Structure-function relationships in substrate binding protein dependent secondary transporters

Tripartite transport systems exhibit increased substrate affinity and transport rates as they depend on substrate binding proteins (SBPs). In contrast to primary transporters, SBP dependent secondary transport systems are under-researched although already discovered in 1996 with TRAPRc-DctPQM from Rhodobacter capsulatus (Madden 2002; Jacobs et al. 1996). Within the family of the TRAP transport systems three subfamilies evolved, the TRAP-TRAP, TRAP-TAXI and TRAP-TPAT transporter (L. T. Rosa, Bianconi, et al. 2018). To reduce the complexity of the naming, we will hereafter use the terms TRAP, TAXI and TPAT. Following the nomenclature of TRAPRc-DctPQM the subunits of TRAP transporter are most often referred to as P for the SBP and Q and M for the two membrane domains (Jacobs et al. 1996). TRAP transport systems are found in bacteria and those of the TAXI family additionally in archaea because of which TAXI transport systems are believed to represent an ancient form of TRAP transporter (Mulligan, Fischer, and Thomas 2011; L. T. Rosa, Bianconi, et al. 2018). Detailed in vitro characterization is limited to three N-acetylneuraminic acid TRAP transport systems, derived from Haemophilus influenzae, Vibrio cholerae and Photobacterium profundum (Mulligan et al. 2012, 2009; Davies, Currie, and North 2023). Within the SBP dependent secondary transport systems, the TAXI family is the least studied group. Structurally only one TAXI SBP was solved, named TtGluBP from Thermus thermophilus (Takahashi et al. 2004) and functional characterization of a TAXI transport system is absent from the literature (Mulligan, Fischer, and Thomas 2011). We selected homologs of this group for functional characterization. Using BLAST, we selected the homologs based on their similarity to TRAPRc-DctPQM from Rhodobacter capsulatus. After the identification of homologs, we amplified TRAP-QM domains to overexpress and detergent-solubilize them in analytical tests. This was successful for transport systems deriving from Desulfotomaculum carboxydivorans, Shimwellia blattae, Natrialba asiatica, Proteus mirabilis and Marinobacter hydrocarbonoclasticus. We then expressed and purified successfully all five corresponding SBPs. To identify potential substrates of the tripartite transport systems, we exposed the SBPs to members of a compound library created from known and similar ligands of TRAP SBPs and studied their thermal melting by differential scanning fluorimetry (DSF). A wide range of mostly C4- and C5-dicarboxylates, amino acids (aa), and sugars were tested. After studying the gene neighborhood of TAXIPm-PQM from Proteus mirabilis, α-ketoglutarate and its direct precursor α-hydroxyglutarate were included into the compound library, motivated by the presence of the IhgO gene, coding for a putative L-2-hydroxyglutarate oxidase, located downstream of the TAXIPm-QM and TAXIPm-P genes in the same operon. For TAXIPm-P from Proteus mirabilis a profound increase in melting temperature was observed in DSF in the presence of the C5-dicarboxylates α-hydroxyglutarate and α-ketoglutarate. The binding appears to be very specific as compounds that are structurally closely related, such as glutarate or glutamate, did not change the thermostability of the protein. When different α-ketoglutarate concentrations were supplied, a destabilization of the protein was observed above 50 µM. For TAXIMh-P from Marinobacter hydrocarbonoclasticus a distinct increase in melting temperature was observed in DSF in the presence of the C4-dicarboxylates fumarate, succinate, and L-malate. Similarly, to the deorphanization of TAXIPm-P, genes located in close proximity to the transporter provided information about the ligand for TAXIMh-P as well. DcuB, located in the operon next to TAXIMh-P and TAXIMh-QM, encodes an antiporter which imports the C4-dicarboxylates fumarate, malate, aspartate, and D-tartrate in exchange for succinate. After the substrates are imported, fumarase, and fumarate reductase convert L-malate to fumarate and fumarate to succinate. L-malate, fumarate, and succinate are all structurally and metabolically connected (Kim 2006) simultaneously to α-hydoxyglutarate and α-ketoglutarate (Brunengraber 2007).