Context-dependent Cryptic Roles of Specific Residues in Substrate Selectivity of the UapA Purine Transporter

Kourkoulou, Anezia; Zantza, Iliana; Foti, Konstantina; Mikros, Emmanuel; Diallinas, George

doi:10.1016/j.jmb.2021.166814

Cited by 9 publications

(6 citation statements)

References 58 publications

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“…In an example from the Nucleobase Ascorbate Transporter (NAT) family, different binding orientations for xanthine and allopurinol were demonstrated for the UapA transporter of Aspergillus nidulans [ 57 ]. Mutations in the binding pocket of this transporter were shown to alter both substrate specificity and substrate orientation [ 58 ]. Similarly, a recent study showed that a series of mutations applied to the Escherichia coli XanQ transporter in order to mimic the ancestral XanQ (AncXanQ) altered it from a xanthine-specific carrier to a xanthine/guanine transporter [ 59 ].…”

Section: Discussionmentioning

confidence: 99%

A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes

Campagnaro

Elati

Balaska

et al. 2022

IJMS

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Toxoplasma gondii is unable to synthesize purines de novo, instead salvages them from its environment, inside the host cell, for which they need high affinity carriers. Here, we report the expression of a T. gondii Equilibrative Nucleoside Transporter, Tg244440, in a Trypanosoma brucei strain from which nucleobase transporters have been deleted. Tg244440 transported hypoxanthine and guanine with similar affinity (Km ~1 µM), while inosine and guanosine displayed Ki values of 4.05 and 3.30 µM, respectively. Low affinity was observed for adenosine, adenine, and pyrimidines, classifying Tg244440 as a high affinity oxopurine transporter. Purine analogues were used to probe the substrate-transporter binding interactions, culminating in quantitative models showing different binding modes for oxopurine bases, oxopurine nucleosides, and adenosine. Hypoxanthine and guanine interacted through protonated N1 and N9, and through unprotonated N3 and N7 of the purine ring, whereas inosine and guanosine mostly employed the ribose hydroxy groups for binding, in addition to N1H of the nucleobase. Conversely, the ribose moiety of adenosine barely made any contribution to binding. Tg244440 is the first gene identified to encode a high affinity oxopurine transporter in T. gondii and, to the best of our knowledge, the first purine transporter to employ different binding modes for nucleosides and nucleobases.

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Section: Discussionmentioning

confidence: 99%

A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes

Campagnaro

Elati

Balaska

et al. 2022

IJMS

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“…The residues modified are located at the interface of the TMS5 helix overlooking the substrate binding site in the sliding elevator domain, and are all extremely conserved in the Nucleobase Ascorbate Transporter (NAT) family (Gournas et al, 2008;Diallinas and Gournas 2008), where UapA belongs (Figure 1B). The mutated UapA versions were introduced and analyzed in a genetic background lacking detectable nucleobaserelated transport activities due to null mutations in all relevant transporter genes (Δ7), as described in Kourkoulou et al, 2021. A. nidulans can grow on purines as N sources and is sensitive to several toxic analogues of nucleobases allowing direct assessment of UapA mutant versions expressed in Δ7 (Figure 1C). Mutants I230A and L234A showed normal growth on media with uric acid and xanthine, similarly to an isogenic control strain expressing wild-type (wt) UapA, while L234S showed moderately reduced growth on these purines, and especially on uric acid.…”

Section: Tms5 Mutationsmentioning

confidence: 99%

“…The inward-facing UapA model PDB ID is 5i6c. The outward-facing model of UapA was generated using as template the Band3 anion exchanger structural homologue crystallized in the outward conformation, as described in Kourkoulou et al, 2021. The models shown here are presented with PyMOL 2.5 (https://pymol.org).…”

Section: Protein Model Constructionmentioning

confidence: 99%

Transmembrane helices 5 and 12 control transport dynamics, substrate affinity, and specificity in the elevator-type UapA transporter

2022

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An increasing number of solute transporters have been shown to function with the so-called sliding-elevator mechanism. Despite structural and functional differences all elevator-type transporters use a common mechanism of substrate translocation via reversible movements of a mobile core domain (the elevator) hosting the substrate binding site along a rigid scaffold domain stably anchored in the plasma membrane via homodimerization. One of the best studied elevator transporters is the UapA uric acid-xanthine/H+ symporter of the filamentous fungus Aspergillus nidulans. Here, we present a genetic analysis for deciphering the role of transmembrane segments (TMS) 5 and 12 in UapA transport function. We show that specific residues in both TMS5 and TMS12 control, negatively or positively, the dynamics of transport, but also substrate binding affinity and specificity. More specifically, mutations in TMS5 can lead to increased rate of transport, but also to an inactive transporter due to high-affinity substrate-trapping, whereas mutations in TMS12 lead to apparently uncontrolled sliding and broadened specificity, leading in specific cases to UapA-mediated purine toxicity. Our findings shed new light on how elevator transporters function and how this knowledge can be applied to genetically modify their transport characteristics.

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“…Based on the detailed mutagenesis studies on the E. coli xanthine transporter XanQ, it was suggested that substrate specificity of SLC23 nucleobase transporters is governed by a few conserved residues, and that changing these residues would also change the specificities of the transporters (Frillingos, 2012;Papakostas and Frillingos, 2012;Karena et al, 2015;Chaliotis et al, 2018;Kourkoulou et al, 2018Kourkoulou et al, , 2021. This hypothesis even led to the development of an algorithm that is supposed to predict the substrate specificity of SLC23 proteins solely based on sequence alignments on 1355 NCS2 homologs from 4655 bacterial and archaeal proteomes (the NAT/NCS2-hound, http://bioinf.bio.uth.gr/nat-ncs2/#/) (Chaliotis et al, 2018).…”

Section: Substrate Specificity Of Slc23 Transportersmentioning

confidence: 99%

“…So far, nevertheless, many SLC23 transporters for which substrate specificities were investigated in more detail mostly are restricted to bind either only purines or only pyrimidines, but the reason for this remains largely unclear. In a recent study, Kourkoulou et al describe a mutant of UapA that is able to recognize a broad variety of purines and pyrimidines (Kourkoulou et al, 2021).…”

Section: Substrate Specificity Of Slc23 Transportersmentioning

confidence: 99%

Substrate translocation in SLC23 and SLC26 transporters

Holzhüter¹

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Specialized transporter proteins facilitate controlled uptake and extrusion of molecules across biological membranes that would otherwise be impermeable to them. The superfamily of solute carriers (SLC) comprises the second largest group of membrane proteins in humans, acting on a variety of small polar and non-polar molecules and ions. Because of their central role in metabolism, malfunctioning of these proteins often is pathogenic. The interest in SLC transporters as drug targets – as well as for drug delivery – has therefore increased in the past years. For many SLC subfamilies, however, structural and functional information remains scarce to date. The here presented data provides important insights into different aspects of the transport mechanism of the SLC23 and SLC26 protein families. Importantly, we show that SLC23 nucleobase transporters, in contrast to what was been previously reported, work as uniporters rather than as proton-coupled symporters. In order to do so, we developed the first and only in vitro transport assay for the SLC23 family, which enables investigation of protein function in a defined environment. Moreover, we provide a hypothesis on the role of the extremely conserved negative charged substrate binding site residue found not only in the SLC23, but also SLC4 and SLC26 families. Based on a detailed analysis of binding and transport we conclude that this conserved negative charged has a relevance for protein stability rather than for substrate binding, which explains its conservation for all three protein families that otherwise differ in their substrate specificities and modes of transport. Lastly, we investigated the relevance of oligomerization for the SLC23 and SLC26 families, highlighting the importance of the STAS domain for forming active dimers in the SLC26 anion transporter family.

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Context-dependent Cryptic Roles of Specific Residues in Substrate Selectivity of the UapA Purine Transporter

Cited by 9 publications

References 58 publications

A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes

A Toxoplasma gondii Oxopurine Transporter Binds Nucleobases and Nucleosides Using Different Binding Modes

Transmembrane helices 5 and 12 control transport dynamics, substrate affinity, and specificity in the elevator-type UapA transporter

Substrate translocation in SLC23 and SLC26 transporters

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