Structural rearrangement of SULT2A1: effects on dehydroepiandrosterone and raloxifene sulfation

Abstract

Background Human cytosoloic sulfotransferase (SULT) 2A1 is a major hepatic isoform and sulfates hydroxyl groups in structurally diverse sterols and xenobiotics. SULT2A1 crystal structures resolved in the presence and absence of 3′,5′-diphosphoadenosine (PAP) or dehydropeiandrosterone (DHEA) suggest a significant rearrangement of the peptide that forms the surface of the active site in the presence of PAP. Materials and methods Molecular modeling was used to examine the effects of the rearrangement in SULT2A1 associated with 3′-phosphoadenosine 5′-phosphosulfate (PAPS) binding on the binding of DHEA and raloxifene. The kinetics of DHEA and raloxifene sulfation was analyzed to investigate the effects of the rearrangement on SULT2A1 activity. Results Molecular models indicate that DHEA is able to bind to SULT2A1 in both conformations (open, without PAP; closed, with PAP) in a catalytic configuration, whereas raloxifene bound in a catalytic conformation only in the open structure. Raloxifene did not bind in the smaller, closed substrate binding pocket. Kinetic analysis of DHEA sulfation was consistent with a random Bi-Bi reaction mechanism, whereas raloxifene sulfation was more indicative of an ordered reaction mechanism with raloxifene binding first. Initial burst kinetics with DHEA yielded similar results after preincubation of SULT2A1 with DHEA or PAPS. Preincubation of SULT2A1 with raloxifene showed a burst of raloxifene sulfate formation with the addition of PAPS. In contrast, little raloxifene sulfate was formed if SULT2A1 was preincubated with PAPS and the reaction initiated with raloxifene. Conclusions The structural rearrangements in SULT2A1 caused by PAPS binding can alter the sulfation mechanism and kinetics of different substrates.

Testing the Sulfotransferase Molecular Pore Hypothesis

“…1) is a dynamic, 30-residue stretch of amino acids (residues 224 -253) that opens and closes in response to nucleotide (20,21). In the closed state, the edge of the cap organizes into a "molecular pore" that sieves from its environment only acceptors whose dimensions lie within the thresholds of the pore (20); in the open state, these thresholds increase dramatically, allowing the enzyme to operate on a far larger set of acceptors (20).…”

“…Reactions were quenched by mixing the solution (10:1) with KOH (0.50 M). Sulfated and non-sulfated acceptors were separated by chloroform extraction using established protocols (2,21). Briefly, KPO 4 (25 mM, pH 8.8) was added to the quenched solution and mixed (1:5) with chloroform.…”

“…The Structure of the Double Mutant-Nucleotide is encapsulated in the closed structure, and its escape requires that the cap open (20,21). Large substrate binding to the wild-type enzyme Table 3.…”

“…SULTs contain an active site cap that "covers" both the nucleotide-and acceptor-binding pockets (20,21). Structures indicate that nucleotide binding closes the cap, producing a small porelike structure at the entrance to the acceptor-binding pocket (20).…”

In Silico Prediction of Human Sulfotransferase 1E1 Activity Guided by Pharmacophores from Molecular Dynamics Simulations

“…It uses a combination of three-dimensional pharmacophores and machine learning models as cornerstones of the prediction process. During model development, flexibility of the active site of the enzyme was taken into consideration as it was shown to be a key factor for substrate selectivity (64). Areas of high flexibility at the binding site, namely the three loops, restructure the shape of the binding pocket and therefore allow structurally different molecules to be accommodated.…”