An ab Initio structural model of a nucleoside permease predicts functionally important residues

Area of Science:

• Structural biology
• Computational modeling
• Membrane transport proteins

Background:

• Equilibrative nucleoside transporters (ENTs) are vital for nucleoside/nucleobase uptake in eukaryotes.
• ENTs facilitate the transport of diverse drugs, including those for cancer, heart disease, AIDS, and parasitic infections.
• No experimental three-dimensional structures exist for ENTs, hindering detailed mechanistic understanding.

Purpose of the Study:

• To generate a high-resolution structural model of a nucleoside permease.
• To investigate the structural basis of transport and substrate specificity in ENTs.
• To validate computational models using site-directed mutagenesis.

Main Methods:

• Ab initio computational modeling was employed to construct a three-dimensional structure of the Leishmania donovani nucleoside permease 1.1 (LdNT1.1).
• Site-directed mutagenesis was performed to probe the functional significance of residues within the modeled structure.
• Comparison with a previously derived threading model was conducted to assess model accuracy.

Main Results:

• The ab initio model identified key residues in the central pore and peripheral regions, correlating with mutagenesis data.
• A potential extracellular gate formed by aromatic residues in transmembrane helices 1, 2, and 7 was proposed.
• Mutating residues in the putative gate abolished transport activity, supporting the model's prediction.

Conclusions:

• The ab initio model provides a detailed structural framework for LdNT1.1, offering insights into nucleoside transport mechanisms.
• The identified extracellular gate may regulate permeation and contribute to substrate specificity within the ENT family.
• The ab initio model represents an improvement over previous threading models, validated by mutagenesis data.