Nanopharmacological Force Sensing to Reveal Allosteric Coupling in Transporter Binding Sites

Abstract

Controversy regardingt he number and function of ligand binding sites in neurotransmitter/sodium symporters arose from conflicting data in crystal structures and molecular pharmacology.Here,wehave designed novel tools for atomic force microscopyt hat directly measure the interaction forces between the serotonin transporter (SERT) and the S-and Renantiomers of citalopram on the single molecule level. This approach is based on force spectroscopy, whichallows for the extraction of dynamic information under physiological conditions thus inaccessible via X-ray crystallography.T wo distinct populations of characteristic binding strengths of citalopram to SERT were revealed in Na + -containing buffer. In contrast, in Li + -containing buffer,S ERT showed only low force interactions.C onversely,t he vestibular mutant SERT-G402H merely displayed the high force population. These observations provide physical evidence for the existence of two binding sites in SERTwhen accessed in aphysiological context. Competition experiments revealed that these two sites are allosterically coupled and exert reciprocal modulation.

Impact of disruption of secondary binding site S2 on dopamine transporter function

“…Crucial in the model is the allosteric interaction between the S 2 and S 1 sites, with ligand occupancy in one affecting the functionality of the other. Such allosterism has been firmly established for the serotonin transporter (Plenge et al 2012;Zhu et al 2016). The results of this study on hDAT provide two lines of evidence in favor of a model with a crucial role of the S 2 site in DA transport.…”

“…; Zhu et al . ). The results of this study on hDAT provide two lines of evidence in favor of a model with a crucial role of the S 2 site in DA transport.…”

“…; Zhu et al . ). The S 2 and S 1 sites are separated by 11–13 Å, and can be bridged by bivalent substrate ligands in our computational models (Schmitt et al .…”

“…The Javitch/ Weinstein group, however, showed destruction of the S 2 site by protein preparation procedures used for crystallography, resulting in a substrate: LeuT stoichiometry of 1 (Quick et al 2012). In a convincing computational study, the two-site substrate transport model has been successfully applied to human (h) DAT (Shan et al 2011), and a functional role for the S 2 site has been firmly established in the serotonin transporter where it allosterically interacts with the S 1 site (Plenge et al 2012;Zhu et al 2016). The S 2 and S 1 sites are separated by 11-13 A, and can be bridged by bivalent substrate ligands in our computational models .…”

Allosteric Modulation of Neurotransmitter Transporters as a Therapeutic Strategy

“…Indeed, SERT-G402H completely ablated allosteric binding of the tested ligands [ 66 ]. Atomic force microscopy (AFM; Box 1 ) experiments using S-citalopram as the ‘probe’ [ 69 ] and the crystal structure of human SERT [ 61 ] confirmed the presence of two citalopram binding sites in SERT. However, by contrast to previous [ 3 H]citalopram dissociation experiments [ 65 ], no sertraline moieties have been found in the S2-site [ 70 ].…”