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

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 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, 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.

Structural determinants of lipid specificity within Ups/PRELI lipid transfer proteins

“…In addition to headgroup recognition, we highlight a role for conformational plasticity and hydrophobicity of the Ω loop gate in the PRELI domain, in regulating membrane targeting and capture of specific lipids. Notably, a similar role of flexible loop regions has been postulated for other lipid transfer proteins of the STARkin superfamily, pointing to similar structural principles of substrate selection [27][28][29][30][31][32] .…”

Aster Proteins Facilitate Nonvesicular Plasma Membrane to ER Cholesterol Transport in Mammalian Cells

“…Despite the relatively low sequence identity, the three-dimensional structure of Aster A broadly resembles the START domain fold, and is similar to the START-like domains in the Lam2 and Lam4 proteins (Horenkamp et al, 2018; Jentsch et al, 2018; Tong et al, 2018) (C-alpha RMSD c.2Å; Supplemental Figure 2B). However, sequence differences within the cholesterol-binding pocket result in a different binding mode for the ligand, such that in Aster-A the sterol is rotated by approximately 120° about the long axis of ligand compared with the ligands in the START domains.…”

“…A simulated annealing composite omit map unambiguously identifies the ligand orientation in mouse Aster A (left panel - contoured at 1.0 sigma). The orientation of the 25-hydroxycholesterol ligand in mouse Aster A is markedly different from that in the distantly related yeast Lam4p (right panel - pdbcode 6bym) (Jentsch et al, 2018). The ligand is rotated by approximately 120° about it long axis such that the axial methyl groups on the cholesterol are orientated very differently.…”

Endoplasmic reticulum-plasma membrane contact sites integrate sterol and phospholipid regulation

“…Lam proteins each have one or two sterol-binding StARkin domains. The purified domains have been shown to catalyze sterol exchange between vesicles in vitro [ 26 – 28 ]. Lam1–Lam4 are integral ER membrane proteins located at the cell cortex, where they might function as sterol transporters, similar to the mammalian StARkin STARD3, which is anchored to endosomal membranes and has been suggested to facilitate endosome–ER cholesterol transfer [ 29 ].…”