Chemical and structural investigation of the paroxetine-human serotonin transporter complex
- Oregon Health and Science University, United States
- Imperial College London, United Kingdom
Abstract
Antidepressants target the serotonin transporter (SERT) by inhibiting serotonin reuptake. Structural and biochemical studies aiming to understand binding of small-molecules to conformationally dynamic transporters like SERT often require thermostabilizing mutations and antibodies to stabilize a specific conformation, leading to questions about relationships of these structures to the bonafide conformation and inhibitor binding poses of wild-type transporter. To address these concerns, we determined the structures of ∆N72/∆C13 and ts2-inactive SERT bound to paroxetine analogues using single-particle cryo-EM and x-ray crystallography, respectively. We synthesized enantiopure analogues of paroxetine containing either bromine or iodine instead of fluorine. We exploited the anomalous scattering of bromine and iodine to define the pose of these inhibitors and investigated inhibitor binding to Asn177 mutants of ts2-active SERT. These studies provide mutually consistent insights into how paroxetine and its analogues bind to the central substrate-binding site of SERT, stabilize the outward-open conformation, and inhibit serotonin transport.
Data availability
The coordinates and associated volumes for the cryo-EM reconstruction of SERT 8B6 Fab paroxetine, Br-paroxetine, and I-paroxetine datasets have been deposited in the PDB (https://www.rcsb.org/) and Electron Microscopy Data Bank (EMDB; https://www.ebi.ac.uk/pdbe/emdb/) under the accession codes 6VRH and 21368, 6VRK and 21369, and 6VRL and 21370, respectively. The half maps for each dataset have also been deposited in the EMDB (https://www.ebi.ac.uk/pdbe/emdb/). The x-ray coordinates for Br-paroxetine and I-paroxetine have been deposited in the PDB (https://www.rcsb.org/) under accession codes 6W2B and 6W2C, respectively.
Article and author information
Author details
Funding
National Institutes of Health (5R37MH070039)
- Eric Gouaux
Howard Hughes Medical Institute (N/A)
- Eric Gouaux
Royal Society (UF140161)
- James A Bull
Royal Society (RG150444)
- James A Bull
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Lucy R Forrest, National Institute of Neurological Disorders and Stroke, National Institutes of Health, United States
Version history
- Received: February 27, 2020
- Accepted: July 2, 2020
- Accepted Manuscript published: July 3, 2020 (version 1)
- Version of Record published: September 3, 2020 (version 2)
Copyright
© 2020, Coleman et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Chemical and structural investigation of the paroxetine-human serotonin transporter complex
eLife 9:e56427.
https://doi.org/10.7554/eLife.56427
Further reading
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- Biochemistry and Chemical Biology
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The ATPase p97 (also known as VCP, Cdc48) has crucial functions in a variety of important cellular processes such as protein quality control, organellar homeostasis, and DNA damage repair, and its de-regulation is linked to neuromuscular diseases and cancer. p97 is tightly controlled by numerous regulatory cofactors, but the full range and function of the p97–cofactor network is unknown. Here, we identify the hitherto uncharacterized FAM104 proteins as a conserved family of p97 interactors. The two human family members VCP nuclear cofactor family member 1 and 2 (VCF1/2) bind p97 directly via a novel, alpha-helical motif and associate with p97-UFD1-NPL4 and p97-UBXN2B complexes in cells. VCF1/2 localize to the nucleus and promote the nuclear import of p97. Loss of VCF1/2 results in reduced nuclear p97 levels, slow growth, and hypersensitivity to chemical inhibition of p97 in the absence and presence of DNA damage, suggesting that FAM104 proteins are critical regulators of nuclear p97 functions.
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- Biochemistry and Chemical Biology
- Epidemiology and Global Health
Background: High levels of circulating adiponectin are associated with increased insulin sensitivity, low prevalence of diabetes, and low body mass index (BMI); however, high levels of circulating adiponectin are also associated with increased mortality in the 60-70 age group. In this study, we aimed to clarify factors associated with circulating high-molecular-weight (cHMW) adiponectin levels and their association with mortality in the very old (85-89 years old) and centenarians.
Methods: The study included 812 (women: 84.4%) for centenarians and 1,498 (women: 51.7%) for the very old. The genomic DNA sequence data were obtained by whole genome sequencing or DNA microarray-imputation methods. LASSO and multivariate regression analyses were used to evaluate cHMW adiponectin characteristics and associated factors. All-cause mortality was analyzed in three quantile groups of cHMW adiponectin levels using Cox regression.
Results: The cHMW adiponectin levels were increased significantly beyond 100 years of age, were negatively associated with diabetes prevalence, and were associated with SNVs in CDH13 (p = 2.21 × 10-22) and ADIPOQ (p = 5.72 × 10-7). Multivariate regression analysis revealed that genetic variants, BMI, and high-density lipoprotein cholesterol (HDLC) were the main factors associated with cHMW adiponectin levels in the very old, whereas the BMI showed no association in centenarians. The hazard ratios for all-cause mortality in the intermediate and high cHMW adiponectin groups in very old men were significantly higher rather than those for all-cause mortality in the low level cHMW adiponectin group, even after adjustment with BMI. In contrast, the hazard ratios for all-cause mortality were significantly higher for high cHMW adiponectin groups in very old women, but were not significant after adjustment with BMI.
Conclusions: cHMW adiponectin levels increased with age until centenarians, and the contribution of known major factors associated with cHMW adiponectin levels, including BMI and HDLC, varies with age, suggesting that its physiological significance also varies with age in the oldest old.
Funding: This study was supported by grants from the Ministry of Health, Welfare, and Labour for the Scientific Research Projects for Longevity; a Grant-in-Aid for Scientific Research (No 21590775, 24590898, 15KT0009, 18H03055, 20K20409, 20K07792, 23H03337) from the Japan Society for the Promotion of Science; Keio University Global Research Institute (KGRI), Kanagawa Institute of Industrial Science and Technology (KISTEC), Japan Science and Technology Agency (JST) Research Complex Program 'Tonomachi Research Complex' Wellbeing Research Campus: Creating new values through technological and social innovation (JP15667051), the Program for an Integrated Database of Clinical and Genomic Information from the Japan Agency for Medical Research and Development (No. 16kk0205009h001, 17jm0210051h0001, 19dk0207045h0001); the medical-welfare-food-agriculture collaborative consortium project from the Japan Ministry of Agriculture, Forestry, and Fisheries; and the Biobank Japan Program from the Ministry of Education, Culture, Sports, and Technology.
