Sandbox: Difference between revisions

Line 149:

The OmcS structure determined by Filman ''et al.'' <ref name="strauss" />was very similar, with '''80%''' loops assigned by the authors (86% by Jmol), having only 3% beta strand but otherwise very similar. We compared OmcS with three other c-type multi-heme cytochrome crystal structures: [[1ofw]], [[3ucp]], and [[3ov0]] had 45%, 49%, and 60% loops respectively.

===~~Hemes~~===

===Mechanism of OAT1 inhibition by probenecid===

The cryo-EM structure of hOAT1 bound to the classic inhibitor probenecid reveals a dual-mechanism of action that goes beyond simple competition, effectively arresting the transporter in a restricted state.

'''1. Binding Mode and Direct Competition'''

*Probenecid binds at the top of the central cavity, parallel to the membrane plane.

*Its binding site overlaps with both Site 1 (partially) and Site 3.

*It engages in specific, high-affinity interactions with key residues:

:*K382 on TM8 forms a hydrogen bond with the carboxylate group of probenecid.

:*Y354 on TM7 forms a hydrogen bond with its sulfonyl group.

:*Crucially, K382 is also the residue that interacts with the counter-substrate α-ketoglutarate (α-KG), establishing a direct competitive inhibition mechanism by blocking α-KG binding.

'''2. Conformational Arrest and Cytoplasmic Path Blockage'''

The primary inhibitory mechanism is a probenecid-induced conformational change that physically blocks substrate access and exit.

*'''Constriction of the Binding Pocket:''' Compared to the apo state, the cytoplasmic opening of the binding pocket narrows from ~15 Å to ~12 Å in the probenecid-bound state.

*'''Dual-Pathway Blockade:''' The cytosolic entrance is split into two paths. Probenecid binding critically affects both:

:*'''Path A''' (between TM2 and TM11) is narrowed from ~5 Å to ~4 Å.

:*'''Path B''' (between TM5 and TM8) is completely blocked.

This structural rearrangement is caused by a slight inward movement of the cytoplasmic ends of TM5, TM8, TM10, and TM11 toward the binding pocket.

'''3. Locked Conformation'''

By constricting the cytoplasmic access routes, probenecid does not just compete for the substrate-binding site; it stabilizes the transporter in an apo-like, inward-facing conformation that is inaccessible to cytosolic substrates. This prevents the entry of new substrates and likely traps the transporter in this non-functional state, effectively "locking" it and preventing the conformational changes necessary for the transport cycle.

Each OmcS monomer <scene name='83/835223/Hemes/10'>contains 6 hemes</scene>:

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@@ Line 149: / Line 149: @@
 The OmcS structure determined by Filman ''et al.'' <ref name="strauss" />was very similar, with '''80%''' loops assigned by the authors (86% by Jmol), having only 3% beta strand but otherwise very similar. We compared OmcS with three other c-type multi-heme cytochrome crystal structures: [[1ofw]], [[3ucp]], and [[3ov0]] had 45%, 49%, and 60% loops respectively.
-===Hemes===
+===Mechanism of OAT1 inhibition by probenecid===
+The cryo-EM structure of hOAT1 bound to the classic inhibitor probenecid reveals a dual-mechanism of action that goes beyond simple competition, effectively arresting the transporter in a restricted state.
+'''1. Binding Mode and Direct Competition'''
+*Probenecid binds at the top of the central cavity, parallel to the membrane plane.
+*Its binding site overlaps with both Site 1 (partially) and Site 3.
+*It engages in specific, high-affinity interactions with key residues:
+:*K382 on TM8 forms a hydrogen bond with the carboxylate group of probenecid.
+:*Y354 on TM7 forms a hydrogen bond with its sulfonyl group.
+:*Crucially, K382 is also the residue that interacts with the counter-substrate α-ketoglutarate (α-KG), establishing a direct competitive inhibition mechanism by blocking α-KG binding.
+'''2. Conformational Arrest and Cytoplasmic Path Blockage'''
+The primary inhibitory mechanism is a probenecid-induced conformational change that physically blocks substrate access and exit.
+*'''Constriction of the Binding Pocket:''' Compared to the apo state, the cytoplasmic opening of the binding pocket narrows from ~15 Å to ~12 Å in the probenecid-bound state.
+*'''Dual-Pathway Blockade:''' The cytosolic entrance is split into two paths. Probenecid binding critically affects both:
+:*'''Path A''' (between TM2 and TM11) is narrowed from ~5 Å to ~4 Å.
+:*'''Path B''' (between TM5 and TM8) is completely blocked.
+This structural rearrangement is caused by a slight inward movement of the cytoplasmic ends of TM5, TM8, TM10, and TM11 toward the binding pocket.
+'''3. Locked Conformation'''
+By constricting the cytoplasmic access routes, probenecid does not just compete for the substrate-binding site; it stabilizes the transporter in an apo-like, inward-facing conformation that is inaccessible to cytosolic substrates. This prevents the entry of new substrates and likely traps the transporter in this non-functional state, effectively "locking" it and preventing the conformational changes necessary for the transport cycle.
 Each OmcS monomer <scene name='83/835223/Hemes/10'>contains 6 hemes</scene>: