Abstract
Signaling lipids are found in specific subcellular membranes, where they recruit and regulate cytosolic proteins and contribute to bilayer structure and dynamics. These interactions are vital for signaling and membrane trafficking pathways and contribute to the organization, growth, and differentiation of the cell. However, the analysis of the physical and chemical mechanisms of membrane interaction and lipid recognition is technically challenging, motivating the development of new NMR methods to study lipid and bilayer binding by peripheral membrane proteins in solution. We describe methods that have been optimized for the FYVE and phox (PX) domains of the EEA1 and Vam7p proteins, respectively, both of which specifically recognize phosphatidylinositol 3-phosphate (PtdIns3P) within endocytic membranes. Solution-state NMR methods were used to characterize the phosphoinositide and membrane interaction sites and affinities and can be used to illustrate protein:micelle structures and phospholipid specificities. The methods are generally applicable and can be used to discover and characterize the phospholipid interactions of other membrane-interacting protein domains.
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Abbreviations
- DHPC::
-
dihexanoyl- or diheptanoyl-phosphatidylcholine;
- DPC::
-
dodecylphosphocholine;
- GST::
-
glutathione S-transferase;
- HSQC::
-
heteronuclear single quantum coherence;
- PC::
-
phosphatidylcholine;
- PI::
-
phosphatidyl inositol;
- TCEP::
-
tris (2-carboxyethyl) phosphine.
References
Lee SA, Eyeson R, Cheever ML, Geng JM, Verkhusha VV, Burd C, Overduin M, Kutateladze TG. Targeting of the FYVE domain to endosomal membranes is regulated by a histidine switch. Proc Natl Acad Sci USA 2005;102:13052–7.
Cheever ML, Sato TK, de Beer T, Kutateladze TG, Emr SD, Overduin M. Phox domain interaction with PtdIns(3)P targets the Vam7 t-SNARE to vacuole membranes. Nat Cell Biol 2001;3:613–8.
Ford MG, Pearse BM, Higgins MK, Vallis Y, Owen DJ, Gibson A, Hopkins CR, Evans PR, McMahon HT. Simultaneous binding of PtdIns(4,5)P 2 and clathrin by AP180 in the nucleation of clathrin lattices on membranes. Science 2001;291:1051–5.
Overduin M, Cheever ML, Kutateladze TG. Signalling with phosphoinositides: Better than binary. Mol Interv 2001;1:150–9.
Lee E, Marcucci M, Daniell L, Pypaert M, Weisz OA, Ochoa GC, Farsad K, Wenk MR, De Camilli P. Amphiphysin 2 (Bin1) and T-tubule biogenesis in muscle. Science 2002;297:1193–6.
Johnson JE, Giorgione J, Newton AC. The C1 and C2 domains of protein kinase C are independent membrane targeting modules, with specificity for phosphatidylserine conferred by the C1 domain. Biochemistry 2000;39:11360–9.
Mikoshiba K, Fukuda M, Ibata K, Kabayama H, Mizutani A. Role of synaptotagmin, a Ca2+ and inositol polyphosphate binding protein, in neurotransmitter release and neurite outgrowth. Chem Phys Lipids 1999;98:59–67.
Stolt PC, Jeon H, Song HK, Herz J, Eck MJ, Blacklow SC. Origins of peptide selectivity and phosphoinositide binding revealed by structures of disabled-1 PTB domain complexes. Structure 2003;11:569–79.
Iyer LM, Koonin EV, Aravind L. Adaptations of the helix-grip fold for ligand binding and catalysis in the START domain superfamily. Proteins 2001;43:134–44.
Zwaal RF, Comfurius P, Bevers EM. Lipid-protein interactions in blood coagulation. Biochim Biophys Acta 1998;1376:433–53.
Tokonzaba E, Capelluto DG, Kutateladze TG, Overduin M. Phosphoinositide, phosphopeptide and pyridone interactions of the Abl SH2 domain. Chem Biol Drug Des 2006;67:230–7.
Boggon TJ, Shan WS, Santagata S, Myers SC, Shapiro L. Implication of tubby proteins as transcription factors by structure-based functional analysis. Science 1999;286:2119–25.
Harlan JE, Hajduk PJ, Yoon HS, Fesik SW. Pleckstrin homology domains bind to phosphatidylinositol-4,5-bisphosphate. Nature 1994;371:168–70.
Peter BJ, Kent HM, Mills IG, Vallis Y, Butler PJ, Evans PR, McMahon HT. BAR domains as sensors of membrane curvature: The amphiphysin BAR structure. Science 2004;303:495–9.
Niggli V, Andreoli C, Roy C, Mangeat P. Identification of a phosphatidylinositol-4,5-bisphosphate-binding domain in the N-terminal region of ezrin. FEBS Lett 1995;376:172–6.
Zimmermann P, Meerschaert K, Reekmans G, Leenaerts I, Small JV, Vandekerckhove J, David G, Gettemans J. PIP(2)-PDZ domain binding controls the association of syntenin with the plasma membrane. Mol Cell 2002;9:1215–25.
Prilusky J, Felder CE, Zeev-Ben-Mordehai T, Rydberg EH, Man O, Beckmann JS, Silman I, Sussman JL. FoldIndex: A simple tool to predict whether a given protein sequence is intrinsically unfolded. Bioinformatics 2005;21:3435–38.
Yang ZR, Thomson R, McNeil P, Esnouf RM. RONN: The bio-basis function neural network technique applied to the detection of natively disordered regions in proteins. Bioinformatics 2005;21:3369–76.
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res 1997;25:3389–402.
Thompson JD, Higgins DG, Gibson TJ. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 1994;22:4673–80.
Lepre CA, Moore JM. Microdrop screening: A rapid method to optimize solvent conditions for NMR spectroscopy of proteins. J Biomol NMR 1998;12:493–9.
Armstrong N, de Lencastre A, Gouaux E. A new protein folding screen: Application to the ligand binding domains of a glutamate and kainate receptor and to lysozyme and carbonic anhydrase. Protein Sci 1999;8:1475–83.
Sheehan D, O'Sullivan S. Fast protein liquid chromatography. Methods Mol Biol 2004;244:253–8.
Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel RD, Bairoch A. ExPASy: The proteomics server for in-depth protein knowledge and analysis. Nucleic Acids Res 2003;31:3784–8.
Cheever ML, Kutateladze TG, Overduin M. Increased mobility in the membrane targeting PX domain induced by phosphatidylinositol 3-phosphate. Protein Sci 2006;15:1873–82.
Vuillard L, Braun-Breton C, Rabilloud T. Non-detergent sulphobetaines: A new class of mild solubilization agents for protein purification. Biochem J 1995;305(Pt 1):337–43.
Golovanov AP, Hautbergue GM, Wilson SA, Lian LY. A simple method for improving protein solubility and long-term stability. J Am Chem Soc 2004;126:8933–9.
Amezcua CA, Harper SM, Rutter J, Gardner KH. Structure and interactions of PAS kinase N-terminal PAS domain: Model for intramolecular kinase regulation. Structure 2002;10:1349–61.
Kay LE. Pulsed field gradient multi-dimensional NMR methods for the study of protein structure and dynamics in solution. Prog Biophys Mol Biol 1995;63:277–99.
Vinogradova O, Sonnichsen F, Sanders CR. On choosing a detergent for solution NMR studies of membrane proteins. J Biomol NMR 1998;11:381–6.
le Maire M, Champeil P, Moller JV. Interaction of membrane proteins and lipids with solubilizing detergents. Biochim Biophys Acta 2000;1508:86–111.
Altieri AS, Hinton DP, Byrd RA. Association of biomolecular systems via pulsed field gradient NMR self-diffusion measurements. J Am Chem Soc 1995;117:7566–7.
Kutateladze TG, Capelluto DG, Ferguson CG, Cheever ML, Kutateladze AG, Prestwich GD, Overduin M. Multivalent mechanism of membrane insertion by the FYVE domain. J Biol Chem 2004;279:3050–7.
Goetz H, Kuschel M, Wulff T, Sauber C, Miller C, Fisher S, Woodward C. Comparison of selected analytical techniques for protein sizing, quantitation and molecular weight determination. J Biochem Biophys Methods 2004;60:281–93.
Lebowitz J, Lewis MS, Schuck P. Modern analytical ultracentrifugation in protein science: A tutorial review. Protein Sci 2002;11:2067–79.
Wilson WW. Light scattering as a diagnostic for protein crystal growth—A practical approach. J Struct Biol 2003;142:56–65.
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Kami, K., Rajesh, S., Overduin, M. (2009). Phospholipid-Interacting Proteins by Solution-State NMR Spectroscopy. In: Larijani, B., Woscholski, R., Rosser, C. (eds) Lipid Signaling Protocols. Methods in Molecular Biology, vol 462. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-115-8_20
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DOI: https://doi.org/10.1007/978-1-60327-115-8_20
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