The autophagy-related Beclin-1 protein requires the coiled-coil and BARA domains to form a homodimer with submicromolar affinity

Author(s)

Matthew J. Ranaghan, Broad Institute of MIT and Harvard
Michael A. Durney, Broad Institute of MIT and Harvard
Michael F. Mesleh, Broad Institute of MIT and Harvard
Patrick R. McCarren, Broad Institute of MIT and Harvard
Colin W. Garvie, Broad Institute of MIT and Harvard
Douglas S. Daniels, University of DaytonFollow
Kimberly L. Carey, Broad Institute of MIT and Harvard
Adam P. Skepner, Broad Institute of MIT and Harvard
Beth Levine, University of Texas Southwestern Medical Center
Jose R. Perez, Broad Institute of MIT and Harvard

Document Type

Article

Publication Date

11-29-2017

Publication Source

Biochemistry

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.

Inclusive pages

6639-6651

ISBN/ISSN

0006-2960

Document Version

Postprint

Comments

The document available for download is the authors' accepted manuscript, provided in compliance with the publisher's policy on self-archiving. To view the version of record, use the DOI provided. Permission documentation is on file.

Copyright

Copyright © 2017 American Chemical Society

Publisher

ACS Publications

Volume

56

Peer Reviewed

yes

Issue

51

Keywords

Vesicles, Inorganic compounds, Genetics, Monomers, Group 13 compounds

Sponsoring Agency

National Institute of Allergy and Infectious Diseases of the National Institutes of Health via Grant U19 AI109725

eCommons Citation

Ranaghan, Matthew J.; Durney, Michael A.; Mesleh, Michael F.; McCarren, Patrick R.; Garvie, Colin W.; Daniels, Douglas S.; Carey, Kimberly L.; Skepner, Adam P.; Levine, Beth; and Perez, Jose R., "The autophagy-related Beclin-1 protein requires the coiled-coil and BARA domains to form a homodimer with submicromolar affinity" (2017). Chemistry Faculty Publications. 99.
https://ecommons.udayton.edu/chm_fac_pub/99