Structural mechanism for sterol sensing and transport by OSBP-related proteins

Abstract

The oxysterol binding protein (OSBP)-related proteins (ORPs) are conserved from yeast to man 1, 2 and are implicated in regulation of sterol pathways 3,4 and in signal transduction 5 . The structure of the full-length yeast ORP Osh4 was determined at 1.5-1.9 Å resolution in complexes with ergosterol, cholesterol, and 7-, 20-, and 25-hydroxycholesterol. A single sterol molecule binds in a hydrophobic tunnel in a manner consistent with a transport function for ORPs. The entrance is blocked by a flexible N-terminal lid and surrounded by functionally critical basic residues. The structure of the open state of a lid-truncated form of Osh4 was determined at 2.5 Å resolution. Structural analysis and limited proteolysis show that sterol binding closes the lid and stabilizes a conformation favoring transport across aqueous barriers and transmitting signals. The unliganded structure exposes potential phospholipid-binding sites that are positioned for membrane docking and sterol exchange. Based on these observations we propose a model in which sterol and membrane binding promote reciprocal conformational changes that facilitate a sterol transfer and signaling cycle.OSBP was first discovered 6,7 as a cytosolic receptor for oxysterols that downregulate cholesterol synthesis 8 . The cloning of OSBP 1 led to the identification of a large family of OSBP-related proteins, the ORPs, with 7 members in S. cerevisiase and 12 in H. sapiens 2 . ORPs are essential for life in eukaryotes. The deletion of all 7 ORPs leads to severe defects in sterol and lipid distribution and endocytosis in yeast 3,4 , and OSBP is essential for embryonic development in mice (M. Brown, personal communication). All ORPs contain a core OSBPrelated domain (ORD), and many also contain pleckstrin homology (PH) domains, transmembrane regions, endoplasmic reticulum (ER)-targeting FFAT motifs, GOLD domains, and/or ankyrin repeats 2 . These additional domains localize ORPs by binding to phosphoinositides 9 , the ER protein VAP 10 , and other targeting signals. The localization of ORPs is dynamic. Oxysterol binding changes the subcellular localization of certain ORPs from the cytosol to the Golgi or ER 9,11 . ORPs can bind lipids other than oxysterols, including phosphoinositides and phosphatidic acid 12,13 . OSBP is a cholesterol sensing regulator of two protein phosphatases, a PTPPBS family member, and Ser/Thr phosphatase PP2A 5 .The structure of full-length Osh4 was determined by multiple isomorphous replacement and refined to a free R-factor of 23 % at 1.5 Å resolution (Supplementary Fig. 1 Table 1). Osh4 is built around a central antiparallel β-sheet of 19 strands (residues 115-293) which form a nearly complete β-barrel (Fig. 1a, Supplementary Fig. 2). The strands of the barrel are pitched at ~45 degrees to its axis. The barrel has structural similarity to the large β-barrels of various bacterial outer membrane transporters 14 as scored by Dali 15 ( Supplementary Fig. 3). There is no PH domain within the ORD 13 . A tunnel with largely hydrophobic walls...

Using HHsearch to tackle proteins of unknown function: A pilot study with PH domains

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The complex that inserts lipopolysaccharide into the bacterial outer membrane forms a two-protein plug-and-barrel

“…To our knowledge, there are no other known examples of such a two-protein architecture in any organism, as every previously described plug-barrel complex consists of a single polypeptide (29)(30)(31). Encoding the plug and barrel as two distinct proteins complicates the assembly of the LPS transport apparatus.…”

Sterol-regulated transport of SREBPs from endoplasmic reticulum to Golgi: Oxysterols block transport by binding to Insig

“…Rather, it may be that the hydroxyl group orients the sterol in relation to the lipid bilayer so that Insig-2 can recognize it. It is also possible that Insig-2 interacts indirectly with the hydroxyl group on an oxysterol through water-mediated interactions as in the case of Osh4, a yeast oxysterol-binding protein whose crystal structures has been determined with bound ligand (37). Whatever the mechanism of binding, it is noteworthy that all sterols that completely inhibit SREBP processing bind to either Scap or Insig, but not to both (Fig.…”