bi7b00936_si_001.pdf (1.16 MB)
The Autophagy-Related Beclin‑1 Protein Requires the Coiled-Coil and BARA Domains To Form a Homodimer with Submicromolar Affinity
journal contribution
posted on 2017-11-29, 00:00 authored by Matthew
J. Ranaghan, Michael A. Durney, Michael F. Mesleh, Patrick R. McCarren, Colin W. Garvie, Douglas S. Daniels, Kimberly L. Carey, Adam P. Skepner, Beth Levine, Jose R. PerezBeclin-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.