The serotonin transporter (SERT) is the primary target for the selective serotonin reuptake inhibitors (SSRIs). However, the structural basis for the extraordinarily high binding affinity of the widely prescribed SSRI, paroxetine, to human SERT (hSERT) has not yet been fully elucidated. Our previous findings unveiled a plausible ambiguity in paroxetine's binding orientations that may constitute an integral component of this SSRI's high affinity for hSERT. Herein, we investigate factors contributing to paroxetine's high affinity by modifying both the ligand and the protein. We generated a series of bromine (Br)-containing derivatives and found that the one in which the 4-F of paroxetine had been replaced with the chemically similar but more electron-rich Br atom (13) had the highest affinity. By comparatively characterizing the binding of paroxetine and 13 to both wild type (WT) and a construct harboring a paroxetine-sensitive mutation in the binding cavity, we identified a mechanistic determinant responsible for the pose ambiguity of paroxetine, which can guide future drug design.

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The sigma 1 receptor (σ1R) has been implicated in cancers, neurological disorders, and substance use disorders. Yet, its molecular and cellular functions have not been well-understood. Recent crystal structures of σ1R reveal a single N-terminal transmembrane segment and C-terminal ligand-binding domain, and a trimeric organization. Nevertheless, outstanding issues surrounding the functional or pharmacological relevance of σ1R oligomerization remain, such as the minimal protomeric unit and the differentially altered oligomerization states by different classes of ligands. Western blot (WB) assays have been widely used to investigate protein oligomerizations. However, the unique topology of σ1R renders several intertwined challenges in WB. Here we describe a WB protocol without temperature denaturization to study the ligand binding effects on the oligomerization state of σ1R. Using this approach, we observed unexpected ladder-like incremental migration pattern of σ1R, demonstrating preserved homomeric interactions in the detergent environment. We compared the migration patterns of intact σ1R construct and the C-terminally tagged σ1R constructs, and found similar trends in response to drug treatments. In contrast, N-terminally tagged σ1R constructs show opposite trends to that of the intact construct, suggesting distorted elicitation of the ligand binding effects on oligomerization. Together, our findings indicate that the N-terminus plays an important role in eliciting the impacts of bound ligands, whereas the C-terminus is amenable for modifications for biochemical studies.