Thermal Unfolding of the Human Serotonin Transporter: Differential Effect by Stabilizing and Destabilizing Mutations and Cholesterol on Thermodynamic and Kinetic Stability

Abstract

Folding-deficient mutants of SLC6 (solute carrier 6) family members have been linked to human diseases. The serotonin transporter (SERT/SLC6A4) is an important drug target in the treatment of depression, anxiety and obsessive-compulsive disorders and -with structural information in several conformational states -one of the best understood transporters. Here, we surmised that thermal unfolding offered a glimpse on the folding energy landscape of SLC6 transporters. We carried out molecular dynamic (MD) simulations to understand the mechanistic basis for enhanced and reduced stability, respectively, of the thermostabilized variant SERT-Y110A/I291A/T439S, which had previously been used for crystallization of human SERT in the outward-facing state, and of the folding-deficient SERT-P601A/G602A.We also examined the hydrophobic mismatch caused by the absence of cholesterol to explore the contribution of cholesterol to protein stability. When compared to wild type SERT, the thermodynamic and kinetic stability of SERT-Y110A/I291A/T439S was enhanced. In the other instances, changes in these two components were not correlated: the mutations in SERT-P601A/G602A led to a drop in thermodynamic but an increase in kinetic stability. The divergence was even more pronounced after cholesterol depletion, which reduced thermodynamic stability but increased the kinetic stability of wild type SERT to a level comparable to that of SERT-Y110A/I291A/T439S. We conclude that the low cholesterol content of the endoplasmic reticulum facilitates progression of the folding trajectory by reducing the energy difference between folding intermediates and the native state.

Integrated UPLC-MS, network pharmacology, and intestinal flora analysis to determine the treatment effect of Qiangzhi decoction on comorbid Tourette syndrome and RRTI

“…SLC6A4 , one member of the solute carrier family, maps to chromosome 17q11.2 ( 27 ). The serotonin transporter ( SERT/SLC6A4 ) is an important drug target in the treatment of multiple disorders, including depression, anxiety, autism, TS, and peripherally-based disorders of cardiovascular and gastrointestinal systems.…”

A mechanism of uncompetitive inhibition of the serotonin transporter

“…The endpoint of the folding trajectory of a protein is a minimum energy conformation. The outward and the inward-facing conformation are equivalent stable folds [45]. The ionic conditions in the endoplasmic reticulum (ER) differ from those at the cell surface.…”

Computational Chemistry in Structure-Based Solute Carrier Transporter Drug Design: Recent Advances and Future Perspectives

“…In addition, SLCs are membrane proteins, and their functions are regulated by the membrane environment. For example, cholesterol, a major component of the synaptic plasma membrane can influence the out-to-inward transition of DAT and the ligand binding to DAT. , Cholesterol can also influence the stability and conformation change of SERT, thereby effecting the substrate transition. , Thus, the effect of membranes on the transporter structure and function should also be considered in future drug design. The next section discusses how computational chemistry can be used to supplement experimental methods for predicting unresolved 3D structures of SLCs.…”

“…104,105 Cholesterol can also influence the stability and conformation change of SERT, thereby effecting the substrate transition. 106,107 Thus, the effect of membranes on the transporter structure and function should also be considered in future drug design. The next section discusses how computational chemistry can be used to supplement experimental methods for predicting unresolved 3D structures of SLCs.…”