Sandbox Reserved 895: Difference between revisions
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Crystal structure of bovine RPE65 (PDB: ''3FSN''), which is 99% similar to human RPE65, (although the crystal structure for human RPE65 is not currently available) is used as the basis of studying RPE65 structure. RPE65 resembles a seven-bladed β-propeller with single-stranded extension on blades VI and VII and two-stranded extension on blade III, generated in PyMol and shown in '''Figure 4'''. The top face of the β-propeller is defined by connecting the outer strand of the β-sheet with the inner strand of the next β-sheet. The iron cofactor is located near the top surface of the propeller which is coordinated by four His residues and three secondary Glu residues. Each blade of the propeller contributes one His residue to coordinate with the iron cofactor. A hydrophobic tunnel leads the protein exterior to the active site which is defined by the iron ion to accommodate the passage of retinoids (which are conjugated to a fatty acid tail) from the membrane to the RPE65 catalytic site. The mouth of the tunnel is surrounded by three groups of nonpolar residues that contribute to the overall hydrophobicity of the tunnel and the integration with the lipid bilayer. There are also a few aromatic amino acid side chains that reside in this portion of the enzyme. This suggest that the depth of RPE65 membrane is restricted to the proximal portions of the phospholipid acyl chains with respect to the polar head groups. Arg and Lys residues within this region also contribute to the association with the negatively charged phospholipid head groups. <ref> DOI 19805034 </ref> | Crystal structure of bovine RPE65 (PDB: ''3FSN''), which is 99% similar to human RPE65, (although the crystal structure for human RPE65 is not currently available) is used as the basis of studying RPE65 structure. RPE65 resembles a seven-bladed β-propeller with single-stranded extension on blades VI and VII and two-stranded extension on blade III, generated in PyMol and shown in '''Figure 4'''. The top face of the β-propeller is defined by connecting the outer strand of the β-sheet with the inner strand of the next β-sheet. The iron cofactor is located near the top surface of the propeller which is coordinated by four His residues and three secondary Glu residues. Each blade of the propeller contributes one His residue to coordinate with the iron cofactor. A hydrophobic tunnel leads the protein exterior to the active site which is defined by the iron ion to accommodate the passage of retinoids (which are conjugated to a fatty acid tail) from the membrane to the RPE65 catalytic site. The mouth of the tunnel is surrounded by three groups of nonpolar residues that contribute to the overall hydrophobicity of the tunnel and the integration with the lipid bilayer. There are also a few aromatic amino acid side chains that reside in this portion of the enzyme. This suggest that the depth of RPE65 membrane is restricted to the proximal portions of the phospholipid acyl chains with respect to the polar head groups. Arg and Lys residues within this region also contribute to the association with the negatively charged phospholipid head groups. <ref> DOI 19805034 </ref> | ||
[[Image:RPE65_Figure4_S7_blades.jpg|thumb|center|512 px|alt=Figure 4: RPE65 structure| '''Figure 4:''' Crystal structure of bovine RPE65 viewed from the bottom face of the seven-bladed β-propeller labeled in roman numerals from I to VII. Figure generated using PyMol. <ref> DOI 19805034 </ref> | [[Image:RPE65_Figure4_S7_blades.jpg|thumb|center|512 px|alt=Figure 4: RPE65 structure| '''Figure 4:''' Crystal structure of bovine RPE65 viewed from the bottom face of the seven-bladed β-propeller labeled in roman numerals from I to VII. Figure generated using PyMol. <ref> DOI 19805034 </ref> <ref> The PyMOL Molecular Graphics System, Version 2.3.3, Schrödinger, LLC. </ref>]] | ||
==== '''[1.2.2.2] Active Site Structural Analysis of RPE65''' ==== | ==== '''[1.2.2.2] Active Site Structural Analysis of RPE65''' ==== | ||
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The main tunnel and its interior cavity contain two regions of strong residual electron density that is not accounted for by the atoms located in the protein. This suggest that the substrate would interact with these electron dense regions. The first electron dense region is linear with a triangular shape on one end, suggesting the presence of a linear molecule containing a terminal functional group with trigonal planar geometry (such as an ester in the retinyl esters or a carboxylic acid functional group in fatty acids). The triangular portion is in proximity to the iron ion and when positioned correctly fulfills one or both open coordination sites set forth by the octahedral geometry. The linear portion of the molecule then occupies the main tunnel. The second electron dense region is linear and bent in shape which is located within the interior cavity of the protein. This region could be represented by a bound PEG 200 molecule or a string of partially ordered water molecules. However, the electron dense region cannot accommodate longer compound such as (Hydroxyethyloxy)tri(ethyloxy)octane (C8E4). | The main tunnel and its interior cavity contain two regions of strong residual electron density that is not accounted for by the atoms located in the protein. This suggest that the substrate would interact with these electron dense regions. The first electron dense region is linear with a triangular shape on one end, suggesting the presence of a linear molecule containing a terminal functional group with trigonal planar geometry (such as an ester in the retinyl esters or a carboxylic acid functional group in fatty acids). The triangular portion is in proximity to the iron ion and when positioned correctly fulfills one or both open coordination sites set forth by the octahedral geometry. The linear portion of the molecule then occupies the main tunnel. The second electron dense region is linear and bent in shape which is located within the interior cavity of the protein. This region could be represented by a bound PEG 200 molecule or a string of partially ordered water molecules. However, the electron dense region cannot accommodate longer compound such as (Hydroxyethyloxy)tri(ethyloxy)octane (C8E4). | ||
[[Image:RPE65_Binding_Pocket.jpg|thumb|center|512 px|alt=Figure 5: RPE65 Binding Pocket| '''Figure 5:''' RPE65 iron ion binding pocket coordination sites. Shown in olive is the iron ion, shown in red are the bond lengths between the iron atom and the His residues, shown in green are the bond lengths between the His residues and the hydrogen bond interactions with Glu and Val. The bond length interactions are shown in angstroms from the iron atom. Figure generated using PyMol. <ref> DOI 19805034 </ref> | [[Image:RPE65_Binding_Pocket.jpg|thumb|center|512 px|alt=Figure 5: RPE65 Binding Pocket| '''Figure 5:''' RPE65 iron ion binding pocket coordination sites. Shown in olive is the iron ion, shown in red are the bond lengths between the iron atom and the His residues, shown in green are the bond lengths between the His residues and the hydrogen bond interactions with Glu and Val. The bond length interactions are shown in angstroms from the iron atom. Figure generated using PyMol. <ref> DOI 19805034 </ref> <ref> The PyMOL Molecular Graphics System, Version 2.3.3, Schrödinger, LLC. </ref>]] | ||
=== ''' [1.2.3] Proposed Enzymatic Mechanism of RPE65''' === | === ''' [1.2.3] Proposed Enzymatic Mechanism of RPE65''' === | ||
