@article{3032697,
    title = "Identification of New Specificity Determinants in Bacterial Purine
Nucleobase Transporters based on an Ancestral Sequence Reconstruction
Approach",
    author = "Tatsaki, Ekaterini and Anagnostopoulou, Eleni and Zantza, Iliana and and Lazou, Panayiota and Mikros, Emmanuel and Frillingos, Stathis",
    journal = "JOURNAL OF MOLECULAR BIOLOGY",
    year = "2021",
    volume = "433",
    number = "24",
    publisher = "ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD",
    issn = "0022-2836",
    doi = "10.1016/j.jmb.2021.167329",
    abstract = "The relation of sequence with specificity in membrane transporters is
challenging to explore. Most relevant studies until now rely on
comparisons of present-day homologs. In this work, we study a set of
closely related transporters by employing an evolutionary,
ancestral-reconstruction approach and reveal unexpected new specificity
determinants. We analyze a monophyletic group represented by the
xanthinespecific XanQ of Escherichia coli in the Nucleobase-Ascorbate
Transporter/Nucleobase-Cation Symporter-2 (NAT/NCS2) family. We
reconstructed AncXanQ, the putative common ancestor of this clade,
expressed it in E. coli K-12, and found that, in contrast to XanQ, it
encodes a high-affinity permease for both xanthine and guanine, which
also recognizes adenine, hypoxanthine, and a range of analogs. AncXanQ
conserves all binding-site residues of XanQ and differs substantially in
only five intramembrane residues outside the binding site. We subjected
both homologs to rationally designed mutagenesis and present evidence
that these five residues are linked with the specificity change. In
particular, we reveal Ser377 of XanQ (Gly in AncXanQ) as a major
determinant. Replacement of this Ser with Gly enlarges the specificity
of XanQ towards an AncXanQ-phenotype. The ortholog from Neisseria
meningitidis retaining Gly at this position is also a xanthine/guanine
transporter with extended substrate profile like AncXanQ. Molecular
Dynamics shows that the S377G replacement tilts transmembrane helix 12
resulting in rearrangement of Phe376 relative to Phe94 in the XanQ
binding pocket. This effect may rationalize the enlarged specificity. On
the other hand, the specificity effect of S377G can be masked by G27S or
other mutations through epistatic interactions. (C) 2021 The Author(s).
Published by Elsevier Ltd."
}