5 years ago

Characterization of the Escherichia coli Concentrative Nucleoside Transporter NupC Using Computational, Biochemical, and Biophysical Methods

Characterization of the Escherichia coli Concentrative Nucleoside Transporter NupC Using Computational, Biochemical, and Biophysical Methods
Ryan J. Hope, Hao Xie, Simon G. Patching, Stephen A. Baldwin, Jean C. Ingram, Lijie Sun
Members of the concentrative nucleoside transporter (CNT) family of proteins mediate uptake of nucleosides into cells driven by a cation gradient, which then enter salvage pathways for nucleic acid synthesis. In humans, they also transport hydrophilic anticancer and antiviral nucleoside analogue drugs into cells and tissues where they exert their pharmacological effects. Escherichia coli CNT NupC (400 residues) is pyrimidine-specific and driven by a proton gradient. We have used computational, biochemical, and biophysical methods to characterize evolutionary relationships, conservation of residues, structural domains, transmembrane helices, and residues involved in nucleoside binding and/or transport activity in NupC compared with those of sodium-driven Vibrio cholerae CNT (vcCNT) and human CNTs (hCNT1–3). As in the crystal structure of vcCNT, NupC appears to contain eight transmembrane-spanning α-helices. Wild-type NupC and single-cysteine-containing mutants were tested for transport activity in energized E. coli whole cells and for binding of nucleosides in non-energized native inner membranes using novel cross-polarization magic-angle spinning solid-state nuclear magnetic resonance methods. Wild-type NupC had an apparent affinity of initial rate transport (Kmapp) for [14C]uridine of 22.2 ± 3.7 μM and an apparent binding affinity (Kdapp) for [1′-13C]uridine of 1.8–2.6 mM. Mutant S142C retained transport and binding affinities similar to those of the wild type. Mutants G146C and E149C had no transport activity but retained varying degrees of partial binding activity with affinities decreasing in the following order: wild type > S142C > G146C > E149C. Results were explained with respect to a homology model of NupC based on the structure of vcCNT and a hypothetical elevator-type mechanism of alternating access membrane transport in NupC.

Publisher URL: http://dx.doi.org/10.1021/acs.biochem.7b00172

DOI: 10.1021/acs.biochem.7b00172

You might also like
Discover & Discuss Important Research

Keeping up-to-date with research can feel impossible, with papers being published faster than you'll ever be able to read them. That's where Researcher comes in: we're simplifying discovery and making important discussions happen. With over 19,000 sources, including peer-reviewed journals, preprints, blogs, universities, podcasts and Live events across 10 research areas, you'll never miss what's important to you. It's like social media, but better. Oh, and we should mention - it's free.

  • Download from Google Play
  • Download from App Store
  • Download from AppInChina

Researcher displays publicly available abstracts and doesn’t host any full article content. If the content is open access, we will direct clicks from the abstracts to the publisher website and display the PDF copy on our platform. Clicks to view the full text will be directed to the publisher website, where only users with subscriptions or access through their institution are able to view the full article.