A Sec14-like Phosphatidylinositol Transfer Protein Paralog Defines a Novel Class of Heme-binding Proteins With An Unusual Heme Coordination Mechanism

Abstract

Yeast Sfh5 is an unusual member of the Sec14-like phosphatidylinositol transfer protein (PITP) family. Whereas PITPs are defined by their abilities to transfer phosphatidylinositol between membranes in vitro, and to stimulate phosphoinositide signaling in vivo, Sfh5 does not exhibit these activities. Rather, Sfh5 is a redox-active penta-coordinate high spin Fe III heme-binding protein with an unusual heme-binding arrangement that involves a co-axial tyrosine/histidine coordination strategy and a complex electronic structure connecting the open shell iron d-orbitals with three aromatic ring systems. That Sfh5 is not a PITP is supported by demonstrations that heme is not a readily exchangeable ligand, and that phosphatidylinositol-exchange activity is resuscitated in heme binding-deficient Sfh5 mutants. The collective data identify Sfh5 as the prototype of a new class of fungal hemoproteins, and emphasize the versatility of the Sec14-fold as scaffold for translating the binding of chemically distinct ligands to the control of diverse sets of cellular activities. Huang et al., 2018), to plants (Peterman et al., 2004;Vincent et al., 2005;Ghosh et al, 2015;Huang et al., 2016), and to vertebrates (Hamilton et al., 1997;Alb et al., 2003;Kono et al., 2013). Sec14-like PITPs potentiate activities of PtdIns 4-OH kinases via a heterotypic lipid exchange cycle between a membrane-docked PITP molecules and the bilayer where the dynamics of an abortive exchange of a 'priming' lipid for PtdIns exposes the PtdIns to the lipid kinase catalytic site. In this way, the lipid exchange cycle renders PtdIns a superior substrate for the enzyme. Structure-based predictions that PtdIns-binding is conserved across the Sec14 superfamily, while the nature of the priming lipid is diversified, suggests a mechanism where the PITP acts as a metabolic sensor for activating PtdIns4P signaling in response to a metabolic input (Schaaf et al., 2008;Bankaitis et al., 2010;Grabon et al., 2019).As all characterized Sec14-like PITPs are lipid-binding proteins, interrogation of the priming ligand question has focused on lipids as it is widely presumed that lipid exchange/transfer activity is the common feature amongst these proteins. Recent work suggests the ligand menu for Sec14-like proteins might be broader than previously thought, however.Cavity mapping studies identified the yeast Sec14-like protein Sfh5 as exhibiting an electropositive surface and a uniquely basic 'lipid-binding' cavity -thus suggesting Sfh5 might bind a ligand that is not a lipid (Tripathi et al., 2019). Herein, we describe a biophysical and structural characterization of Sfh5. We report Sfh5 is a novel penta-coordinate high spin Fe 3+ heme-binding protein conserved across the fungal kingdom that exhibits unusual heme-binding properties. While the biological functions of Sfh5 remains to be determined, chemogenomic analyses forecast Sfh5 plays a role in organic oxidant-induced stress responses. As such, Sfh5 is the founding member of a new and conserved class of fungal hemoprotei...