Structural basis of sterol binding and transport by a yeast StARkin domain

Julian-Alexander Jentsch,Irene Kiburu,Kalpana Pandey,Michael Timme,Trudy Ramlall,Bodo Levkau,Jin Wu,David Eliezer,Olga Boudker,Anant K Menon

doi:10.1074/jbc.ra118.001881

Abstract

The StARkin superfamily comprises proteins with steroidogenic acute regulatory protein-related lipid transfer (StART) domains that are implicated in intracellular, non-vesicular lipid transport. A new family of membrane-anchored StARkins was recently identified, including six members, Lam1-Lam6, in the yeast Saccharomyces cerevisiae. Lam1-Lam4 are anchored to the endoplasmic reticulum (ER) membrane at sites where the ER is tethered to the plasma membrane and proposed to be involved in sterol homeostasis in yeast. To better understand the biological roles of these proteins, we carried out a structure-function analysis of the second StARkin domain of Lam4, here termed Lam4S2. NMR experiments indicated that Lam4S2 undergoes specific conformational changes upon binding sterol, and fluorescence-based assays revealed that it catalyzes sterol transport between vesicle populations in vitro, exhibiting a preference for vesicles containing anionic lipids. Using such vesicles, we found that sterols are transported at a rate of ∼50 molecules per Lam4S2 per minute. Crystal structures of Lam4S2, with and without bound sterol, revealed a largely hydrophobic but surprisingly accessible sterol-binding pocket with the 3-OH group of the sterol oriented toward its base. Single or multiple alanine or aspartic acid replacements of conserved lysine residues in a basic patch on the surface of Lam4S2 near the likely sterol entry/egress site strongly attenuated sterol transport. Our results suggest that Lam4S2 engages anionic membranes via a basic surface patch, enabling "head-first" entry of sterol into the binding pocket followed by partial closure of the entryway. Reversal of these steps enables sterol egress.

Highlights

The StARkin superfamily comprises proteins with steroidogenic acute regulatory protein–related lipid transfer (StART) domains that are implicated in intracellular, non-vesicular lipid transport
Similar results were obtained for a number of point mutants in which specific lysine resides in the StARkin domain were replaced with alanine or aspartic acid (Figs. 1B and 2)
Förster resonance energy transfer (FRET) experiments demonstrated proximity between tryptophan residue(s) and the naturally fluorescent sterol dehydroergosterol (DHE) in protein samples that had been incubated with DHE-containing liposomes

Summary

To whom correspondence may be addressed

Sterols play an important role in eukaryotic cells because of their ability to regulate the fluidity and barrier function of the plasma membrane (PM) over a wide range of temperatures and lipid compositions [1, 2]. Oxysterol-binding protein and some of its homologs as well as members of the steroidogenic acute regulatory protein–related lipid transfer (StART) protein superfamily (termed StARkins) have been implicated as sterol transport proteins [11,12,13,14]. The latter are characterized by an ␣/␤ helix-grip fold with a ␤ sheet forming the wall of a binding pocket for sterols or other lipids [15]. Replacement of conserved lysine residues in this region severely attenuates the ability of the protein to transport sterols in vitro

Results and discussion

Conclusion

Experimental procedures