Academic journal article Genetics

Substrate Specificity of the FurE Transporter Is Determined by Cytoplasmic Terminal Domain Interactions

Academic journal article Genetics

Substrate Specificity of the FurE Transporter Is Determined by Cytoplasmic Terminal Domain Interactions

Article excerpt

Years of research have led to a model where solute transmembrane transporters operate by an alternating access mechanism that exhibits at least two structurally distinct conformations, outward (extracellular)-facing and inward (cytoplasmic)-facing, the alternation of which is promoted by substrate binding and release (Forrest et al. 2011; Kaback et al. 2011). More recent genetic, functional, and structural evidence has supported the idea that during transport catalysis, transporters acquire multiple distinct conformations, a distinction based not only on whether the binding site is facing the extracellular or cytoplasmic side of the cell, but also on whether specific domains of the protein allow or occlude substrate access to, or release from, the binding site. These distinct domains seem to function as gates, gating elements, or selectivity filters (Diallinas 2008, 2016). Thus, current models consider that during substrate translocation, transporters obtain at least four structurally distinct sequential conformations: an outward-facing "open" conformation that provides access to substrates; an outward-facing "closed" conformation, where the substrate is bound in the major substrate-binding site while a domain (e.g., an external gate) moves to occlude further access of substrates from the extracellular side or leakage of the substrate from the binding site; an inward-facing closed conformation, where the substrate is still bound in the major substrate-binding site while a distinct domain (e.g., an internal gate) occludes substrate release into the cytoplasm; and an inward-facing open conformer, where the internal gate is displaced to allow the release of the substrate into the cytoplasm (Shi 2013; Diallinas 2014, 2016; Penmatsa and Gouaux 2014; Colas et al. 2016; Quistgaard et al. 2016).

Interestingly, genetic, functional, and structural approaches have shown that mutations altering the specificity of transporters are preferably located at gating elements or selectivity filters, rather than within the major binding site. Such gating or selectivity elements can be located at the periphery of the major binding site, but also at flexible transmembranea-helices or hydrophilic loops that act as dynamic hinges (Papageorgiou et al 2008; Weyand et al. 2008; Kosti et al 2010; Shimamura et al. 2010; Adelman et al. 2011; Kaźmier etai. 2014; Simmons etai. 2014; Alguel etai. 2016a; Diallinas 2016). The independent action of gating or selectivity elements has been further supported by the fact that specific mutations in these elements often do not alter the transport kinetics for physiological substrates, and may lead to additive or synthetic transport activities and specificities when combined with substrate-binding mutations (Papageorgiou et al 2008; Kosti et al. 2010; Alguel et al. 2016a). Overall, these findings strongly suggest that transporter specificity might be determined through molecular and functional synergy of multiple domains (Diallinas 2014, 2016). Recently, homooligomeriźation was also found to be important for transporter specificity (Alguel et al. 2016a,b; Diallinas 2016).

In this work, we present genetic and biochemical evidence supporting the idea that transporter specificity in the NCS1 (Nucleobase Cation Symporter 1) transporter family (Pantazopoulou and Diallinas 2007; Weyand et al. 2008; Krypotou et al 2012, 2015; Sioupouli et al. 2017) is determined via dynamic intramolecular interactions of multiple domains. More specifically, we show that cytoplasmic N- and C-terminal segments of FurE, an allantoin-uric acid-uracil transporter of the fungus Aspergillus nidulans (Krypotou et al. 2015), interact with each other and that this interaction is critical for substrate specificity. Our results are discussed in the context of available knowledge from crystallographic and in silico approaches to study NSC1 transporters.

Materials and Methods

Media, strains, and growth conditions

Standard complete (CM) and minimal media (MM) for A. …

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