HOAT1: Difference between revisions

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*'''Cavity Borders and Cytosolic Gate:'''
*'''Cavity Borders and Cytosolic Gate:'''


::*The top border (extracellular side) of the cavity is formed by residues including N35, Y230, Y353, and Y354.
::*The top border (extracellular side) of the cavity is formed by residues including N35, Y230, Y353, and Y354 and are involved in substrate recognition


::*The bottom border (cytosolic side) features a narrow "thin bottom gate" formed by residues M207 and F442. The interaction between these two residues splits the cytosolic entrance into two distinct pathways:
::*The bottom border (cytosolic side) features a narrow "thin bottom gate" formed by residues M207 and F442. The interaction between these two residues splits the cytosolic entrance into two distinct pathways:
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:::*Path B: Located between TM5 and TM8.
:::*Path B: Located between TM5 and TM8.
::*This suggests that aromatic
residues located at the top border are important for extracellular
anion binding, while residues at the bottom play a role in
exporting extracellular anions to the cytoplasmic side.


*'''Conformational State:'''
*'''Conformational State:'''


::*In the apo state, the transporter is in a relaxed, inward-open conformation, providing access for substrates from the cytoplasm.
::*In the apo state, the transporter is in a relaxed, inward-open conformation, providing access for substrates from the cytoplasm.
::*The structure serves as a baseline for understanding the conformational changes that occur upon substrate or inhibitor binding.


===Olmesartan recognition by hOAT1===
===Olmesartan recognition by hOAT1===
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'''2. Key Interacting Residues'''
'''2. 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:
:*Olmesartan occupies Site 3 of
 
the binding pocket and is located within 5A˚ distance of residues
*'''Aromatic and Hydrophobic Cage:'''
of TM1, TM4, TM5, TM7, TM10, and TM11, namely N35, M207,
 
G227, Y230, W346, Y353, Y354, F438, F442, S462, and R466.
::*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.
 
'''3. 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
biphenyl group and tetrazole ring of olmesartan rely on interactions
with hydrophobic residues close to the bottom gate in
the binding pocket.  


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


::*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.




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