Structural basis of receptor-mediated cellular vitamin A uptake

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ABSTRACT Vitamin A is an essential nutrient for all mammals. Many biological processes, including and foremost vision, are crucially dependent on its adequate supply for proper function. Alterations of vitamin A metabolism can result in a wide spectrum of ocular defects and lead to blindness. Retinol (vitamin A alcohol) is the predominant circulating vitamin A form in the fasting state. In times of need (i.e. in the absence of dietary vitamin A intake), in order to distribute vitamin A to the target peripheral tissues, retinol is released in the bloodstream from the liver, the main body storage site of the vitamin, bound to retinol-binding protein (RBP). Inside the cells, retinol binds specific intracellular carriers, namely cellular retinol-binding proteins, and it serves as a precursor for the active vitamin A forms: retinaldehyde, critical for vision, and retinoic acid, the ligand for specific nuclear receptors that regulate the transcription of hundreds of target genes. How retinol is released from the retinol-RBP complex and internalized by the cell has been subject of debate for decades. STRA6, the putative plasma membrane receptor for RBP, was identified in 2007. We first determined the structure of STRA6, from Danio rerio reconstituted in amphipol, by single-particle cryo- electron microscopy to 3.9 Å resolution. Our structure revealed a possible mechanism for retinol to transition from RBP across the membrane, in a STRA6-mediated manner. It also showed an unexpected association of STRA6 with calmodulin, setting forth the hypothesis of a correlation between retinol metabolism and calcium homeostasis, which we investigated in a biophysical and cellular context. Here we propose to understand how the system works mechanistically at a molecular level. In particular, we aim to investigate how RBP and STRA6 interact to mediate retinol exchange, the role the membrane plays in the process, and how the entire process is impacted by varying calcium levels. To do so, we plan to adopt an integrated approach comprising structural biology, molecular dynamics (MD) simulations, biophysical experiments and retinol-uptake assays. We will switch to a mammalian system so as to better correlate our results with cell-based observations, and to perform structural experiments in the close to native environment of a lipid-filled nanodisc. In support of the proposed experiments, we present preliminary data on the structure of a mammalian STRA6 both in the apo form and in complex with RBP, on the MD simulations focusing on the molecular-detailed mechanistic aspects of the release of retinol from RBP into STRA6, and from STRA6 into the membrane, and evidence that all the biophysical and biochemical assays we will utilize are in place. The multifaceted, synergistic mechanistic studies we propose to carry out in this application will enable detailed structure-based understanding of STRA6-mediated retinol uptake. This is a process of extreme importance, in particular in organs such as the eye, that rely on tight control of retinol levels for proper physiological function.