Structure and Backbone Dynamics of Apo- and Holo-cellular Retinol-binding Protein in Solution

Abstract

Retinoid-binding proteins play an important role in regulating transport, storage, and metabolism of vitamin A and its derivatives. The solution structure and backbone dynamics of rat cellular retinol-binding protein type I (CRBP) in the apo-and holo-form have been determined and compared using multidimensional high resolution NMR spectroscopy. The global fold of the protein is consistent with the common motif described for members of the intracellular lipid-binding protein family. The most relevant difference between the NMR structure ensembles of apo-and holoCRBP is the higher backbone disorder, in the ligand-free form, of some segments that frame the putative entrance to the ligandbinding site. These comprise ␣-helix II, the subsequent linker to ␤-strand B, the hairpin turn between ␤-strands C and D, and the ␤E-␤F turn. The internal backbone dynamics, obtained from 15 N relaxation data (T 1 , T 2 , and heteronuclear nuclear Overhauser effect) at two different fields, indicate several regions with significantly higher backbone mobility in the apoprotein, including the ␤C-␤D and ␤E-␤F turns. Although apoCRBP contains a binding cavity more shielded than that of any other retinoid carrier, conformational flexibility in the portal region may assist retinol uptake. The stiffening of the backbone in the holoprotein guarantees the stability of the complex during retinol transport and suggests that targeted retinol release requires a transiently open state that is likely to be promoted by the acceptor or the local environment.Vitamin A derivatives play important roles in a variety of biological processes including vision, cell growth, cell differentiation, and morphogenesis (1). Plasma transport of retinol to target cells and intracellular transport for either storage or metabolic conversion are performed by binding proteins that belong to the calycin superfamily. The cytosolic carriers are members of the intracellular lipid-binding protein (i-LBP) 1 family, characterized by molecular masses of around 15 kDa. Their structure consists of a 10-stranded ␤-barrel, formed by two orthogonal ␤-sheets, and two short ␣-helices (2). The two best known intracellular carriers of retinol are cellular retinolbinding protein type I (CRBP), widely distributed in various tissues (3, 4), and cellular retinol-binding protein type II (CRBP-II), present in the enterocytes of the small intestine and in neonatal hepatocytes (5, 6). The structures of rat apo-and holoCRBP-II have been solved both in the crystal (7) and in solution (8, 9), whereas the only structure of CRBP available to date was that of the holoprotein in the crystal (10). More recently, two other retinol carriers have been identified as follows: murine CRBP-III, expressed primarily in heart, muscle, and adipose tissue (11); and human CRBP-III, most abundant in liver and kidney, whose structure in the retinol-free form has been solved by x-ray crystallography (12).In the cell, the poorly water-soluble retinol is stored within membranes as a retinyl-ester derivative of long-cha...

New insights on the protein-ligand interaction differences between the two primary cellular retinol carriers

“…The overall structure of CRBP-I complexed with all-trans -retinol is essentially identical in the crystal ( 24 ) and in solution ( 25 ). In particular, the binding cavity is fully shielded from the outside medium, as is the case also for the ligand-free form in solution, the only state in which the apo structure has been determined ( 25 ).…”

“…In the case of CRBP-I in solution, the topology of the entry portal is essentially identical in the presence and absence of retinol ( 25 ). Similarly, apo and holo CRBP-II in the crystal exhibit only small differences in the backbone fold ( 29 ), with a very low total C ␣ trace root mean square deviation (0.23 Å), while in solution the helix ␣ II segment of the typical helix-turn-helix motif has been described as unwound in the apo form ( 28 ).…”

“…In particular, the binding cavity is fully shielded from the outside medium, as is the case also for the ligand-free form in solution, the only state in which the apo structure has been determined ( 25 ). Studies of the protein dynamics indicated that merely a few residues exhibit increased mobility on the picosecond-to-nanosecond timescale in apo with respect to holo CRBP-I ( 25 ), while a high degree of conformational exchange on the microsecondto-millisecond timescale has been revealed ( 26 ), which is markedly reduced upon retinol binding.…”

“…In particular, the binding cavity is fully shielded from the outside medium, as is the case also for the ligand-free form in solution, the only state in which the apo structure has been determined ( 25 ). Studies of the protein dynamics indicated that merely a few residues exhibit increased mobility on the picosecond-to-nanosecond timescale in apo with respect to holo CRBP-I ( 25 ), while a high degree of conformational exchange on the microsecondto-millisecond timescale has been revealed ( 26 ), which is markedly reduced upon retinol binding. Using line shape analysis of NMR signals, we derived a mechanism of ligand binding that suggests that retinol, after an initial nonspecifi c encounter with the surface of apo CRBP-I, modulates site-specifi c mobility of the protein by inducing the formation of long-lived (millisecond to second) transiently open conformers in the portal region ( 27 ).…”

Indirect assessment of small hydrophobic ligand binding to a model protein using a combination of ESI MS and HDX/ESI MS

“…This notion is often used when studying protein-protein interactions [31,32], however, it can also be very useful when protein interaction with small organic ligands is considered. A common assumption here is that binding reduces flexibility [33,34], which has been proven experimentally in the case of several retinoid carriers [19,35,36]. Therefore, changes in protein backbone dynamics upon ligand introduction can be used as indicators of the protein-ligand interaction.…”