Abstract
Beclin 1 (BECN1) is a key regulator of autophagy, a critical catabolic homeostasis pathway that involves sequestration of selected cytoplasmic components by multilayered vesicles called autophagosomes, followed by lysosomal fusion and degradation. BECN1 is a core component of class III phosphatidylinositol-3-kinase complexes responsible for autophagosome nucleation. Without heterologous binding partners, BECN1 forms an antiparallel homodimer via its coiled-coil domain (CCD). However, the last 16 CCD residues, composing an "overlap helix" (OH), have been crystallized in two mutually exclusive states: either as part of the CCD or packed against the C-terminal β-α repeated, autophagy-specific domain (BARAD). Here, using CD spectroscopy, isothermal titration calorimetry, and small-angle X-ray scattering, we show that in the homodimeric state, the OH transitions between these two different packing states, with the predominant state comprising the OH packed against the BARAD, contrary to expectations based on known BECN1 interactions with heterologous partners. We confirmed this observation by comparing the impact of mutating four residues that mediate packing of the OH against both the CCD and BARAD on structure and stability of the CCD, the OH+BARAD, and the two-domain CCD-BARAD. Last, we used cellular assays to demonstrate that mutation of these OH-interface residues abrogates starvation-induced up-regulation of autophagy but does not affect basal autophagy. In summary, we have identified a BECN1 helical region that transitions between packing as part of either one of two conserved domains (i.e. the CCD or the BARAD). Our findings have important implications for the relative stability of autophagy-inactive and autophagy-active BECN1 complexes.
Highlights
Beclin 1 (BECN1) is a key regulator of autophagy, a critical catabolic homeostasis pathway that involves sequestration of selected cytoplasmic components by multilayered vesicles called autophagosomes, followed by lysosomal fusion and degradation
1359 Å2 of surface area are buried upon the overlap helix” (OH) packing against the BARAD, significantly more than the 937 Å2 buried when the OH packs within the coiled-coil domain (CCD)
The secondary structure and melting temperature estimations by CD, homodimer association affinity quantified by isothermal titration calorimetry (ITC), solution size and shape parameter determinations by size-exclusion chromatography (SEC)-small angle X-ray scattering (SAXS), and assessment of flexibility by the Kratky plot and ensemble optimization method (EOM) all indicate that CCD secondary, tertiary, and quaternary structure stability is significantly disrupted by the OH tetrad mutation
Summary
The BECN1 OH cannot simultaneously pack against the CCD homodimer partner helix and the BARAD. Analysis of the OHϩ BARAD crystal structure (PDB entry 4DDP) indicates that the OHϩBARAD molecules are arranged in a head-to-tail manner in the crystal lattice, stabilized by the aromatic finger, consisting of Phe359, Phe360, and Trp361, of one OHϩBARAD molecule being buried within a hydrophobic pocket formed partly by the OH in the OHϩBARAD molecule [33] (supplemental Fig. S2) We hypothesized that this interaction may be the cause of protein aggregation during purification of WT OHϩBARAD– containing constructs. The AFM, wherein the aromatic finger residues, Phe359, Phe360, and Trp361, are mutated to Asp, has no effect on the secondary structure content estimated from CD (supplemental Fig. S3 and Table S1) or the SEC-SAXS profiles (supplemental Fig. S4 and Table S2) of the CCDϩBARAD constructs This is consistent with previously published analyses that showed that the AFM does not change the overall secondary structure or thermal stability of the BECN1 OHϩBARAD [33]. A narrower Rg and Dmax distribution for the selected ensemble compared with the random pool suggests that the molecule is rigid, whereas an Rg and Dmax distribution for the ensemble that is as broad as the pool suggests
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