Recognition Of Inhibitors By ORP Family

The oxysterol binding protein(OSBP)and OSBP-related proteins(ORPs)constitute ORP family,which is conserved in yeast,plants and mammals.They play an important role in signal transduction,lipid metabolism and non-vesicular transport.The family belongs to lipid transfer protein(LTP)superfamily and these proteins share a conserved functional OSBP-related domain(ORD),which has been shown to bind and transfer sterols and glycerophospholipids.Previous studies have shown that oxysterols can inhibit the lipids binding and transfer ability by certain ORPs,besides there are multiple anti-proliferative and anti-fungal natural compounds targeting the first subfamily of the ORP family.However,it is unclear the molecular mechanism of these compounds inhibiting on the function of ORP and their selectivity for different ORPs,so confirming the binding selectivity of different inhibitors by ORP members and determining the complex of specific compounds with certain members of ORP family would elucidate the molecular mechanism of recognition and it will provide basis for structure-based rational design of specific and high affinity drugs targeting ORPs.Since these two small molecules have been reported to target the first subfamily of the human ORP family(OSBP and ORP4L),and affect cholesterol binding and transfer ability by OSBP and ORP4L,we first aligned ORP family members based on structures and constructed a large number of ORD fragments of OSBP and ORP4 L,but we did not obtain the high-purity soluble proteins,so relevant biochemical and structural studies could not be performed.Previous studies in our laboratory have shown that ORP 1-ORD and ORP2-ORD can bind and transport cholesterol,and both processes are regulated by PI(4,5)P2,so we explored the effect of oxysterols and natural compounds on cholesterol and PI(4,5)P2 binding ability by ORP1-ORD and ORP2-ORD,and conducted structural biology experiments to elucidate the specific molecular mechanism.We selected each one of this two possible types of cholesterol inhibitors,oxysterols and natural compounds as reported,which are named as Compound 1(C1)and Compound 2(C2)respectively.The results of gel filtration chromatography indicated that Compound2 could induce the tetramerization of ORP 1-ORD,while oxysterol Compound 1 hardly affected the oligomerization state of ORP1-ORD.Neither of these two small molecules affect the oligomerization state of ORP2-ORD.In vitro lipid binding assay results showed that C1 can inhibit the binding ability of PI(4,5)P2 by ORP1-ORD,while C2 inhibited the cholesterol binding ability by ORP1-ORD,but neither of them affected the lipid binding ability by ORP2-ORD.We have also attempted to crystal the complexes of ORP1-ORD with these two types of inhibitors,and microcrystals of two complexes have been obtained.