Structural basis of receptor-mediated cellular vitamin A uptake - 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.