Insights into the molecular basis for substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC

Abstract

Pathogenic enterobacteria need to survive the extreme acidity of the stomach to successfully colonize the human gut. Enteric bacteria circumvent the gastric acid barrier by activating extreme acidresistance responses, such as the arginine-dependent acid resistance system. In this response, L-arginine is decarboxylated to agmatine, thereby consuming one proton from the cytoplasm. In Escherichia coli, the L-arginine/agmatine antiporter AdiC facilitates the export of agmatine in exchange of L-arginine, thus providing substrates for further removal of protons from the cytoplasm and balancing the intracellular pH. We have solved the crystal structures of wild-type AdiC in the presence and absence of the substrate agmatine at 2.6-Å and 2.2-Å resolution, respectively. The high-resolution structures made possible the identification of crucial water molecules in the substrate-binding sites, unveiling their functional roles for agmatine release and structure stabilization, which was further corroborated by molecular dynamics simulations. Structural analysis combined with site-directed mutagenesis and the scintillation proximity radioligand binding assay improved our understanding of substrate binding and specificity of the wild-type L-arginine/agmatine antiporter AdiC. Finally, we present a potential mechanism for conformational changes of the AdiC transport cycle involved in the release of agmatine into the periplasmic space of E. coli. membrane protein | scintillation proximity assay | substrate binding | transporter | X-ray structure

Asc-1 Transporter (SLC7A10): Homology Models And Molecular Dynamics Insights Into The First Steps Of The Transport Mechanism

“…The human amino acid sequence of Asc-1 (SLC7A10) used in this study was retrieved from UniProt Database (http://www.uniprot.org) under the code Q9NS82. At the time our studies were conducted, a multiple sequence alignment with NCBI BLAST on the protein data bank (PDB) identified bacterial transporters AdiC (PDB ID: 5J4I), GadC (PDB ID: 4DJI), MjApcT (PDB ID: 3GIA) and the recent release of the bacterial cationic amino acid transporter GkApcT (PDB ID: 5OQT) as the most homologous templates [22][23][24][25] .…”

“…Also, d-serine is formed from l-serine by Serine Racemase (SR, green). Transport of d-serine including the efflux of the substrate (indicated by M1 mechanism) along with the uptake of the substrate (indicated by M2 mechanism) across the membrane involves roughly three states substrate-free in the outward state, but also co-crystallized with different substrates (PDB ID: 3LRB, 3NCY, 30B6, 3L1L, 5J4I, 5J4N) 22,[26][27][28][29] . On the contrary, no crystal structure of GkApcT was solved in the apo outward-open state, and the substrate-bound GkApcT complex (PDB ID: 5OQT) was crystallized in the inward occluded state with the intracellular side noticeably more open 25 .…”

Metabolic engineering of Escherichia coli for agmatine production

“…The AST pathway contributes to arginine degradation only during nitrogen limited growth. For the transport of agmatine, only the arginine: agmatine antiporter encoded by the adiC gene is identified [26], other transport reactions mediating the uptake or secretion of agmatine is still unknown in E. coli. In addition to l-glutamate, l-glutamine, and l-aspartate participate in agamtine synthesis, and improvement of the contents of l-glutamine and l-aspartate could positively affect agmatine production in engineered E. coli strains.…”

“…The supply of cofactor NADPH generated from the pentose phosphate pathway can become a critical factor for the efficient biosynthesis of fermentative products when the enzyme levels are no longer limiting [24,25]. For the transport of agmatine, only the arginine: agmatine antiporter encoded by the adiC gene is identified [26], other transport reactions mediating the uptake or secretion of agmatine is still unknown in E. coli. The arginine: agmatine antiporter AdiC may also be considered as a target for improving agmatine production.…”

Cytosolic termini of the FurE transporter regulate endocytosis, pH-dependent gating and specificity

“…TMSs 1-10 are arranged as a 5-helix intertwined inverted repeat (5HIRT), the so called LeuT-fold, also found in other transporter families involved in neurotransmitter, sugar, amino acid, and drug transport (Shi, 2013; Västermark and Saier, 2014; Drew and Boudker, 2016). The last two TMSs (11 and 12) seem to be crucial for the oligomerization state of some NCS1-like transporters, rather than being involved in the mechanism of transport (Ilgü et al, 2016).…”