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The past two years have witnessed a revolution in structural biology. Single particle cryo-electron microscopy (cryo-EM) was a method once limited to determining structures at low resolutions, where chemical features cannot be distinguished. Now, with the advent of directelectron detectors, single-particle cryo-EM has begun to reach the atomic resolutions formerly only available through X-ray crystallography – where chemistry can be related to both structure and sequence. Importantly, however, cryo-EM does not require the formation of crystals and only minuscule amounts of homogeneous sample are necessary. In principle any biological problem is within reach, and sample preparation (i.e. biochemistry) is the primary if not only limiting factor. We present here a recent example from my lab, where we used cryo-EM to determine the structure of a novel eukaryotic membrane protein, and place our results in a biological context.
Many biological processes, including the visual cycle and embryonic development are crucially dependent on an adequate supply of Vitamin A. A cell receives Vitamin A either directly from food intake, or from the liver, released as retinol (vitamin A alcohol; ROH) bound to its carrier retinol-binding protein (RBP, also termed RBP4), which allows the highly hydrophobic retinol to circulate in plasma. Once inside the cell, retinol binds specific intracellular carriers, specifically cellular retinol-binding proteins (CRBPs).
How retinol is released from RBP and internalized by target cells has been the subject of intense debate. In a landmark study in 2007, the RBP receptor was cloned and found to be a protein encoded by a gene previously identified and classified as stimulated by retinoid acid gene 6 (STRA6). STRA6 is a 75 kDa protein with 9 predicted TM segments, showing no sequence similarity to any known transporter, channel or receptor. However, since then progress in understanding how this system works at a molecular level has been hampered by the absence of an atomic model of STRA6.
We determined the structure of zebrafish STRA6 to 3.9Å resolution by cryo-EM. STRA6 displays one intramembrane and nine transmembrane helices in an intricate dimeric assembly.
Unexpectedly, calmodulin is bound tightly to STRA6 in a non-canonical arrangement.
Residues identified with RBP binding map to an arch-like structure that covers a deep lipophilic cleft. This cleft is open to the membrane, suggesting a possible mode for internalization of retinol via direct diffusion into the lipid bilayer.
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