Structure of the STRA6 receptor for retinol uptake

INTRODUCTION Vitamin A is an essential nutrient for all mammals, being vital for vision and for transcription of a wide array of genes. Retinol (vitamin A alcohol) is the predominant circulating retinoid. In the fasting state, retinol from liver stores is mobilized bound to retinol-binding protein (RBP), which transports this highly hydrophobic molecule in the bloodstream. How retinol is released from RBP and internalized by target cells has been the subject of intense debate. The RBP receptor, STRA6, was cloned in 2007. STRA6 was predicted to be a 75-kDa multipass transmembrane (TM) protein without sequence similarity to any known transporter, channel, or receptor. STRA6 is expressed widely, with particular abundance in the eye and placenta. Mutations in the human STRA6 gene have been linked to Matthew-Wood syndrome, which presents with ocular abnormalities and developmental defects. RATIONALE Despite a wealth of biochemical work aimed at investigating how STRA6 mediates internalization of retinol from RBP, progress at the molecular level has been hindered by the absence of structural information. Purified STRA6 from zebrafish was a detergent-stable dimer in an unexpected association with calmodulin (CaM), forming a 180-kDa complex. RESULTS Using cryo-electron microscopy, we determined the structure of zebrafish STRA6 in complex with CaM to 3.9 A resolution. The protein is assembled as an intricate dimer with a topology that includes 18 TM helices (nine per protomer) and two long horizontal intramembrane (IM) helices interacting at the dimer core. Each STRA6 protomer comprises an N-terminal domain (NTD) of the first five TM helices, connected by a linker containing the first CaM-binding peptide to a central domain at the dimer interface that includes TMs 6 to 9 and the IM helices, and a cytoplasmic C-terminal segment that interacts with CaM through two additional helices. Each protomer is compactly associated with one molecule of CaM, adopting an unconventional arrangement in which it is bound to three helical regions of STRA6. We characterized the STRA6-CaM interaction biophysically by isothermal titration calorimetry, showing that the affinity of CaM for one STRA6 peptide alone is subnanomolar, and structurally by x-ray crystallography. We also demonstrated that the STRA6-CaM association is physiological by performing immunoprecipitation experiments on native zebrafish tissue. Both the extra- and intracellular surfaces of the NTD feature conserved polar pockets. The outer NTD pocket spans half the bilayer. The central domain of STRA6 defines a large ~20,000 A 3 cleft on the extracellular side, which encompasses the space between previously characterized binding sites for RBP, ~25 A above the membrane surface, and the IM helices located down at the mid-bilayer level. This outer cleft is hydrophobic, contains two ordered putative cholesterols, and is exposed to the membrane through two symmetry-related lateral windows defined by TMs 8 and 9 and the IM helices. CONCLUSIONS The structure of STRA6 suggests a mechanism for retinol release from RBP into the hydrophobic environment of the outer cleft and direct diffusion into the membrane through the lateral window. Our work also sets the basis for future experiments aimed at investigating how the system is regulated, whether STRA6 also has a role in signaling, and the functional relevance of its association with CaM.