Redefining the specificity of a human protein: the plasma retinol-binding protein case

RBP4 (plasma retinol-binding protein) is the 21 kDa transporter of all-trans retinol that circulates in serum as a moderately tight 1:1 molar complex of the vitamin with the protein. RBP4 is primarily synthesised in the liver but is also produced by adipose tissue (about 20-40% of the amounts released by the liver) and circulates bound to a larger protein, transthyretin, TTR, that serves to increase its molecular mass to about 80,000 and thus avoid its elimination by glomerular filtration. The RBP-TTR complex dissociates readily upon interaction with the RBP receptor, STRA6, that removes the vitamin from the transporter and facilitates its entrance into the cell. When retinol is not present in the complex, RBP dissociates from TTR and is eliminated in urine. In 1993 the X-ray structure of human holo RBP4 and of what was expected to be the apo form, i.e. after the loss of retinol, to 2.5 Å resolution has been reported. A couple of years ago we tested crystals from the same batch at the ESRF and we collected a full data set of both the liganded and unliganded crystal forms at a resolution of 1.5 Å and 2.0 Å respectively. The result was that we identified a fatty acid in the ligand-binding site of the protein believed to be apo. We also prepared crystals of BP4 purified from human urine and amniotic fluid, two sources that contain non fluorescent RBP4, i.e. not bound to retinol, and for that reason believed to be the apo form of the protein. In every case we found a fatty acid in the central cavity of the RBP4 molecule, a result that we confirmed by GC-MS analysis of the samples used in the crystallization experiments. This result changes substantially our perception of this protein that has so far been considered to be specific for retinol and is a good example of how simply increasing the quality of the diffraction data can change the perception of the function of a protein.