Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK.
Department of Molecular Biology and Biotechnology, The University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK.
J Mol Biol. 2019 Jan 18;431(2):351-367. doi: 10.1016/j.jmb.2018.11.016. Epub 2018 Nov 22.
C4-dicarboxylates play a central role in cellular physiology as key metabolic intermediates. Under aerobic conditions, they participate in the citric acid cycle, while in anaerobic bacteria, they are important in energy-conserving fermentation and respiration processes. Ten different families of secondary transporters have been described to participate in C4-dicarboxylate movement across biological membranes, but only one of these utilizes an extracytoplasmic solute binding protein to achieve high-affinity uptake. Here, we identify the MatBAC system from the photosynthetic bacterium Rhodopseudomonas palustris as the first member of the tripartite tricarboxylate transport family to be involved in C4-dicarboxylate transport. Tryptophan fluorescence spectroscopy showed that MatC, the periplasmic binding protein from this system, binds to l- and d-malate with K values of 27 and 21 nM, respectively, the highest reported affinity to date for these C4-dicarboxylates, and to succinate (K = 110 nM) and fumarate (K = 400 nM). The 2.1-Å crystal structure of MatC with bound malate shows a high level of substrate coordination, with participation of two water molecules that bridge hydrogen bonds between the ligand proximal carboxylic group and the main chain of two conserved loops in the protein structure. The substrate coordination in MatC correlates with the binding data and explains the protein's selectivity for different substrates and respective binding affinities. Our results reveal a new function in C4-dicarboxylate transport by members of the poorly characterized tripartite tricarboxylate transport family, which are widely distributed in bacterial genomes but for which details of structure-function relationships and transport mechanisms have been lacking.
C4-二羧酸在细胞生理学中起着核心作用，是关键的代谢中间产物。在有氧条件下，它们参与柠檬酸循环，而在厌氧细菌中，它们在节能发酵和呼吸过程中很重要。已经描述了 10 种不同的次级转运蛋白家族参与 C4-二羧酸穿过生物膜的运动，但只有一种利用细胞外溶质结合蛋白实现高亲和力摄取。在这里，我们鉴定来自光合细菌沼泽红假单胞菌的 MatBAC 系统是第一个参与 C4-二羧酸转运的三聚体三羧酸转运家族成员。色氨酸荧光光谱表明，该系统的周质结合蛋白 MatC 分别以 27 和 21 nM 的 K 值结合 l-和 d-苹果酸，这是迄今为止报道的这些 C4-二羧酸的最高亲和力，并结合琥珀酸（K = 110 nM）和富马酸（K = 400 nM）。与结合的苹果酸的 MatC 的 2.1-Å 晶体结构显示出高水平的底物配位，其中两个水分子参与配体近端羧酸基团和蛋白质结构中两个保守环的主链之间的氢键桥接。MatC 中的底物配位与结合数据相关，并解释了该蛋白对不同底物的选择性和各自的结合亲和力。我们的结果揭示了三聚体三羧酸转运家族成员在 C4-二羧酸转运中的新功能，该家族在细菌基因组中广泛分布，但结构-功能关系和转运机制的细节尚不清楚。
