Identification of New Specificity Determinants in Bacterial Purine Nucleobase Transporters based on an Ancestral Sequence Reconstruction Approach
| Autor: | Panayiota Lazou, Ekaterini Tatsaki, Emmanuel Mikros, Eleni Anagnostopoulou, Iliana Zantza, Stathis Frillingos |
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| Rok vydání: | 2021 |
| Předmět: |
Guanine
Molecular Dynamics Simulation Neisseria meningitidis medicine.disease_cause Xanthine Protein Structure Secondary Substrate Specificity chemistry.chemical_compound Bacterial Proteins Structural Biology medicine Escherichia coli Nucleobase Transport Proteins Binding site Molecular Biology Hypoxanthine Phylogeny Genetics Permease Chemistry Mutagenesis Transporter Transmembrane domain |
| Zdroj: | Journal of molecular biology. 433(24) |
| ISSN: | 1089-8638 |
| Popis: | 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 xanthine-specific 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. |
| Databáze: | OpenAIRE |
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