distribution of virulence factors and AFLP typing of Bacillus cereus food isolates. Biologia, 2002. 57(3): p. 313-319.

5.          Griffith, O.H., Ryan, M., Bacterial phosphatidylinositol-specific phospholipase C: structure, function, and interaction with lipids. Biochimica et biophysica acta., 1999. 1441(2-3): p. 237-54.

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.