Robert V. Stahelin, Ph.D.
Associate Professor, Biochemistry and Molecular Biology
Indiana University School of Medicine - South Bend
Adjunct Assistant Professor, Chemistry and Biochemistry
University of Notre Dame
Ph.D. in Chemistry (2003), University of Illinois at Chicago
Postdoctoral Research Associate (2003-2005), University of Illinois at Chicago
Visiting Research Assistant Professor (2005-2006), University of Illinois at Chicago
Wonhwa Cho , University of Illinois at Chicago
Diana Murray , Weill Medical College of Cornell University
Charles Chalfant , Virginia Commonwealth University
Tatiana Kutateladze , University of Colorado at Denver and Health Sciences Center
Tom Hope, Northwestern University
A large number of cytoplasmic proteins involved in cell signaling and membrane trafficking reversibly translocate to different cellular membranes in response to specific stimuli. Many of these peripheral proteins contain one or more modular domains specialized in lipid binding. These lipid-binding structural modules, also known as membrane-targeting domains, include C1, C2 (see Fig. 1), PH, FYVE, PX, ENTH, ANTH, BAR, FERM, and tubby domains. My laboratory’s work aims to progress our understanding of the mechanisms by which reversible binding of membrane targeting domains and their host proteins to different cell membranes is mediated and regulated, with an emphasis on how kinetics and energetics of their membrane-protein interactions are modulated by different factors. The long-term objective of this research is to apply principles learned from biochemical and biophysical studies to the generation of novel therapeutics to combat cancer, arthritis, asthma, and infections diseases such as HIV.
Figure 1X-ray structures of C2 domains at the membrane interface. Also shown is the membrane penetration of the C1 domain of PKCdelta.
Lipid bilayers have a highly polarized structure that consists of a central hydrocarbon core region and two flanking interfacial regions (see Fig. 2). The hydrocarbon region and the combined interfacial regions have comparable width (ca. 30Å each) so that the interfaces account for roughly 50% of the total thickness of the bilayer. The interfacial regions consist of a complex mixture of water, lipid backbone phosphate groups, headgroups, and the polar portion of the acyl chains and the polarity profile changes dramatically over the 15 Å span of an interface, from the hydrocarbon region to the aqueous solution. Due to this complex nature of the lipid bilayer, the location of the protein in the bilayer is a critical factor that governs the kinetics and energetics of its membrane interactions. Based on their membrane location, membrane targeting proteins can be arbitrarily subdivided into three groups; (1) S-type proteins that are localized at the membrane surface and in the shallow interfacial region (i.e., outside of the level of the backbone phosphate group; see Fig. 2) and interact predominantly with the polar headgroups, (2) I-type proteins that penetrate significantly into the interfacial region (i.e., inside the level of the phosphate), and (3) H-type proteins that penetrate into the hydrocarbon core region of the lipid bilayer. Both I- and H-type peripheral proteins interact with both the polar headgroups and the hydrocarbon of the bilayer.
Figure 2 Depiction of membrane penetration behavior of different proteins described above.
All intracellular membranes contain a varying degree of anionic lipids and a majority of peripheral proteins (and membrane targeting domains) contain cationic surfaces, at least locally. Recent biophysical studies of membrane-protein interactions using a large number of membrane targeting domains, their host proteins, and respective mutants have revealed that binding of these proteins to anionic membranes also follows a two-step mechanism, in which the initial formation of nonspecific collisional complexes, driven by diffusion and electrostatic forces, is followed by the formation of tightly bound complexes, which are stabilized by specific interactions and/or membrane penetration (see Fig. 2 and 3). The initial membrane adsorption of peripheral proteins enhances the effective concentration of the protein at the membrane increasing the probability the protein is able to interact with both effectors and substrates.
Figure 3 Two-step membrane binding mechanism of a peripheral protein.
The initial membrane attachment can also facilitate the penetration of hydrophobic and aromatic residues on the surfaces of peripheral proteins (mainly H/I-types) into the interfacial and hydrocarbon core regions of the lipid bilayer. Since hydrophobic side chains of proteins are not normally exposed to the molecular surface, membrane penetration of membrane targeting domains and peripheral proteins often involves the conformational change of proteins at the membrane interface that exposes the buried hydrophobic side chains. Biological activities of some peripheral proteins depend heavily on their partial membrane insertion. Specific lipid binding and membrane penetration are not mutually exclusive, as it has been shown that specific lipid binding of some phosphoinositide-binding domains, such as FYVE, PX, and ENTH domains, also induces their membrane penetration. Resulting specific interactions and/or hydrophobic interactions provide the proteins with extra binding energy that is necessary for their membrane recruitment and activity.
1) Elucidation of the Membrane-Targeting Mechanisms of Human Nedd4 Proteins: Ubiquitination mediates diverse cellular functions being essential to protein degradation and trafficking. This process is crucial in the proteasomal degradation and is an essential part of regulating protein function, protein trafficking, DNA repair, gene transcription, and misfolded proteins. Neuronal precursor cell-expressed developmentally down-regulated 4 (Nedd4) proteins are called E3 ligases, and have the critical role of determining substrate specificity of the ubiquitination process. Nedd4 proteins are comprised of an N-terminal C2 domain (membrane-binding/Ca2+-binding), multiple WW domains (substrate recognition and intracellular localization), and a catalytic homologous E6-AP carboxyl terminus (HECT) domain. To date, little is known regarding how the C2 domains are involved in the subcellular targeting and regulation of different Nedd4 proteins. We are using are biophysical tools and mammalian cell culture to demonstrate how C2 domains of Nedd4 proteins regulate the targeting and activation of this important class of proteins.
2) Investigation of the Differential Mechanisms of Activation of cPLA2 Isoforms: Phospholipases A2 (PLA2) is a superfamily of enzymes that catalyze the hydrolysis of fatty acid ester at the sn-2 position of phospholipids (See Fig. 4). The PLA2-catalyzed hydrolysis of some membrane phospholipids liberates arachidonic acid (AA) that can be converted to potent inflammatory lipid mediators, prostaglandins (PG) and leukotrienes (LT) (collectively known as eicosanoids), through the cyclooxygenase (COX) and lipoxygenase (LO) pathways, respectively. Because free AA levels are very low in cells, its formation by PLA2 generally limits the synthesis of AA metabolites. Because an imbalance in the production of these potent mediators of allergic and inflammatory reactions could lead to acute and chronic inflammatory diseases, including sepsis, rheumatoid arthritis, asthma and heart disease, understanding the regulation of PLA2 is an important step toward the elucidation of the pathogenesis of inflammatory diseases. Among many forms of mammalian secretory PLA2s (sPLA2s) and intracellular PLA2s identified so far, secretory group V PLA2 (gVPLA2) and group IVA cytosolic PLA2s (cPLA2s) (See Fig. 5), have been implicated in eicosanoid biosynthesis and inflammation. Recent evidence suggests different cellular lipids, such as phosphoinositides (PIs) or sphingolipids may be responsible for the membrane targeting and activation of cPLA2 isoforms under different conditions. We are investigating the role and regulatory mechanisms of PIs and sphingolipids in the membrane targeting and activation of the group IV cPLA2 isoforms (a, b, d, e, and z) using a biophysical and cellular approach. Click here to see a movie of cPLA2a translocation in response to Ca2+ release.
Fig. 4 above, Fig. 5 Below
Figure 4 Hydrolysis of phospholipid by PLA2.
Figure 5 Domain architecture of cytosolic PLA2 alpha and beta.
Movie Translocation of cPLA2-alpha in response to calcium release.
3) Evaluation of Retroviral Matrix Proteins Interacting with the Membrane: Retroviruses (i.e., HIV and Leukemia virus) are enveloped viruses that cause a number of diseases in humans and animals. When viruses are newly assembled, they acquire their lipid coats by budding through the plasma membrane of host cells, this coat is then shed when the virus is mature and fuses with the membrane of a target cell. The Gag polyprotein harbors structural proteins of the retroviral particles as domains, which are cleaved by the viral protease during budding and maturation. Gag regulates the assembly of new virions and is targeted to the plasma membrane by its matrix domain (MA). The mature MA remains at the membrane until the virus infects a cell and its components disassemble. MA-membrane interactions play a critical role throughout the retroviral life cycle. To better understand how these interactions regulate retroviral targeting, we are investigating how different MA interact differentially with biological membranes and how one retroviral MA can function in many roles. Lipid affinity, specificity, and penetration are being explored along with structure-function analysis.
Stahelin, R.V. and Cho, W. “Membrane Binding Analysis of C1 and C2 Domains Using Surface Plasmon Resonance” (2001) Biophys. J., 80:1, 534a.
Bittova, L., Stahelin, R.V., and Cho, W. “Roles of Ionic Residues of the C1 Domain in Protein Kinase Ca Activation and the Origin of Phosphatidylserine Specificity” (2001) J. Biol. Chem., 276, 4218-4226.
Stahelin, R.V. and Cho, W. “Differential Roles of Ionic, Aliphatic, and Aromatic Residues in Membrane-Protein Interactions: A Surface Plasmon Resonance Study on Phospholipases A2” (2001) Biochemistry, 40, 4672-4678 .
Cho, W., Bittova, L., and Stahelin, R.V. “In vitro Membrane Binding Assays for Peripheral Proteins” (2001) Anal. Biochem., 296, 153-161.
Stahelin, R.V. and Cho, W. “Roles of Ca2+ Ions in the Membrane Binding of C2 Domains” (2001) Biochem. J., 359, 679-685 .
Stahelin, R.V., Long, F., Diraviyam, K., Bruzik, K.S., Murray, D., and Cho, W. “Phosphatidylinositol-3-Phosphate Induces the Membrane Penetration of the FYVE Domains of Vps27p and Hrs” (2002) J. Biol. Chem., 277, 26379-26388.
Karathanassis, D., Stahelin, R.V., Bravo, J., Perisic, O., Pacold, C.M., Cho, W., and Williams, R.L. “Binding of the PX Domain of p47phox to Phosphatidylinositol-3,4-bisphosphate and Phosphatidic Acid is Masked by an Intramolecular Interaction (2002) EMBO J., 21, 5057-5068.
Cho, W., Digman, M.A., Ananthanarayanan, B., and Stahelin, R.V. “Bacterial Expression and Purification of C1 and C2 Domains of Protein Kinase C Isoforms” (2003) Methods in Molecular Biology, vol. 233: Protein Kinase C Protocols, Ed., Newton, A.C., K. Humana Press, Totowa, New Jersey 291-298.
Stahelin, R.V., Forslund, R.E., Wink, D.J., and Cho, W. “Development of a Biochemistry Laboratory Course with a Project-Oriented Goal” (2003) Biochemistry and Molecular Biology Education, 31, 106-112 .
Stahelin, R.V., Rafter, J.D., Das, S., and Cho, W. “A Molecular Basis for Differential Subcellular Localization of C2 Domains of Protein Kinase C-a and Cytosolic Phospholipase A2” (2003) J. Biol. Chem., 278, 12452-12460 .
Diraviyam, K., Stahelin, R.V., Cho, W., and Murray, D. “Computer Modeling of the Membrane Interactions of FYVE Domains”(2003) J. Mol. Biol., 328, 721-736 .
Stahelin, R.V., Burian, A., Murray, D., Bruzik, K.S., and Cho, W. “Membrane Binding Mechanisms of the NADPH Oxidase PX Domains” (2003) J. Biol. Chem., 278, 14469-14479 .
Stahelin, R.V., Long, F., Peter, B.J., Murray, D., De Camilli, P., McMahon, H.T., and Cho, W. “Contrasting Membrane Interaction Mechanisms of AP180 and N-terminal Homology (ANTH) and Epsin N-terminal Homology (ENTH) Domains”(2003) J. Biol. Chem., 278, 28993-28999 .
Ananthanarayanan, B., Stahelin, R.V., Digman, M.A., and Cho, W. “Activation Mechanisms of Conventional Protein Kinase C Isoforms are Determined by the Ligand Affinity and Conformational Flexibility of Their C1 Domains” (2003) J. Biol. Chem., 278, 46886-46894 .
Malkova, S., Stahelin, R.V., Long, F., Pingali, S.V., Cho, W., and Schlossman, M.L. “X-ray Reflectivity Studies of cPLA2-C2 Domains Adsorbed onto Langmuir Monolayers of SOPC” (2004) Biophys. J., 86:1, 377a .
Stahelin, R.V., Digman, M.A., Medkova, M., Ananthanarayanan, B., Rafter, J.D., Melowic, H.R., and Cho, W. “Mechanism of Diacylglycerol-Induced Membrane Targeting and Activation of Protein Kinase Cd” (2004) J. Biol. Chem., 279, 29501-29512 .
Blatner, N.R., Stahelin, R.V., Diraviyam, K., Hawkins, P.T., Hong, W., Murray, D., and Cho, W. “The Molecular Basis of the Differential Subcellular Localization of FYVE Domains” (2004) J. Biol. Chem., 279, 53818-53827 .
Stahelin, R.V., Ananthanarayanan, B., Blatner, N.R., Singh, S., Bruzik, K.S., Murray, D., and Cho, W. “Mechanism of Membrane Binding of the Phospholipase D1 PX Domain” (2004) J. Biol. Chem., 279, 54918-54926 .
Cho, W., and Stahelin, R.V. “Membrane-Protein Interactions in Membrane Trafficking and Signal Transduction” (2005) Annu. Rev. Biophys. Biomol. Struct. 34:119-151.
Subramanian, P., Stahelin, R.V., Szulc, Z., Bielawska, A., Cho, W., and Chalfant, C.E. “Ceramide-1-Phosphate Acts as a Positive Allosteric Activator of Cytosolic Phospholipase A2 and Enhances the Interaction of the Enzyme with Phosphatidylcholine” (2005) J. Biol. Chem., 280, 17601-17607 .
Stahelin, R.V., Digman, M.A., Medkova, M., Ananthanarayanan, B., Melowic, H.R., Rafter, J.D., and Cho, W. “Diacylglycerol-Induced Membrane Targeting and Activation of Protein Kinase C?: Mechanistic Differences Between PKCd and e” (2005) J. Biol. Chem., 280, 19784-19793 .
Cho, W. and Stahelin, R.V. “In Vitro and Cellular Membrane Binding Mechanisms of Membrane Targeting Domains” (2005) In “Protein-Lipid Interactions”, Tamm, L.K. Ed., Academic Press, N.Y., 369-401.
Malkova, S., Long, F., Stahelin, R.V., Pingali, S.V., Murray, D., Cho, W., and Schlossman, M.L. “X-ray Reflectivity Studies of cPLA2-C2 Domains Determine its Depth of Penetration and Membrane Orientation” (2005) Biophys. J., 89, 1861-1873 .
Stahelin, R.V., Wang, J., Blatner, N.R., Rafter, J.D., Murray, D., and Cho, W. “The Origin of C1 and C2 Domain Interactions of PKC-a In Vitro and In Vivo” (2005) J. Biol. Chem., 280, 36452-36463 .
Stahelin, R.V., Hwang, J.H., Kim, J.H., Park, Z.Y., Johnson, K.R., Obeid, L., and Cho, W. “The Mechanism of the Subcellular Localization of Human Sphingosine Kinase 1” (2005) J. Biol. Chem., 280, 43030-43038 .
Bhardwaj, N., Stahelin, R.V., Langois, R.E., Cho, W., and Lui, H. “Structural Bioinformatics Prediction of Membrane-Binding Proteins” (2006) J. Mol. Biol., 359, 486-495 .
Mallikaratchy, P., Stahelin, R.V., Cao, Z., Cho, W., and Tan, W. “Selection of High Affinity DNA Ligands for Protein Kinase C-d” (2006) Chem. Commun., 2006, 3229-3231 .
Cho, W. and Stahelin, R.V. “Membrane Binding and Subcellular Localization of C2 Domains”(2006) Biochim. Biophys. Acta, in press .
Mohsin Vora, B.S., Research Scientist
Sean Cullen, undergraduate, University of Notre Dame
Keaton Jones, undergraduate, University of Notre Dame
Sachi Seilie, undergraduate, University of Notre Dame