Biochemical Characterization of ThiT from Lactococcus lactis: A Thiamin Transporter with Picomolar Substrate Binding Affinity

Abstract

The putative thiamin transporter ThiT from Lactococcus lactis was overproduced in the membrane of lactococcal cells. In vivo transport assays using radiolabeled thiamin demonstrated that ThiT indeed was involved in thiamin transport. The protein was solubilized from the membranes and purified in detergent solution. Size exclusion chromatography coupled to static light scattering, refractive index, and UV absorbance measurements (SEC-MALLS) showed that ThiT is a monomer of 22.7 kDa in detergent solution. When the cells overexpressing ThiT had been cultivated in complex growth medium, all binding sites of the purified protein were occupied with substrate, which had copurified with the protein. MALDI-TOF mass spectrometry analysis confirmed that the copurified substance was thiamin. Substrate-depleted ThiT was obtained by expressing the protein in cells that were cultivated in chemically defined growth medium without thiamin. The intrinsic tryptophan fluorescence of substrate-depleted ThiT was strongly quenched upon thiamin binding. The quenching of the fluorescence was used to determine dissociation constants for thiamin and related compounds. ThiT had an unusually high affinity for thiamin (K(D) = 122 +/- 13 pM) and bound the substrate with a 1:1 (protein:ligand) stoichiometry. TPP, TMP, and pyrithiamin bound to ThiT with nanomolar affinity. A multiple sequence alignment of ThiT homologues revealed that well-conserved residues were clustered in a tryptophan-rich stretch comprising the loop between the predicted membrane spanning segments 5 and 6. Mutational analysis of the conserved residues in this region combined with binding assays of thiamin and related compounds was used to build a model of the high-affinity binding site. The model was compared with thiamin binding sites of other proteins and interpreted in terms of the transport mechanism.

Structural and mechanistic insights into prokaryotic energy-coupling factor transporters

“…In addition, some of the free energy that is released by the hydrolysis of ATP could be used to lower the affinity of the S component for the substrate when the toppled state is reached, thereby enabling the release of the substrate. In complete ECF complexes, substrate affinity seems to be much lower than in solitary S components 9,11,24,26 , probably as a result of rearrangements in the substrate-binding site upon toppling 21 . A reduction in substrate affinity is likely to be important as it would enable the substrate to diffuse into the cytoplasm.…”

“…Alternatively, it is also possible that the solitary BioY proteins are nonspecifically assisted by a protein other than an ECF module to enable toppling, or that they form oligomers to facilitate toppling. Oligomerization of R. capsulatus BioY has been observed when it is heterologously expressed in E. coli cells, although this protein and other solitary S components are monomeric when purified 24,26,35,50 .…”

“…It is likely that this fold is shared by all S components (with the possible exception of the S components for Co 2+ and Ni 2+ ; BOX 1). A conserved functional feature of S components is their high affinity for substrates, with dissociation constants in the low to sub-nanomolar range 9,11,24,26 .…”

Challenges of ligand identification for riboswitch candidates

“…Furthermore, understanding the biological triggers for gene expression may also allow natural ligands for many of the transporters to be identified, similarly to the way thiamin was confirmed as the ligand for transporters regulated by TPP riboswitches. 19 Two additional features elevate interest in ykkC RNAs. First, several examples have been found in tandem with guanine riboswitches in various species of Firmicutes.…”

Dynamic Combinatorial Chemistry to Identify Binders of ThiT, an S‐Component of the Energy‐Coupling Factor Transporter for Thiamine

“…Up to date, there are only a few reports in which DCC has been applied to identify binders of transmembrane proteins 4, 5, 6. Here, we have applied DCC to identify possible binders for ThiT, the S‐component of the energy‐coupling factor (ECF) transporter for thiamine (Figure 1 A) in Lactococcus lactis 7, 8. ECF transporters represent an interesting target for the development of antibacterial agents with a novel mode of action by blocking vitamin transport 9…”