Structural, Thermodynamic and Phosphatidylinositol 3-Phosphate Binding Properties of Phafin2

Abstract

Phafin2 is a phosphatidylinositol 3-phosphate (PtdIns(3)P)-binding protein involved in the regulation of endosomal cargo trafficking and in the lysosomal induction of autophagy. Binding of Phafin2 to PtdIns(3)P is mediated by both its PH and FYVE domains. However, the structural basis for understanding how Phafin2 promotes signaling at endomembrane compartments is unknown. Here, we show that the human recombinant Phafin2 is a moderately elongated monomer of ∼28 kDa. Circular dichroism (CD) analysis indicates that Phafin2 exhibits an α/β structure and predicts ∼40% of random coil content in the protein. Heteronuclear NMR data indicates that a unique conformation of Phafin2 is present in solution and that dispersion of resonances suggests that the protein exhibit random coil regions, in agreement with CD data. Dynamic light scattering studies show that Phafin2 is relatively stable, displaying a melting temperature of 35°C. The folding-unfolding curves, obtained by the use of urea- and guanidine hydrochloride-mediated denaturation, indicate that Phafin2 undergoes a two-state native to denatured transition. Analysis of these transitions shows that the free energy change for urea- and guanidine hydrochloride-induced Phafin2 denaturation in water is ∼4 kcal mol−1. Using SPR analysis, we show that PtdIns(3)P binding to Phafin2 occurs with moderate affinity. Preliminary data shows that Phafin2 promotes membrane tethering and tubulation in vitro in a PtdIns(3)P-dependent manner, suggesting that the protein plays a role in inducing membrane curvature changes, a pre-requisite to trigger autophagy. Taken together, these studies represent a platform for establishing the structural basis of Phafin2 membrane interactions and the role of its two potentially redundant PtdIns(3)P-binding domains in endomembrane compartments.