Target of rapamycin FATC domain as a general membrane anchor: The FKBP‐12 like domain of FKBP38 as a case study

• Published in: Protein science Vol. 27; no. 2; pp. 546 - 560
• Main Authors: De Cicco, Maristella, Milroy, Lech‐G., Dames, Sonja A.
• Format: Journal Article
• Language: English
• Published: United States Wiley Subscription Services, Inc 01.02.2018; John Wiley and Sons Inc
• Subjects: Anchoring; Calcium chloride; Case studies; Cell Membrane - metabolism; Chemical synthesis; Circular Dichroism; Data processing; Humans; Liposomes; Liposomes - metabolism; Localization; Magnetic resonance spectroscopy; membrane mimetic; Membranes; Methods and Applications; Micelles; Models, Molecular; Molecular Dynamics Simulation; Mutation; NMR spectroscopy; Nuclear Magnetic Resonance, Biomolecular; Peptides; pH effects; Phosphatidylinositol 3-Kinases - chemistry; Phosphatidylinositol 3-Kinases - genetics; Phosphatidylinositol 3-Kinases - metabolism; Protein Conformation; Protein Domains; protein membrane anchoring; Proteins; protein–lipid interactions; protein–membrane interactions; Rapamycin; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Reducing agents; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - chemistry; Saccharomyces cerevisiae Proteins - genetics; Saccharomyces cerevisiae Proteins - metabolism; Salts; Signaling; Sodium chloride; Spectrum analysis; Tacrolimus Binding Proteins - chemistry; Tacrolimus Binding Proteins - genetics; Tacrolimus Binding Proteins - metabolism; Tacrolimus-binding protein; Titration; TOR protein; Yeast; protein-membrane interactions; protein membrane anchoring; membrane mimetic; NMR spectroscopy; protein-lipid interactions
• DOI: 10.1002/pro.3321
• ISSN: 0961-8368

Increased efforts have been undertaken to better understand the formation of signaling complexes at cellular membranes. Since the preparation of proteins containing a transmembrane domain or a prenylation motif is generally challenging an alternative membrane anchoring unit that is easy to attach, water‐soluble and binds to different membrane mimetics would find broad application. The 33‐residue long FATC domain of yeast TOR1 (y1fatc) fulfills these criteria and binds to neutral and negatively charged micelles, bicelles, and liposomes. As a case study, we fused it to the FKBP506‐binding region of the protein FKBP38 (FKBP38‐BD) and used 1H–15N NMR spectroscopy to characterize localization of the chimeric protein to micelles, bicelles, and liposomes. Based on these and published data for y1fatc, its use as a C‐terminally attachable membrane anchor for other proteins is compatible with a wide range of buffer conditions (pH circa 6–8.5, NaCl 0 to >150 mM, presence of reducing agents, different salts such as MgCl2 and CaCl2). The high water‐solubility of y1fatc enables its use for titration experiments against a membrane‐localized interaction partner of the fused target protein. Results from studies with peptides corresponding to the C‐terminal 17–11 residues of the 33‐residue long domain by 1D 1H NMR and CD spectroscopy indicate that they still can interact with membrane mimetics. Thus, they may be used as membrane anchors if the full y1fatc sequence is disturbing or if a chemically synthesized y1fatc peptide shall be attached by native chemical ligation, for example, unlabeled peptide to 15N‐labeled target protein for NMR studies.