Abstract
Phafin2 is a phosphatidylinositol 3‐phosphate (PtdIns(3)P) binding protein involved in the regulation of endosomal cargo trafficking and lysosomal induction of autophagy. Binding of Phafin2 to PtdIns(3)P is mediated by both its PH and FYVE domains. However, there are no studies on the structural basis, conformational stability, and lipid interactions of Phafin2 to better understand how this protein participates in signaling at the surface of endomembrane compartments. Here, we show that human Phafin2 is a moderately elongated monomer of ∼28 kDa with an intensity‐average hydrodynamic diameter of ∼7 nm. Circular dichroism (CD) analysis indicates that Phafin2 exhibits an α/β structure and predicts ∼40% random coil content in the protein. Heteronuclear NMR data indicates that a unique conformation of Phafin2 is present in solution and dispersion of resonances suggests that the protein exhibits random coiled regions, in agreement with the CD data. Phafin2 is stable, displaying a melting temperature of 48.4°C. The folding‐unfolding curves, obtained using 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. PtdIns(3)P binding to Phafin2 occurs with high affinity, triggering minor conformational changes in the protein. Taken together, these studies represent a platform for establishing the structural basis of Phafin2 molecular interactions and the role of the two potentially redundant PtdIns(3)P‐binding domains of the protein in endomembrane compartments.
Highlights
IntroductionTerminal FYVE (Fab[1], YOTB, Vac[1], and EEA1) domains [Fig. 1(A)].1
There are no studies on the structural basis, conformational stability, and lipid interactions of Phafin[2] to better understand how this protein participates in signaling at the surface of endomembrane compartments
Heteronuclear NMR data indicates that a unique conformation of Phafin[2] is present in solution and dispersion of resonances suggests that the protein exhibits random coiled regions, in agreement with the Circular dichroism (CD) data
Summary
Terminal FYVE (Fab[1], YOTB, Vac[1], and EEA1) domains [Fig. 1(A)].1. Both PH and FYVE domains of Abbreviations: AUC, analytical ultracentrifugation; CD, circular dichroism; DLS, dynamic light scattering; ƒ/ƒ0, frictional ratio; GuHCl, guanidine hydrochloride; PtdIns(3)P, phosphatidylinositol 3-phosphate; s, sedimentation coefficient; S20,w, sedimentation coefficient under standard conditions; SPR, surface plasmon resonance. Phafin2-mediated autophagy requires the presence of PtdIns(3)P at the lysosomal surface and the serine/threonine kinase Akt.[2] Binding of Phafin[2] to Akt requires both the PH and FYVE domains. The action of Phafin[2] favors a more rapid increase of the cytosolic Ca21 levels, which enhances TNF-a-mediated apoptosis and suppresses the unfolding protein response at the ER.[7]
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