@article{53f8fded497b471f976c5bba8e375fc5,
title = "The Autophagy-Related Beclin-1 Protein Requires the Coiled-Coil and BARA Domains to Form a Homodimer with Submicromolar Affinity",
abstract = "Beclin-1 (BECN1) is an essential component of macroautophagy. This process is a highly conserved survival mechanism that recycles damaged cellular components or pathogens by encasing them in a bilayer vesicle that fuses with a lysosome to allow degradation of the vesicular contents. Mutations or altered expression profiles of BECN1 have been linked to various cancers and neurodegenerative diseases. Viruses, including HIV and herpes simplex virus 1 (HSV-1), are also known to specifically target BECN1 as a means of evading host defense mechanisms. Autophagy is regulated by the interaction between BECN1 and Bcl-2, a pro-survival protein in the apoptotic pathway that stabilizes the BECN1 homodimer. Disruption of the homodimer by phosphorylation or competitive binding promotes autophagy through an unknown mechanism. We report here the first recombinant synthesis (3-5 mg/L in an Escherichia coli culture) and characterization of full-length, human BECN1. Our analysis reveals that full-length BECN1 exists as a soluble homodimer (KD ∼ 0.45 μM) that interacts with Bcl-2 (KD = 4.3 ± 1.2 μM) and binds to lipid membranes. Dimerization is proposed to be mediated by a coiled-coil region of BECN1. A construct lacking the C-terminal BARA domain but including the coiled-coil region exhibits a homodimer KD 3.5-fold weaker than that of full-length BECN1, indicating that both the BARA domain and the coiled-coil region of BECN1 contribute to dimer formation. Using site-directed mutagenesis, we show that residues at the C-terminus of the coiled-coil region previously shown to interact with the BARA domain play a key role in dimerization and mutations weaken the interface by ∼5-fold.",
author = "Ranaghan, {Matthew J.} and Durney, {Michael A.} and Mesleh, {Michael F.} and McCarren, {Patrick R.} and Garvie, {Colin W.} and Daniels, {Douglas S.} and Carey, {Kimberly L.} and Skepner, {Adam P.} and Beth Levine and Perez, {Jose R.}",
note = "Funding Information: The authors thank Vamsi Mootha for the use of his ultracentrifuge and Jason McCoy for helpful discussions regarding the liposome float experiments. The authors also thank Stuart Schreiber for the use of his DLS instrument and Zarko Boskovic for his help with collecting the DLS data. The authors thank Deborah Pheasant and the Biophysical Instrumentation Facility for the Study of Complex Macromolecular Systems (National Science Foundation Grant 0070319) for their help with the ultracentrifugation experiments. The authors also thank Huaying Zhao for her helpful discussions regarding the sedimentation velocity data. Funding Information: *Phone: 1-617-714-7350. E-mail: jrperez@broadinstitute.org. ORCID Matthew J. Ranaghan: 0000-0003-3572-5127 Author Contributions M.J.R. cloned and purified all BECN1 proteins, purified Bcl-2, and performed all in vitro experiments. K.L.C. cloned the Bcl-2 construct. A.P.S. generated various BECN1 clones. M.A.D. and M.F.M. helped design, conduct, and analyze all NMR experiments. P.R.M. performed the molecular modeling of the BECN1 BARA domain. C.W.G., D.S.D., B.L., and J.R.P. initiated and supervised the project. All authors contributed to writing and proofreading the manuscript. Funding This work was supported by the National Institute of Allergy and Infectious Diseases of the National Institutes of Health via Grant U19 AI109725. Notes The authors declare no competing financial interest. Publisher Copyright: {\textcopyright} 2017 American Chemical Society.",
year = "2017",
month = dec,
day = "26",
doi = "10.1021/acs.biochem.7b00936",
language = "English (US)",
volume = "56",
pages = "6639--6651",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "51",
}