Journal:Protein Science:1: Difference between revisions
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The photosensitizer, <scene name='Journal:Protein_Science:1/Cv/3'>methylene blue (MB)</scene> <font color='darkmagenta'><b>(colored in darkmagenta)</b></font>, generates singlet oxygen that irreversibly inhibits Torpedo californica acetylcholinesterase (''Tc''AChE). In the dark, it inhibits reversibly. | The photosensitizer, <scene name='Journal:Protein_Science:1/Cv/3'>methylene blue (MB)</scene> <font color='darkmagenta'><b>(colored in darkmagenta)</b></font>, generates singlet oxygen that irreversibly inhibits Torpedo californica acetylcholinesterase (''Tc''AChE). In the dark, it inhibits reversibly. | ||
The ''Tc''AChE active site consists of two binding subsites. One of them is the '''"catalytic anionic site" (CAS)''', which involves the catalytic triad <scene name='Journal:Protein_Science:1/Cv/6'>Ser200, His440, and Glu327</scene> <span style="color:orange;background-color:black;font-weight:bold;">(colored in orange)</span> and the conserved residues <scene name='Journal:Protein_Science:1/Cv/8'>Trp84 and Phe330</scene> which also participate in ligand recognition. Another conserved residue <scene name='Journal:Protein_Science:1/Cv/9'>Trp279</scene> <span style="color:cyan;background-color:black;font-weight:bold;">(colored in cyan)</span> is situated at the second binding subsite, termed the '''"peripheral anionic site" (PAS)''', ~14 Å from CAS. <scene name='Journal:Protein_Science:1/Cv/10'>Thioflavin T</scene> ([[2j3q]]) is a good example of the '''PAS-binding''' AChE inhibitors. <scene name='Journal:Protein_Science:1/Cv/11'>Superposition</scene> of the structure of known '''CAS-binding''' inhibitor <font color='crimson'><b>edrophonium</b></font>/''Tc''AChE ([[2ack]]) on the <font color='magenta'><b>thioflavin T</b></font>/''Tc''AChE complex structure ([[2j3q]]) shows that these <scene name='Journal:Protein_Science:1/Cv/12'>ligands' positions do not overlap</scene><ref name="Ravelli">PMID:10089512</ref> <ref name="Sonoda">PMID:18512913</ref>. | The ''Tc''AChE active site consists of two binding subsites. One of them is the '''"catalytic anionic site" (CAS)''', which involves the catalytic triad <scene name='Journal:Protein_Science:1/Cv/6'>Ser200, His440, and Glu327</scene> <span style="color:orange;background-color:black;font-weight:bold;">(colored in orange)</span> and the conserved residues <scene name='Journal:Protein_Science:1/Cv/8'>Trp84 and Phe330</scene> which also participate in ligand recognition. Another conserved residue <scene name='Journal:Protein_Science:1/Cv/9'>Trp279</scene> <span style="color:cyan;background-color:black;font-weight:bold;">(colored in cyan)</span> is situated at the second binding subsite, termed the '''"peripheral anionic site" (PAS)''', ~14 Å from CAS. <scene name='Journal:Protein_Science:1/Cv/10'>Thioflavin T</scene> ([[2j3q]]) is a good example of the '''PAS-binding''' AChE inhibitors. <scene name='Journal:Protein_Science:1/Cv/11'>Superposition</scene> of the structure of known '''CAS-binding''' inhibitor <font color='crimson'><b>edrophonium</b></font>/''Tc''AChE ([[2ack]]) on the <font color='magenta'><b>thioflavin T</b></font>/''Tc''AChE complex structure ([[2j3q]]) shows that these <scene name='Journal:Protein_Science:1/Cv/12'>ligands' positions do not overlap</scene><ref name="Ravelli">PMID:10089512</ref> <ref name="Sonoda">PMID:18512913</ref>. | ||
MB is a noncompetitive inhibitor of ''Tc''AChE, competing with reversible inhibitors directed at both ‘‘anionic’’ subsites, but a single site is involved in inhibition. The crystal structure reveals a <scene name='Journal:Protein_Science:1/Cv1/2'>single MB stacked against Trp279 in the PAS</scene>, oriented down the gorge toward the CAS ([[2w9i]]); it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge. Superposition of the '''PAS regions''' of the <font color='darkmagenta'><b>MB</b></font>/''Tc''AChE ([[2w9i]]) and <font color='magenta'><b>thioflavin T</b></font>/''Tc''AChE ([[2j3q]]) complexes reveals <scene name='Journal:Protein_Science:1/Cv1/4'>similarity between positions of these ligands</scene>. As the conformation of ''Tc''AChE in the crystal structures of the two complexes is practically identical, only that of the <font color='darkmagenta'><b>MB</b></font>/''Tc''AChE structure ([[2w9i]]) is shown. The kinetic and spectroscopic data showing that inhibitors binding at the '''CAS''' can impede binding of MB are reconciled by docking studies showing that the <scene name='Journal:Protein_Science:1/Cv2/5'>conformation adopted by Phe330</scene>, midway down the gorge, in the MB/''Tc''AChE crystal structure, precludes simultaneous binding of a second MB at the CAS (<font color='blueviolet'><b>2nd MB is colored blueviolet</b></font>, <span style="color:orange;background-color:black;font-weight:bold;">Phe330 of the crystal structure is in orange</span> and <font color='indigo'><b>Phe330 of the modeled structure is in indigo</b></font>. | MB is a noncompetitive inhibitor of ''Tc''AChE, competing with reversible inhibitors directed at both ‘‘anionic’’ subsites, but a single site is involved in inhibition. The crystal structure reveals a <scene name='Journal:Protein_Science:1/Cv1/2'>single MB stacked against Trp279 in the PAS</scene>, oriented down the gorge toward the CAS ([[2w9i]]); it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge. Superposition of the '''PAS regions''' of the <font color='darkmagenta'><b>MB</b></font>/''Tc''AChE ([[2w9i]]) and <font color='magenta'><b>thioflavin T</b></font>/''Tc''AChE ([[2j3q]]) complexes reveals <scene name='Journal:Protein_Science:1/Cv1/4'>similarity between positions of these ligands</scene>. As the conformation of ''Tc''AChE in the crystal structures of the two complexes is practically identical, only that of the <font color='darkmagenta'><b>MB</b></font>/''Tc''AChE structure ([[2w9i]]) is shown. The kinetic and spectroscopic data showing that inhibitors binding at the '''CAS''' can impede binding of MB are reconciled by docking studies showing that the <scene name='Journal:Protein_Science:1/Cv2/5'>conformation adopted by Phe330</scene>, midway down the gorge, in the MB/''Tc''AChE crystal structure, precludes simultaneous binding of a second MB at the CAS (<font color='blueviolet'><b>2nd MB is colored blueviolet</b></font>, <span style="color:orange;background-color:black;font-weight:bold;">Phe330 of the crystal structure is in orange</span> and <font color='indigo'><b>Phe330 of the modeled structure is in indigo</b></font>. Conversely, binding of ligands at the '''CAS''' dislodges MB from its preferred locus at the '''PAS'''. The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage. | ||
</StructureSection> | </StructureSection> | ||
<references/> | <references/> | ||
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