4.7 Article

Thermostabilisation of the Serotonin Transporter in a Cocaine-Bound Conformation

Journal

JOURNAL OF MOLECULAR BIOLOGY
Volume 425, Issue 12, Pages 2198-2207

Publisher

ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/j.jmb.2013.03.025

Keywords

membrane protein; thermostability; structure; neurotransmitter transporter; thermostabilisation

Funding

  1. Medical Research Council [MRC U105197215]
  2. European Union (EDICT consortium) [HEALTH-201924]
  3. National Institutes of Health [R01 GM095685]
  4. Medical Research Council
  5. MRC [MC_U105197215] Funding Source: UKRI
  6. Medical Research Council [MC_U105197215] Funding Source: researchfish

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Structure determination of mammalian integral membrane proteins is challenging due to their instability upon detergent solubilisation and purification. Recent successes in the structure determination of G-protein-coupled receptors (GPCRs) resulted from the development of GPCR-specific protein engineering strategies. One of these, conformational thermostabilisation, could in theory facilitate structure determination of other membrane proteins by improving their tolerance to detergents and locking them in a specific conformation. We have therefore used this approach on the cocaine-sensitive rat serotonin transporter (SERT). Out of a panel of 554 point mutants throughout SERT, 10 were found to improve its thermostability. The most stabilising mutations were combined to make the thermostabilised mutants SAH6 (L99A + G278A + A505L) and SAH7 (L405A + P499A + A505L) that were more stable than SERT by 18 degrees C and 16 degrees C, respectively. Inhibitor binding assays showed that both of the thermostabilised SERT mutants bound [I-125]RT155 (beta-CIT) with affinity similar to that of the wild-type transporter, although cocaine bound with increased affinity (17- to 56-fold) whilst ibogaine, imipramine and paroxetine all bound with lower affinity (up to 90-fold). Neither SAH6 nor SAH7 was capable of transporting [H-3]serotonin into HEK293 cell lines stably expressing the mutants, although serotonin bound to them with an apparent K-i of 155 mu M or 82 mu M, respectively. These data combined suggest that SAH6 and SAH7 are thermostabilised in a specific cocaine-bound conformation, making them promising candidates for crystallisation. Conformational thermostabilisation is thus equally applicable to membrane proteins that are transporters in addition to those that are GPCRs. (c) 2013 Elsevier Ltd. All rights reserved.

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