Purification and properties of the Escherichia coli nucleoside transporter NupG, a paradigm for a major facilitator transporter sub-family

Abstract

NupG from Escherichia coli is the archetype of a family of nucleoside transporters found in several eubacterial groups and has distant homologues in eukaryotes, including man. To facilitate investigation of its molecular mechanism, we developed methods for expressing an oligohistidine-tagged form of NupG both at high levels (>20% of the inner membrane protein) in E. coli and in Xenopus laevis oocytes. In E. coli recombinant NupG transported purine (adenosine) and pyrimidine (uridine) nucleosides with apparent K_m values of ∼20–30 μM and transport was energized primarily by the membrane potential component of the proton motive force. Competition experiments in E. coli and measurements of uptake in oocytes confirmed that NupG was a broad-specificity transporter of purine and pyrimidine nucleosides. Importantly, using high-level expression in E. coli and magic-angle spinning cross-polarization solid-state nuclear magnetic resonance, we have for the first time been able directly to measure the binding of the permeant ([1′-¹³C]uridine) to the protein and to assess its relative mobility within the binding site, under non-energized conditions. Purification of over-expressed NupG to near homogeneity by metal chelate affinity chromatography, with retention of transport function in reconstitution assays, was also achieved. Fourier transform infrared and circular dichroism spectroscopy provided further evidence that the purified protein retained its 3D conformation and was predominantly α-helical in nature, consistent with a proposed structure containing 12 transmembrane helices. These findings open the way to elucidating the molecular mechanism of transport in this key family of membrane transporters.

• Nucleoside
• transport
• bacteria
• major facilitator superfamily
• solid-state NMR

AZA, 3′-azido-2′,3′-dideoxyadenosine; AZT, 3′-azido-3′-deoxythymidine; BLAST, basic local alignment search tool; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CD, circular dichroism; CP, cross-polarization; CNT, concentrative nucleoside transporter; DDM, dodecyl-β-D-maltopyranoside; E, expect (value); ESI-MS, electrospray ionization mass spectrometry; IPTG, isopropyl β-D-thiogalactoside; MAS SS NMR, magic-angle spinning solid-state nuclear magnetic resonance; NBMPR, nitrobenzylthioinosine (6-[(4-nitrobenzyl)thio]-9-β-D-ribofuranosylpurine); NCBI, National Centre for Biotechnology Information; Ni-NTA, Ni²⁺-nitrilotriacetate; OG, n-octyl-β-D-glucoside; PCR, polymerase chain reaction; pmf, proton-motive force; PMS, phenazine methosulphate; PSSM, position-specific score matrix; TM, transmembrane

AZA, 3′-azido-2′,3′-dideoxyadenosine; AZT, 3′-azido-3′-deoxythymidine; BLAST, basic local alignment search tool; CCCP, carbonyl cyanide m-chlorophenylhydrazone; CD, circular dichroism; CP, cross-polarization; CNT, concentrative nucleoside transporter; DDM, dodecyl-β-D-maltopyranoside; E, expect (value); ESI-MS, electrospray ionization mass spectrometry; IPTG, isopropyl β-D-thiogalactoside; MAS SS NMR, magic-angle spinning solid-state nuclear magnetic resonance; NBMPR, nitrobenzylthioinosine (6-[(4-nitrobenzyl)thio]-9-β-D-ribofuranosylpurine); NCBI, National Centre for Biotechnology Information; Ni-NTA, Ni²⁺-nitrilotriacetate; OG, n-octyl-β-D-glucoside; PCR, polymerase chain reaction; pmf, proton-motive force; PMS, phenazine methosulphate; PSSM, position-specific score matrix; TM, transmembrane