Invariant BECN1 CXXC motifs bind Zn2+ and regulate structure and function of the BECN1 intrinsically disordered region

Abstract

ABSTRACT Autophagy is an essential catabolic cellular homeostasis process conserved in all eukaryotes. BECN1/Beclin 1, a key autophagy protein involved in autophagosome nucleation, contains two invariant CxxC motifs within a large intrinsically disordered region (IDR) at the BECN1 N terminus. We used inductively coupled plasma mass spectrometry to demonstrate that BECN1 binds Zn2+ in a 1:1 molar ratio, and that mutation of the invariant cysteines abrogated Zn2+ binding, demonstrating that the invariant CxxC motifs were responsible for binding Zn2+. We used circular dichroism spectroscopy to show that Zn2+-binding increases IDR helicity, and likely also binding-associated helical transitions. Further, we used small angle X-ray scattering and multi-angle light scattering to show that Zn2+-binding made the BECN1 IDR more compact, and HDX-MS to show that the Zn2+-bound state of the IDR had reduced solvent accessibility. We also used isothermal titration calorimetry to show that the BECN1 IDR did not self-associate and abrogation of Zn2+ binding does not affect binding of a viral BCL2 homolog. Last, we showed that both CxxC motifs are required, but the intervening IDR is less important, for starvation-triggered upregulation of autophagy. Thus, Zn2+-binding via the CxxC motifs likely facilitates binding-associated helical conformational transitions of BECN1 important for up-regulating BECN1-mediated autophagy. Abbreviations AFM: aromatic finger mutant; BH3D: BCL2 homology 3 domain; CCD: coiled-coil domain; CD: circular dichroism spectroscopy; [CysDM1]: C18S and C21S double mutant; [CysDM2]: C137S, and C140S double mutant; [CysTM], C18S, C21S, C137S, and C140S tetrad mutant; Dmax: maximum particle diameter; dRI, differential refractive index; EFA: evolving factor analysis; FHD: flexible helical domain; FL: full length; GFP: green fluorescent protein; HDX-MS: hydrogen/deuterium exchange mass spectrometry; ICP-MS: inductively coupled plasma mass spectrometry; IDR: intrinsically disordered region; ITC, isothermal titration calorimetry; MALS, multi angle light scattering; MBP: maltose-binding protein; MoRFs: molecular recognition features; P(r): pairwise-distance distribution; PtdIns3K: class III phosphatidylinositol 3-kinase; Rg: radius of gyration; SASBDB: small angle scattering biological data bank; SEC: size-exclusion chromatography; SEC-SAXS: size-exclusion chromatography in tandem with small angle X-ray scattering; TEV: tobacco-etch virus; TFE: 2,2,2-trifluoroethanol; TPEN: N,N,N,N-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine; Vc: volume of correlation; WT: wild-type.