Differential Mechanisms of Retinoid Transfer from Cellular Retinol Binding Proteins Types I and II to Phospholipid Membranes

Abstract

Cellular retinol-binding proteins types I and II (CRBP-I and CRBP-II) are known to differentially facilitate retinoid metabolism by several membrane-associated enzymes. The mechanism of ligand transfer to phospholipid small unilamellar vesicles was compared in order to determine whether differences in ligand trafficking properties could underlie these functional differences. Unidirectional transfer of retinol from the CRBPs to membranes was monitored by following the increase in intrinsic protein fluorescence that occurs upon ligand dissociation. The results showed that ligand transfer of retinol from CRBP-I was >5-fold faster than transfer from CRBP-II. For both proteins, transfer of the other naturally occurring retinoid, retinaldehyde, was 4 -5-fold faster than transfer of retinol. Rates of ligand transfer from CRBP-I to small unilamellar vesicles increased with increasing concentration of acceptor membrane and with the incorporation of the anionic lipids cardiolipin or phosphatidylserine into membranes. In contrast, transfer from CRBP-II was unaffected by either membrane concentration or composition. Preincubation of anionic vesicles with CRBP-I was able to prevent cytochrome c, a peripheral membrane protein, from binding, whereas CRBP-II was ineffective. In addition, monolayer exclusion experiments demonstrated differences in the rate and magnitude of the CRBP interactions with phospholipid membranes. These results suggest that the mechanisms of ligand transfer from CRBP-I and CRBP-II to membranes are markedly different as follows: transfer from CRBP-I may involve and require effective collisional interactions with membranes, whereas a diffusional process primarily mediates transfer from CRBP-II. These differences may help account for their distinct functional roles in the modulation of intracellular retinoid metabolism.

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

“…Ͼ0.73 Å), involve the N terminus (residues 0 -2), the loop between the two ␣-helices (residues 23-26), the entire ␣-helix II, the subsequent linker to ␤B (residues 36 and 37), and almost all the turns connecting the ␤-strands, namely ␤B-␤C, ␤C-␤D, ␤E-␤F, ␤F-␤G, and ␤G-␤H. Finally, the size of the gap between ␤D and ␤E is the same in apo-and holoCRBP ensembles, when measuring the C␣-C␣ distance between residues Phe 64 and Phe 70 . These two residues are located in the lower part of the gap with an average distance of ϳ7.4 Å.…”

“…However, this does not provide a ligand entrance to the interior of the protein, because the side chains of several adjacent residues (i.e. Tyr 60 , Met 62 , Phe 64 , Phe 70 , Glu 72 , and Thr 75 ) fill the gap, thereby blocking the access to the cavity. The function of this gap is still obscure, but it is a characteristic feature of the i-LBP family that has been postulated as a second portal for the aqueous solvent (52,53).…”

“…In the first instance, the organic solvent might play a direct role in the opening of the portal and the transfer of retinol into the protein, by generating a suitable interface that alters the local micro-environment. In the second instance, a transient proteinprotein interaction, possibly similar to the dimeric structure observed in the crystal of apoCRABP-I (31), might create a route for retinol exchange; alternatively, retinol bound to CRBP-II might dissociate and diffuse through the aqueous medium, as reported for its transfer from this carrier to small unilamellar vesicles (70), and interact with CRBP, inducing itself the local changes that are necessary to permit its access to the binding cavity.…”

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

“…While the binding of retinol to apo CRBP-I is modulated by changes in the protein dynamics around the portal region that are induced by an initial nonspecifi c interaction of the ligand with the protein surface ( 27 ), experiments in progress indicate that retinol binding to CRBP-II does not require such an activation step. Distinct mechanisms of retinol delivery to model membranes have been reported for the holo forms of the two CRBP types ( 46 ). While CRBP-I as well as heart, brain, intestinal, and adipocyte FABP require a "transient collision-based mechanism," CRBP-II and liver FABP operate by an "aqueous diffusion-mediated mechanism" (47)(48)(49).…”

“…The global fold of the portal region in apo CRBP-II is therefore essentially identical to that of the holo form, as in the case of CRBP-I ( 25 ). Hence, rather than fundamental structural differences between the two homologous proteins, variations in restricted i ) to helix ␣ II (residues 28-34), which features high fl exibility and solvent exposure throughout the i-LBP family, ii ) to the linker ␣ II-␤ B (residues [35][36][37], iii ) to the turn ␤ B-␤ C (residues [45][46][47][48], and iv ) to the turn ␤ E-␤ F (residues 75-79), while it does not occur in the ␤ -strand sections (i.e., residues 38-44, 49, 70-74, and 80-84). The more limited regions identifi ed in the present work are all part of the portal, except for the turn ␤ B-␤ C.…”