Cellular Retinol-binding Proteins (CRBP) type I and II are the main cytosolic retinol carriers and are members of the Intracellular Lipid-binding protein family. CRBPs are beta-barrel proteins that show high structural conservation in spite of a moderately low sequence identity and a different tissue distribution. They play different roles in the maintenance of vitamin A homeostasis, exhibiting a different affinity for retinol: the dissociation constant of the retinol-CRBP I complex is 100 fold lower than that of the retinol CRBP II complex. However, the binding site of the two isoforms is highly conserved: Lys40 and Gln108, the residues that give binding specificity, are unchanged. Structural comparison between CRBP I and II, both in the apo and in the holo forms, shows very little differences, that can’t explain the different binding affinity, and does not suggest how retinol enters the cavity. Some NMR studies [1] [2] on the dynamics of these carriers, have led to the identification of a portal region formed by alfa helix II and the loops between CD and between EF strands. However, the uptake mechanism has not yet been clarified. Computational methods, such as molecular dynamics (MD) simulations, are useful techniques to investigate at the atomic detail the mechanism by which the ligand enters the binding pocket. Simulations were carried out using the GROMACS package, with the G53a6 force field, starting from the available crystal structures. We set the temperature at 350 K (at which proteins are stable), to overcome the energy barriers separating the local minima that the ligand encounters at the protein surface, and at the same time to facilitate the opening of the portal region. In this way we were able to observe the ligand uptake in less than 80 ns. We confirm, for both CRBPs, the involvement of the same portal region described in the aforementioned NMR studies with a partial unfolding of the helix II. Nevertheless, a different distribution of polar and hydrophobic residues clusters at the surface of the two proteins, in particular of the lid, favored two different entrance pathways. In CRBP I, the retinol enters the binding cavity through a passage created by the movements of alpha helix II, CD and EF loops, while in CRBP II the ligand has a distinct preferential way of entry: driven by few polar interaction, it reaches a position between the two alpha helix and it sinks in this hydrophobic region. Then, in both cases, several polar interactions attract the retinol deeply inside the cavity, where it finally recovers the position present in the crystal structures of CRBP I and II. Our data suggest that, even if the ligand uptake involves the same region with the partial unfolding and displacement of the same helix II, it can find the better entrance pathway according to the hydropathy features of the protein surface. REFERENCES: 1. FRANZONI ET AL, “STRUCTURE AND BACKBONE DYNAMICS OF APO- AND HOLO-CELLULAR RETINOL-BINDING PROTEIN IN SOLUTION”, THE JOURNAL OF BIOLOGICAL CHEMISTRY 2002; 277(24): 21983-21997. 2. LU ET AL., “THE STRUCTURE AND DYNAMICS OF RAT APO-CELLULAR RETINOL-BINDING PROTEIN II IN SOLUTION: COMPARISON WITH THE X-RAY STRUCTURE” J. MOL. BIOL. 1999; 286: 1179-1195.

Cellular Retinol-binding Proteins: a different ligand uptake triggered by the hydropathy profile of the protein surface / Menozzi, I.; Berni, R.; Polverini, E.. - (2016). ((Intervento presentato al convegno Protein 2016 – Dissecting Biological Complexity at the Molecular Level tenutosi a Bologna nel 30 marzo – 1 aprile 2016.

Cellular Retinol-binding Proteins: a different ligand uptake triggered by the hydropathy profile of the protein surface.

MENOZZI, ILARIA;BERNI, Rodolfo;POLVERINI, Eugenia
2016

Abstract

Cellular Retinol-binding Proteins (CRBP) type I and II are the main cytosolic retinol carriers and are members of the Intracellular Lipid-binding protein family. CRBPs are beta-barrel proteins that show high structural conservation in spite of a moderately low sequence identity and a different tissue distribution. They play different roles in the maintenance of vitamin A homeostasis, exhibiting a different affinity for retinol: the dissociation constant of the retinol-CRBP I complex is 100 fold lower than that of the retinol CRBP II complex. However, the binding site of the two isoforms is highly conserved: Lys40 and Gln108, the residues that give binding specificity, are unchanged. Structural comparison between CRBP I and II, both in the apo and in the holo forms, shows very little differences, that can’t explain the different binding affinity, and does not suggest how retinol enters the cavity. Some NMR studies [1] [2] on the dynamics of these carriers, have led to the identification of a portal region formed by alfa helix II and the loops between CD and between EF strands. However, the uptake mechanism has not yet been clarified. Computational methods, such as molecular dynamics (MD) simulations, are useful techniques to investigate at the atomic detail the mechanism by which the ligand enters the binding pocket. Simulations were carried out using the GROMACS package, with the G53a6 force field, starting from the available crystal structures. We set the temperature at 350 K (at which proteins are stable), to overcome the energy barriers separating the local minima that the ligand encounters at the protein surface, and at the same time to facilitate the opening of the portal region. In this way we were able to observe the ligand uptake in less than 80 ns. We confirm, for both CRBPs, the involvement of the same portal region described in the aforementioned NMR studies with a partial unfolding of the helix II. Nevertheless, a different distribution of polar and hydrophobic residues clusters at the surface of the two proteins, in particular of the lid, favored two different entrance pathways. In CRBP I, the retinol enters the binding cavity through a passage created by the movements of alpha helix II, CD and EF loops, while in CRBP II the ligand has a distinct preferential way of entry: driven by few polar interaction, it reaches a position between the two alpha helix and it sinks in this hydrophobic region. Then, in both cases, several polar interactions attract the retinol deeply inside the cavity, where it finally recovers the position present in the crystal structures of CRBP I and II. Our data suggest that, even if the ligand uptake involves the same region with the partial unfolding and displacement of the same helix II, it can find the better entrance pathway according to the hydropathy features of the protein surface. REFERENCES: 1. FRANZONI ET AL, “STRUCTURE AND BACKBONE DYNAMICS OF APO- AND HOLO-CELLULAR RETINOL-BINDING PROTEIN IN SOLUTION”, THE JOURNAL OF BIOLOGICAL CHEMISTRY 2002; 277(24): 21983-21997. 2. LU ET AL., “THE STRUCTURE AND DYNAMICS OF RAT APO-CELLULAR RETINOL-BINDING PROTEIN II IN SOLUTION: COMPARISON WITH THE X-RAY STRUCTURE” J. MOL. BIOL. 1999; 286: 1179-1195.
Cellular Retinol-binding Proteins: a different ligand uptake triggered by the hydropathy profile of the protein surface / Menozzi, I.; Berni, R.; Polverini, E.. - (2016). ((Intervento presentato al convegno Protein 2016 – Dissecting Biological Complexity at the Molecular Level tenutosi a Bologna nel 30 marzo – 1 aprile 2016.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11381/2828117
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