Defense Date

12-28-2020

Graduation Date

Summer 8-7-2021

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Chemistry and Biochemistry

Committee Chair

Michael Cascio

Committee Member

Mihaela Rita Mihailescu

Committee Member

Michael Van Stipdonk

Committee Member

Christopher K Surratt

Keywords

serotonin transporter, crosslinking, mass spectrometry, selective serotonin re-uptake inhibitors

Abstract

The serotonin transporter (SERT) is a member of the neurotransmitter sodium symporter family of transporters. SERT controls the magnitude and duration of serotonergic neurotransmission by facilitating the reuptake of serotonin back into the pre-synaptic neuron and is thus a target for antidepressants. Selective serotonin re-uptake inhibitors (SSRIs) such as fluoxetine, are commonly prescribed to treat depression. SSRIs act by blocking reuptake and prolonging serotonin signaling. However, significant problems regarding selectivity and mechanisms of action of these drugs remain unresolved. The structures of SERT and related transporters have been determined and serve as useful structural models. However, they are typically mutated for thermostability and typically lack significant regions of the extracellular loops and the amino- and carboxy-termini and are characterized in the absence of a native-like lipid bilayer. To further understand SERT structure and the interactions involved in fluoxetine binding, herein we present two studies. Aim 1 investigates fluoxetine binding sites of SERT using a photo-activatable fluoxetine analog and LC-MS/MS in crosslinking studies. Some of the crosslinks observed are consistent with regions forming the central and allosteric sites in the crystal structure. While other crosslinks are novel in that they are in areas of SERT that are unresolved such as the terminal tails. Consistent crosslinks were observed on both N and C terminals, suggesting that they are in close association with the lipid bilayer. Given that fluoxetine partitions in bilayers and might interact non-specifically with membrane proteins, control studies with photo-activatable lipids were also conducted to map lipid accessible sites. Thus, allowing us to differentiate between specific and nonspecific fluoxetine binding. Aim 2 directly interrogates the topology of full-length SERT in lipid vesicles in its resting state by coupling photoaffinity labeling with mass spectrometry (MS). The network of identified crosslinks from introduced single cysteine mutants in the extracellular loops (EL) provides distance constraints and topological information regarding SERT structure in the apo state. Some identified crosslinks were consistent with the recently published crystal structure of human SERT such as crosslinks between EL2 and EL4, which suggest proximity between the two loops. Other crosslinks revealed novel structure information, such as consistent crosslinks to the amino and carboxy terminal domains, thus suggesting that both terminal tails are in close association with the lipid bilayer. Other novel structure information includes modest movements in the N-terminus upon dimerization, and intermolecular crosslinks in mutation S252C which suggest it is located near the dimer interface. Taken together, the studies show that crosslinking mass spectrometry allows for the investigation of local protein structure which can serve as a unique compliment to high resolution structures and for which the benefits will bring us closer to the development of efficient and improved therapeutics.

Language

English

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Included in

Biochemistry Commons

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