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

Abstract

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 H- N 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 MgCl and CaCl ). 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 H 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 N-labeled target protein for NMR studies.

NMR– and MD simulation–based structural characterization of the membrane-associating FATC domain of ataxia telangiectasia mutated

“…Finally, acetylation of lysine 3016 (54) N-terminal of the highly conserved FATC region of ATM may affect the described localization at the plasma membrane, mitochondria, peroxisomes, microsomes, and other cytosolic vesicles (12,14,15,(23)(24)(25). As suggested for the FATC domain of TOR, the one of ATM may also be fused to other proteins or peptides or other substances to tether them to membrane mimetics (64).…”

FKBP8 inhibits virus‐induced RLR‐VISA signaling

“…Recent confocal microscopy results have showed that FKBP8 and NS5A protein of the classical swine fever virus colocalize in the cytoplasm, suggesting a strong interaction between the proteins . Furthermore, RNAi‐mediated silencing of FKBP8 gene can induce the activation of mTOR signaling in goat fetal fibroblasts via protein‐protein interaction through its ankyrin‐repeat domain . FKBP8 can interact with the antiapoptotic protein B‐cell lymphoma 2 (Bcl‐2) and protect it from degradation …”

“…14 Furthermore, RNAi-mediated silencing of FKBP8 gene can induce the activation of mTOR signaling in goat fetal fibroblasts via protein-protein interaction through its ankyrin-repeat domain. [30][31][32][33] FKBP8 can interact with the antiapoptotic protein B-cell lymphoma 2 (Bcl-2) and protect it from degradation. 34 The spatial localization data between FKBP8 and RIG-I, VISA, and TBK1 indicate that FKBP8 translocates from the cytoplasm to the mitochondrial outer membrane and endoplasmic reticulum membrane to participate in the RLR-VISA signaling pathway and thus plays a crucial role in the antiviral process.…”

Unraveling the complex genetics of neural tube defects: From biological models to human genomics and back

“…During morphogenesis, Fkbp8 controls neural cell fate through antagonism of SHH signaling, which is critical for proper neural tube closure (Bulgakov, Eggenschwiler, Hong, Anderson, & Li, 2004;Cho, Ko, & Eggenschwiler, 2008;Fong et al, 2003). Fkbp8 also contains functional tetratricopeptide-repeat (TPR) domains, a leucine zipper repeat, and a C-terminal transmembrane (TM) domain (De Cicco, Milroy, & Dames, 2018). The C-terminal TM domain facilitates FKBP8 protein anchorage to mitochondrial membranes (Shirane & Nakayama, 2003).…”