Sandbox: Difference between revisions

Line 85:

::*The structure serves as a baseline for understanding the conformational changes that occur upon substrate or inhibitor binding.

===~~OmcS Structure~~===

===Olmesartan recognition by hOAT1===

The structural and functional analysis of hOAT1 in complex with the high-affinity antihypertensive drug olmesartan provides a detailed blueprint for substrate specificity and binding.

*'''Binding Location and Pose'''

:*Olmesartan binds within the central cavity of hOAT1 in an inward-facing conformation.

:*It occupies Site 3 of the binding pocket, which is the primary polyspecific site for anionic substrates.

:*The drug adopts a diagonal orientation relative to the membrane plane, a pose that requires more space than the smaller inhibitor probenecid. This orientation is similar to its conformation when bound to the angiotensin receptor.

*''' Key Interacting Residues'''

:*Olmesartan is surrounded by residues from multiple transmembrane helices (TM1, TM4, TM5, TM7, TM10, TM11) within a 5 Å distance. The critical interactions involve:

:*Aromatic and Hydrophobic Cage:

:*The biphenyl group of olmesartan is nestled near residue F438.

:*The tetrazole ring is positioned between the bottom-gate residues M207 and F442.

:*The imidazole moiety is located close to Y354.

*'''Critical Role of Y230:'''

:*Upon olmesartan binding, the side chain of Y230 undergoes a vertical rotation to accommodate and interact with the substrate.

:*Mutagenesis studies confirm its importance: the Y230F mutation increased the IC₅₀ for olmesartan inhibition from 845.3 nM (Wild Type) to 2.36 µM, indicating a reduction in binding affinity.

*'''The Bottom Gate Residues (M207 and F442):'''

:*These residues are crucial for high-affinity olmesartan binding.

:*The M207A mutant caused a 4-fold reduction in affinity (IC₅₀ = 3.78 µM).

:*The F442A mutant caused a dramatic 12-fold reduction in affinity (IC₅₀ = 10.32 µM).

:*This suggests these residues not only form a gate but also directly interact with large, transportable substrates like olmesartan.

*''' Chloride Ion Coordination is Essential'''

:*A key finding is the role of a chloride ion in stabilizing the olmesartan-bound state.

:*The Chloride-Binding Site: A chloride ion (or bromide, used for confirmation) is observed coordinated between residues S203, Y230, and R466.

'''Indirect Role of S203:''' While S203 does not directly contact olmesartan, it is critical for chloride coordination. This is a major species-specific difference, as rat OAT1 has an alanine at this position.

*'''Functional Evidence of Chloride Dependence:'''

The IC₅₀ of olmesartan is 2.01 µM in chloride-rich conditions but improves to 0.91 µM in chloride-depleted conditions, suggesting a more complex relationship where chloride may facilitate transport.

The S203A mutant shows a severe ~5-fold reduction in olmesartan binding affinity specifically in the presence of chloride (IC₅₀: WT = 2.47 µM; S203A = 29.52 µM).

The S203A-Y230F double mutant has an even more profound effect, increasing the IC₅₀ to 93.30 µM in chloride conditions, highlighting their synergistic role in chloride-dependent substrate binding.

The OmcS monomer has <scene name='83/835223/Secondary_structure/2'>remarkably little secondary structure</scene>.

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@@ Line 85: / Line 85: @@
 ::*The structure serves as a baseline for understanding the conformational changes that occur upon substrate or inhibitor binding.
-===OmcS Structure===
+===Olmesartan recognition by hOAT1===
+The structural and functional analysis of hOAT1 in complex with the high-affinity antihypertensive drug olmesartan provides a detailed blueprint for substrate specificity and binding.
+*'''Binding Location and Pose'''
+:*Olmesartan binds within the central cavity of hOAT1 in an inward-facing conformation.
+:*It occupies Site 3 of the binding pocket, which is the primary polyspecific site for anionic substrates.
+:*The drug adopts a diagonal orientation relative to the membrane plane, a pose that requires more space than the smaller inhibitor probenecid. This orientation is similar to its conformation when bound to the angiotensin receptor.
+*''' Key Interacting Residues'''
+:*Olmesartan is surrounded by residues from multiple transmembrane helices (TM1, TM4, TM5, TM7, TM10, TM11) within a 5 Å distance. The critical interactions involve:
+:*Aromatic and Hydrophobic Cage:
+:*The biphenyl group of olmesartan is nestled near residue F438.
+:*The tetrazole ring is positioned between the bottom-gate residues M207 and F442.
+:*The imidazole moiety is located close to Y354.
+*'''Critical Role of Y230:'''
+:*Upon olmesartan binding, the side chain of Y230 undergoes a vertical rotation to accommodate and interact with the substrate.
+:*Mutagenesis studies confirm its importance: the Y230F mutation increased the IC₅₀ for olmesartan inhibition from 845.3 nM (Wild Type) to 2.36 µM, indicating a reduction in binding affinity.
+*'''The Bottom Gate Residues (M207 and F442):'''
+:*These residues are crucial for high-affinity olmesartan binding.
+:*The M207A mutant caused a 4-fold reduction in affinity (IC₅₀ = 3.78 µM).
+:*The F442A mutant caused a dramatic 12-fold reduction in affinity (IC₅₀ = 10.32 µM).
+:*This suggests these residues not only form a gate but also directly interact with large, transportable substrates like olmesartan.
+*''' Chloride Ion Coordination is Essential'''
+:*A key finding is the role of a chloride ion in stabilizing the olmesartan-bound state.
+:*The Chloride-Binding Site: A chloride ion (or bromide, used for confirmation) is observed coordinated between residues S203, Y230, and R466.
+'''Indirect Role of S203:''' While S203 does not directly contact olmesartan, it is critical for chloride coordination. This is a major species-specific difference, as rat OAT1 has an alanine at this position.
+*'''Functional Evidence of Chloride Dependence:'''
+The IC₅₀ of olmesartan is 2.01 µM in chloride-rich conditions but improves to 0.91 µM in chloride-depleted conditions, suggesting a more complex relationship where chloride may facilitate transport.
+The S203A mutant shows a severe ~5-fold reduction in olmesartan binding affinity specifically in the presence of chloride (IC₅₀: WT = 2.47 µM; S203A = 29.52 µM).
+The S203A-Y230F double mutant has an even more profound effect, increasing the IC₅₀ to 93.30 µM in chloride conditions, highlighting their synergistic role in chloride-dependent substrate binding.
 The OmcS monomer has <scene name='83/835223/Secondary_structure/2'>remarkably little secondary structure</scene>.
 <center>