Vaziri Hamidreza, Baldwin Stephen A, Baldwin Jocelyn M, Adams David G, Young James D, Postis Vincent L G
Astbury Centre for Structural Molecular Biology, School of Biomedical Sciences, University of Leeds, Leeds, UK.
Mol Membr Biol. 2013 Mar;30(2):114-28. doi: 10.3109/09687688.2012.748939. Epub 2012 Dec 21.
Nucleosides play key roles in biology as precursors for salvage pathways of nucleotide synthesis. Prokaryotes import nucleosides across the cytoplasmic membrane by proton- or sodium-driven transporters belonging to the Concentrative Nucleoside Transporter (CNT) family or the Nucleoside:H(+) Symporter (NHS) family of the Major Facilitator Superfamily. The high resolution structure of a CNT from Vibrio cholerae has recently been determined, but no similar structural information is available for the NHS family. To gain a better understanding of the molecular mechanism of nucleoside transport, in the present study the structures of two conformations of the archetypical NHS transporter NupG from Escherichia coli were modelled on the inward- and outward-facing conformations of the lactose transporter LacY from E. coli, a member of the Oligosaccharide:H(+) Symporter (OHS) family. Sequence alignment of these distantly related proteins (∼ 10% sequence identity), was facilitated by comparison of the patterns of residue conservation within the NHS and OHS families. Despite the low sequence similarity, the accessibilities of endogenous and introduced cysteine residues to thiol reagents were found to be consistent with the predictions of the models, supporting their validity. For example C358, located within the predicted nucleoside binding site, was shown to be responsible for the sensitivity of NupG to inhibition by p-chloromercuribenzene sulphonate. Functional analysis of mutants in residues predicted by the models to be involved in the translocation mechanism, including Q261, E264 and N228, supported the hypothesis that they play important roles, and suggested that the transport mechanisms of NupG and LacY, while different, share common features.
核苷作为核苷酸合成补救途径的前体，在生物学中发挥着关键作用。原核生物通过属于主要易化子超家族的集中核苷转运体（CNT）家族或核苷：H(+)同向转运体（NHS）家族的质子或钠驱动转运体，将核苷转运穿过细胞质膜。最近已确定了霍乱弧菌一种CNT的高分辨率结构，但尚无关于NHS家族的类似结构信息。为了更好地理解核苷转运的分子机制，在本研究中，基于大肠杆菌乳糖转运体LacY（寡糖：H(+)同向转运体（OHS）家族的成员）的内向和外向构象，对大肠杆菌典型NHS转运体NupG的两种构象结构进行了建模。通过比较NHS和OHS家族内残基保守模式，促进了这些远缘相关蛋白质（序列同一性约为10%）的序列比对。尽管序列相似性较低，但发现内源性和引入的半胱氨酸残基对硫醇试剂的可及性与模型预测一致，支持了模型的有效性。例如，位于预测核苷结合位点内的C358被证明是NupG对对氯汞苯磺酸盐抑制敏感的原因。对模型预测参与转运机制的残基（包括Q261、E264和N228）的突变体进行功能分析，支持了它们发挥重要作用的假设，并表明NupG和LacY的转运机制虽然不同，但具有共同特征。
