Autophagy, a conserved catabolic process required for cellular homeostasis in eukaryotes, is regulated by many proteins. The central goal of my doctoral research is to investigate conformational flexibility of autophagy proteins, with a special focus on BECN1, a core component of the class III phosphatidylinositol-3 kinase autophagosome nucleation complex that may serve as an autophagy interaction hub. Our rigorous bioinformatics analysis predicts that 57% of 59 key human autophagy proteins contain intrinsically disordered regions (IDRs), which lack stable secondary and tertiary structure. The prevalence of IDRs suggests that IDRs play an important, yet hitherto uninvestigated, role in autophagy. We confirm disorder of selected IDRs via biophysical methods, and use additional bioinformatics tools to predict protein-protein interaction and phosphorylation sites within IDRs, identifying potential biological functions. We experimentally investigate four distinct BECN1 domains: (i) The IDR, which includes a functional BCL2 homology 3 domain (BH3D) that binds BCL2 proteins, undergoing a binding-associated disorder-to-helix transition and enabling BCL2s to inhibit autophagy. (ii) The flexible helical domain (FHD) which has an unstructured N-terminal half and structured Cterminal half forming a 2.5-turn helix in our 2.0 Å X-ray crystal structure. Our molecular dynamics simulations and circular dichroism spectroscopy analyses indicate the FHD transiently samples more helical conformations and likely undergoes a binding-associated disorder-to-helix transition. We also show that the FHD bears conserved residues critical for AMBRA1 interaction and for starvation-induced autophagy. (iii) A coiled-coil domain (CCD) which forms an antiparallel homodimer in our 1.46 Å X-ray crystal structure. We have also built a atomistic model of an optimally packed, parallel BECN1:ATG14 CCD heterodimer that agrees with our experimental SAXS data. Further, we show that BECN1:ATG14 heterodimer interface residues identified from this model are important for heterodimer formation and starvation-induced autophagy. (iv) A β-α repeated autophagy-specific domain which bears invariant residues that we show are important for starvation-induced autophagy. Thus, we demonstrate that conformational flexibility is a key BECN1 feature. Lastly, we show that multi-domain BECN1 constructs have extended conformations with no intra-domain interactions that impact structure of other domains, suggesting that BECN1 structure and conformational flexibility enable its function as an autophagy interaction hub.