Molecular basis for the interaction of cellular retinol binding protein 2 (CRBP2) with nonretinoid ligands

Josie A Silvaroli,Jacqueline Plau,Charlie H Adams,Surajit Banerjee,Made Airanthi K Widjaja-Adhi,William S Blaner,Marcin Golczak

doi:10.1016/j.jlr.2021.100054

Abstract

Present in the small intestine, cellular retinol binding protein 2 (CRBP2) plays an important role in the uptake, transport, and metabolism of dietary retinoids. However, the recent discovery of the interactions of CRBP2 with 2-arachidonoylglycerol and other monoacylglycerols (MAGs) suggests the broader involvement of this protein in lipid metabolism and signaling. To better understand the physiological role of CRBP2, we determined its protein-lipid interactome using a fluorescence-based retinol replacement assay adapted for a high-throughput screening format. By examining chemical libraries of bioactive lipids, we provided evidence for the selective interaction of CRBP2 with a subset of nonretinoid ligands with the highest affinity for sn-1 and sn-2 MAGs that contain polyunsaturated C18-C20 acyl chains. We also elucidated the structure-affinity relationship for nonretinoid ligands of this protein. We further dissect the molecular basis for this ligand's specificity by analyzing high-resolution crystal structures of CRBP2 in complex with selected derivatives of MAGs. Finally, we identify T51 and V62 as key amino acids that enable the broadening of ligand selectivity to MAGs in CRBP2 as compared with retinoid-specific CRBP1. Thus, our study provides the molecular framework for understanding the lipid selectivity and diverse functions of CRBPs in controlling lipid homeostasis.

Highlights

Supplementary key words lipid transport retinoids lipid transfer proteins retinol binding protein monoacylglycerols cellular retinol binding protein 2 Rbp2 acyl chain highthroughput screening of lipids
After the elimination of chemicals with spectral properties interfering with the fluorescence assay, the high-throughput screening (HTS) performed on human recombinant cellular retinol binding protein 2 (CRBP2) revealed nine independent hits
In addition to the binding of atROL, our data unambiguously establish the preferential interaction of CRBP2 with MAGs

Summary

Introduction

These widespread and diverse proteins provide a uniform scaffold that can be adapted to provide specificity for a variety of lipid substrates Because of their high affinity for all-trans-retinol (atROL) and all-trans-retinal, a subclass of mammalian intracellular lipocalins has been designated as cellular retinol binding proteins (CRBPs) [4, 5]. It has been shown that in the absence of CRBP2, mice were more susceptible to developing obesity and metabolic disorders This phenotype was associated with variety of metabolic abnormalities, including decreased energy expenditure, elevated levels of small intestinal MAGs, impaired response to glucose challenge, and an altered regulation of gastric inhibitory polypeptide (GIP) synthesis and/or release from enteroendocrine cells.

Methods

Results

Conclusion