Dimeric human sulfotransferase 1B1 displays cofactor‐dependent subunit communication

Abstract

The cytosolic sulfotransferases (SULTs) are dimeric enzymes that catalyze the transformation of hydrophobic drugs and hormones into hydrophilic sulfate esters thereby providing the body with an important pathway for regulating small molecule activity and excretion. While SULT dimerization is highly conserved, the necessity for the interaction has not been established. To perform its function, a SULT must efficiently bind the universal sulfate donor, 3′-phosphoadenosine-5′-phosphosulfate (PAPS), and release the byproduct, 3′, 5′-diphosphoadenosine (PAP), following catalysis. We hypothesize this efficient binding and release of PAPS/PAP may be connected to SULT dimerization. To allow for the visualization of dynamic protein interactions critical for addressing this hypothesis and to generate kinetically testable hypotheses, molecular dynamic simulations (MDS) of hSULT1B1 were performed with PAPS and PAP bound to each dimer subunit in various combinations. The results suggest the dimer subunits may possess the capability of communicating with one another in a manner dependent on the presence of the cofactor. PAP or PAPS binding to a single side of the dimer results in decreased backbone flexibility of both the bound and unbound subunits, implying the dimer subunits may not act independently. Further, binding of PAP to one subunit of the dimer and PAPS to the other caused increased flexibility in the subunit bound to the inactive cofactor (PAP). These results suggest SULT dimerization may be important in maintaining cofactor binding/release properties of SULTs and provide hypothetical explanations for SULT half-site reactivity and substrate inhibition, which can be analyzed in vitro.

A high frequency missense SULT1B1 allelic variant (L145V) selectively expressed in African descendants exhibits altered kinetic properties

“…This destabilization may disrupt hydrogen bonding between M146, directly adjacent to L145, and aspartic acid (D) 250, anchoring the active site “lid” (Loop 3) to the “floor”, helping to preserve Loop 3’s location (Cook et al, 2013b). This bond acts much like a hinge for the Loop 3 lid, separating it into two portions: one portion overlays the substrate binding domain and the other overlays the PAPS binding domain (Cook et al, 2013a; Tibbs & Falany, 2015). Alteration of the M146-Loop 3 hydrogen bond by mutation of an adjacent residue could feasibly alter both the PAPS and substrate affinities, as was observed in the L145V isoform’s kinetics (Pan, 2008).…”

An engineered heterodimeric model to investigate SULT1B1 dependence on intersubunit communication

“…Arg258 is critical for mediating structural shift of the SULT backbone upon PAPS binding via its direct interaction with the cofactor’s 3’ phosphate group [21, 25, 26]. Arg258 is thought to “sense” the orientation of PAP(S) within the active site of the enzyme, possibly allowing communication to the partnering subunit [17]. Mutation of Arg258 to a Lys was confirmed to heavily diminish PAPS binding by SULT1B1 prior to its use in the heterodimer system (data not shown).…”

“…Recent evidence suggests the binding of cofactor (PAPS) to the SULT induces structural asymmetry that drives communication between dimeric SULT subunits, contributing to SULT half-site reactivity. [17]. The aim of this investigation is to test the capacity of cofactor binding and catalysis to drive such intersubunit communication in vitro by engineering a SULT1B1 dimer with interchangeable functional and dysfunctional subunits.…”