Current Research

Secondary (ion-gradient driven) active transport proteins represent a core group of functional processes which are crucial in both microbial adaptations and human physiology and disease. Although transport protein families tend to display high evolutionary conservation in sequence and overall structure, they also display high functional variations between homologs, implying that relatively few side chain changes may account for key local effects on the active-site conformation and function.

We are interested to gain insight on side chains and sequence motifs that dictate substrate selectivity and uptake in such transporter families by using bacterial homologs as study models. We currently focus on the evolutionarily broad family of nucleobase-ascorbate transporters (NAT/NCS2) which includes proteins responsible for the uptake of several frontline purine-related drugs or analogues but is structurally unknown at high resolution.

A major challenge for the study of NAT/NCS2 family is that very few of the >2000 predicted members have been identified functionally, albeit indicating high heterogeneity in specificities, while high-resolution structures or models thereof were missing until recentrly. Currently, the first x-ray structure for a NAT homolog (the uracil permease UraA) has become available; UraA represents a novel structural fold (Lu et al., 2011) and is already used in our lab to build high-resolution models of XanQ for evaluating and refining mutagenesis designs. To understand the molecular basis of the conformational and substrate-specificity divergence in NAT family, we have launched three major experimental routes:

  • (1) Systematic structure-function analysis of a representative NAT homolog, the xanthine permease XanQ (YgfO) from E. coli K-12, which we have cloned and characterized recently (Karatza & Frillingos, 2005). This approach is based mainly on Cys-scanning mutagenesis and site-directed alkylation methods and has yielded a comprehensive view of the important XanQ residues, including residues of the substrate-binding site (Georgopoulou et al., 2010), and a preliminary helix packing model (Karena & Frillingos, 2009), which we interrogate with cross-linking and dynamic functional interaction experiments.
  • (2) Cloning and functional characterization of a series of NAT homologs to reveal and/or systematize correlation patterns between residues present at important positions and substrate selectivity profiles. These data will offer a basis to understand mechanisms of conformational and substrate-profile divergence between different transporters and design efficient strategies to modulate specificity to novel directions.
  • (3) Construction and analysis of cross-homolog chimeras shuffling helical “domains” between closely related NAT homologs with different specificities, to elucidate structural elements and interactions which allow acquisition of new functions and substrate-recognition profiles.

Our work on nucleobase transporters of the NAT/NCS2 family has included development of specific site-directed alkylation protocols (Fig. 1), formulation and refinement of systematic topology-functional models (Fig. 2), and initiation of collaborations for the biophysical and crystallographic analysis of NAT transporters (Fig. 3).

Apart from the mechanistic and the evolutionary implications, our work is expected to confer to efficient, transporter-based application designs for novel nucleobase synthetic drugs and targeted antimicrobial therapies.