Protein-Membrane Interactions Regulating Cell Signaling.

Water soluble proteins or enzymes involved in signal transduction become attached to membrane interfaces as a result of protein acylation and or electrostatic interactions with negatively charged lipid interfaces. For example, MARCKS (the myristolated alanine-rich C-kinase substrate), requires both acylation and electrostatic interactions to become associated with the plasma membrane, it appears to regulate phosphoinositides (such as PI(4,5)P2) within bilayers (1-3).

Proteins also become attached as a result of the interaction of protein domains, such as C2 domains (4) or PH domains (5) with the membrane interface. C2 domains bind membranes in a Ca2+-dependent fashion and appear to be one of the most abundant Ca2+-regulatory motifs found in eucaryotic systems. PH (plexstrin homology) domains bind to polyphosphoinosites and show specificity for specific phosphorylated inositol headgroups.

Membrane attachment and defects in membrane binding domains are involved in many disease states. Dysferlin is a protein that contains 6 C2 domains and it has been implicated in membrane repair in muscle. A deficiency in this protein or mutations in one of its C2 domains appear to be responsible for certain forms of muscular dystrophy (6,7). Otoferlin also has 6 C2 domains and is likely also involved in membrane repair. A mutation in the third C2 domain of human otoferlin is believed to alter its Ca2+ binding ability and has been linked to an inherited form of deafness (8). Many oncogenes express aberrant proteins involved in cell-signaling, and in some cases membrane binding has been shown to be of central importance. For example, the viral form of the src tyrosine kinase will transform cells and promote cancer, but variants of the protein that fail to bind membranes do not transform cells (9). An Arg to Gln mutation in the PX domain of p47phox prevents targeting of the NADPH oxidase to membranes containing PI(3)P. This is responsible for chronic granulomatous, an immunodeficiency disease associated with reduced superoxide production (10).

Our work is addressing the following questions regarding the function of these domains:

How are membrane binding domains oriented on the membrane interface, how deeply do they penetrate into the bilayer, and what are the forces involved in attachment?

What are the mechanisms for Ca+2-induced translocation of C2 domains to the membrane interface. How do electrostatic, hydrophobic and entropic factors contribute to the binding?

What are the membrane bound structures for C2 domains. Does Ca2+-binding alter the structure of the domain or create a binding site for phospholipids?

How do the C2 domains from synaptotagmin dimerize, interact with components of the SNARE complex, and how do they regulate neuronal exocytosis?

We have been addressing these questions with site-directed spin labeling and high-resolution NMR. For example, we recently determined the orientation and depth of penetration of C2 domains from cPLA2 and synaptotagmin at the membrane interface (11-13). The interactions made by the first synaptotagmin domain (C2A) is in contrast to that made by the C2 domain from cPLA2, which has a similar orientation but is translocated about 5Å deeper into the membrane hydrocarbon. This difference in depth is consistent with the results of computational studies, which predict that long-range electrostatic interactions and desolvation will make different contributions to the membrane binding of these domains.

Our current work on C2 domains includes measurements on the second C2 domain from synaptotagmin (C2B) and measurements on larger fragments of synaptotagmin including both C2 domains and the transmembrane anchor. Synaptotagmin appears to be the primary protein mediating the Ca2+-triggered exocytosis in neurons. We are now setting up experiments to map protein-protein interactions between synaptotagmin and proteins that make up the SNARE complex and to determine how C2 domains might modulate bilayer curvature strain. Some of these experiments will make use of EPR distance measurements between labeled sites in the interacting proteins, and will cover medium and longer range distances (7 to 40Å).

In addition to these EPR based experiments, we are making novel NMR measurements on the electrostatic surface potential of the synaptotagmin C2A domain by examining paramagnetic enhancements of nuclear relaxation produced by charged nitroxides or metal ion complexes. These measurements will be compared with the predictions of computational approaches. We are also exploring the use of NMR and bicelle systems to recover residual dipolar couplings for surface associated C2 domains. These data will be used to provide information on the orientation of the membrane associated domain as well as its membrane bound structure.

References:

1. Rauch, M.E., C.G. Ferguson, G.D. Prestwich, and D.S. Cafiso, Myristoylated alanine-rich C kinase substrate (MARCKS) sequesters spin-labeled phosphatidylinositol 4,5-bisphosphate in lipid bilayers. J Biol Chem, 2002. 277(16): p. 14068-76.
2. Wang, J., A. Gambhir, G. Hangyas-Mihalyne, D. Murray, U. Golebiewska, and S. McLaughlin, Lateral sequestration of phosphatidylinositol 4,5-bisphosphate by the basic effector domain of myristoylated alanine-rich C kinase substrate is due to nonspecific electrostatic interactions. J Biol Chem, 2002. 277(37): p. 34401-12.
3. Gambhir, A., G. Hangyas-Mihalyne, I. Zaitseva, D.S. Cafiso, J. Wang, D. Murray, S.N. Pentyala, S.O. Smith, and S. McLaughlin, Electrostatic sequestration of PIP2 on phospholipid membranes by basic/aromatic regions of proteins. Biophys J, 2004. 86(4): p. 2188-207.
4. Rizo, J. and T.C. Sudhof, C2-domains, structure and function of a universal Ca2+-binding domain. J. Biol. Chem., 1998. 273: p. 15879-15882.
5. Lemmon, M.A. and K.M. Ferguson, PH domains: diverse sequences with a common fold recruit signaling molecules to the cell surface. Cell, 1996. 85: p. 621-624.
6. Davis, D.B., K.R. Doherty, A.J. Delmonte, and E.M. McNally, Calcium-sensitive phospholipid binding properties of normal and mutant ferlin C2 domains. J Biol Chem, 2002. 277(25): p. 22883-8.
7. Bansal, D. and K.P. Campbell, Dysferlin and the plasma membrane repair in muscular dystrophy. Trends Cell Biol, 2004. 14(4): p. 206-13.
8. Mirghomizadeh, F., M. Pfister, F. Apaydin, C. Petit, S. Kupka, C.M. Pusch, H.P. Zenner, and N. Blin, Substitutions in the conserved C2C domain of otoferlin cause DFNB9, a form of nonsyndromic autosomal recessive deafness. Neurobiol Dis, 2002. 10(2): p. 157-64.
9. Sigal, C.T., W. Zhou, C.A. Buser, and S.A. McLaughlin, Amino-terminal basic residues of Src mediate membrane binding through electrostatic interaction with acidic phospholipids. PNAS, 1994. 91: p. 12253.
10. Noack, D., J. Rae, A.R. Cross, B.A. Ellis, P.E. Newburger, J.T. Curnutte, and P.G. Heyworth, Autosomal recessive chronic granulomatous disease caused by defects in NCF-1, the gene encoding the phagocyte p47-phox: mutations not arising in the NCF-1 pseudogenes. Blood, 2001. 97(1): p. 305-11.
11. Frazier, A.A., M.A. Wisner, N.J. Malmberg, K.G. Victor, G.E. Fanucci, E.A. Nalefski, J.J. Falke, and D.S. Cafiso, Membrane orientation and position of the C2 domain from cPLA2 by site-directed spin labeling. Biochemistry, 2002. 41: p. 6282-6292.
12. Frazier, A.A., C.R. Roller, J.J. Havelka, A. Hinderliter, and D.S. Cafiso, Membrane-bound orientation and position of the synaptotagmin I C2A domain by site-directed spin labeling. Biochemistry, 2003. 42(1): p. 96-105.
13. Rufener, E., A. Frazier, C.M. Wieser, A. Hinderliter, and D.S. Cafiso, Membrane bound orientation and position of the synaptotagmin C2B domain determined by site-directed spin labeling. Biochemistry, 2005. 44: p. 18-28.