Department of Molecular Biology and Biotechnology, The University of Sheffield, Sheffield, UK.
FEBS J. 2017 Dec;284(24):4262-4277. doi: 10.1111/febs.14304. Epub 2017 Nov 16.
The tripartite tricarboxylate transporter (TTT) family is a poorly characterised group of prokaryotic secondary solute transport systems, which employ a periplasmic substrate-binding protein (SBP) for initial ligand recognition. The substrates of only a small number of TTT systems are known and very few SBP structures have been solved, so the mechanisms of SBP-ligand interactions in this family are not well understood. The SBP RPA4515 (AdpC) from Rhodopseudomonas palustris was found by differential scanning fluorescence and isothermal titration calorimetry to bind aliphatic dicarboxylates of a chain length of six to nine carbons, with K values in the μm range. The highest affinity was found for the C6-dicarboxylate adipate (1,6-hexanedioate). Crystal structures of AdpC, either adipate or 2-oxoadipate bound, revealed a lack of positively charged amino acids in the binding pocket and showed that water molecules are involved in bridging hydrogen bonds to the substrate, a conserved feature in the TTT SBP family that is distinct from other types of SBP. In AdpC, both of the ligand carboxylate groups and a linear chain conformation are needed for coordination in the binding pocket. RT-PCR showed that adpC expression is upregulated by low environmental adipate concentrations, suggesting adipate is a physiologically relevant substrate but as adpC is not genetically linked to any TTT membrane transport genes, the role of AdpC may be in signalling rather than transport. Our data expand the known ligands for TTT systems and identify a novel high-affinity binding protein for adipate, an important industrial chemical intermediate and food additive.
Protein structure co-ordinates are available in the PDB under the accession numbers 5OEI and 5OKU.
三羧酸转运蛋白（TTT）家族是一个特征不明显的原核次级溶质转运系统家族，该家族使用周质基质结合蛋白（SBP）进行初始配体识别。只有少数 TTT 系统的底物被了解，并且很少有 SBP 结构被解决，因此这个家族中 SBP-配体相互作用的机制还不是很清楚。从沼泽红假单胞菌中发现的 SBP RPA4515（AdpC）通过差示扫描荧光和等温滴定微量热法被发现可以结合具有 6 到 9 个碳原子的链长的脂肪族二羧酸，K 值在 μm 范围内。对 C6-二羧酸己二酸（1,6-己二酸）的亲和力最高。AdpC 的晶体结构，无论是与己二酸还是 2-氧代己二酸结合，都显示出结合口袋中缺乏带正电荷的氨基酸，并且表明水分子参与桥接氢键与底物结合，这是 TTT SBP 家族中的一个保守特征，与其他类型的 SBP 不同。在 AdpC 中，配体的两个羧酸基团和线性链构象都需要在结合口袋中进行配位。RT-PCR 显示，低环境己二酸浓度会上调 adpC 的表达，这表明己二酸是一种生理相关的底物，但由于 adpC 与任何 TTT 膜转运基因都没有遗传联系，AdpC 的作用可能在于信号传递而不是运输。我们的数据扩展了 TTT 系统的已知配体，并确定了一种新的高亲和力己二酸结合蛋白，己二酸是一种重要的工业化学中间体和食品添加剂。
蛋白质结构坐标可在 PDB 中以访问号 5OEI 和 5OKU 的形式获得。
