A nucleotide-gated molecular pore selects sulfotransferase substrates

Area of Science:

• Biochemistry
• Enzymology
• Structural Biology

Background:

• Human SULT2A1 is a key liver sulfotransferase catalyzing the transfer of sulfuryl groups from PAPS to various acceptors.
• Sulfation modifies biological activity by altering receptor interactions, but in vivo substrate selectivity remains poorly understood.
• Raloxifene and DHEA are SULT2A1 substrates in vitro, yet only DHEA is sulfated in vivo, indicating a selectivity mechanism.

Purpose of the Study:

• To elucidate the molecular basis of SULT2A1 substrate selectivity, particularly the differential in vivo sulfation of raloxifene and DHEA.
• To investigate the role of enzyme structure and ligand binding in controlling substrate access to the SULT2A1 active site.

Main Methods:

• Initial rate and equilibrium binding studies to assess substrate and cofactor interactions.
• Analysis of crystal structures to understand enzyme-ligand complex conformations.
• In silico docking to predict substrate binding modes.
• Equilibrium and pre-steady-state ligand binding assays to validate structural predictions.

Main Results:

• Significant binding antisynergy (21-fold) was observed between PAPS and raloxifene, but not DHEA.
• Crystal structures and docking revealed that PAP binding induces an isomerization, constricting the active site pore.
• Raloxifene, larger than DHEA, binds only the open enzyme form, while DHEA binds both open and closed forms.
• Experimental data confirmed that nucleotide-binding-induced isomerization controls acceptor pocket access.

Conclusions:

• A nucleotide-driven isomerization mechanism regulates SULT2A1 acceptor pocket accessibility, acting as a gate for substrate entry.
• This conformational control is crucial for SULT2A1's substrate selectivity in vivo, explaining differential sulfation of raloxifene and DHEA.
• The findings suggest this isomerization mechanism may be important for substrate selection in other sulfotransferases as well.