Structural, thermodynamic, and phosphatidylinositol 3-phosphate binding properties of Phafin2.
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.
Protein Signaling Domains Laboratory, Department of Biological Sciences, Biocomplexity Institute, and Center for Soft Matter and Biological Physics, Virginia Tech, Blacksburg, Virginia, 24061.
Tuo-Xian
TX
Tang
Department of Chemical Engineering, Virginia Tech, Blacksburg, Virginia, 24061.
Ami
A
Jo
Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, 24061.
Jingren
J
Deng
Biomolecular Magnetic Resonance Facility, University of Virginia, Charlottesville, Virginia, 22904.
Jeffrey F
JF
Ellena
Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia, 24061.
Iulia M
IM
Lazar
Department of Chemical Engineering, Virginia Tech, Blacksburg, Virginia, 24061.
Richey M
RM
Davis
Protein Signaling Domains Laboratory, Department of Biological Sciences, Biocomplexity Institute, and Center for Soft Matter and Biological Physics, Virginia Tech, Blacksburg, Virginia, 24061.
Daniel G S
0000-0002-0412-4508
DG
Capelluto
D018129:Phosphatidylinositol Phosphates / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D000072417:Protein Domains; D013329:Structure-Activity Relationship; D013816:Thermodynamics; D033921:Vesicular Transport Proteins / Q000737:chemistry* / Q000378:metabolism
C547687:PLEKHF2 protein, human; D018129:Phosphatidylinositol Phosphates; D033921:Vesicular Transport Proteins; C055525:phosphatidylinositol 3-phosphate
Phafin2; conformation; phosphatidylinositol 3-phosphate; protein structure