Structural and biophysical characterization of the tandem substrate-binding domains of the ABC importer GlnPQ

Abstract

The ATP-binding cassette transporter GlnPQ is an essential uptake system that transports glutamine, glutamic acid and asparagine in Gram-positive bacteria. It features two extra-cytoplasmic substrate-binding domains (SBDs) that are linked in tandem to the transmembrane domain of the transporter. The two SBDs differ in their ligand specificities, binding affinities and their distance to the transmembrane domain. Here, we elucidate the effects of the tandem arrangement of the domains on the biochemical, biophysical and structural properties of the protein. For this, we determined the crystal structure of the ligand-free tandem SBD1-2 protein from Lactococcus lactis in the absence of the transporter and compared the tandem to the isolated SBDs. We also used isothermal titration calorimetry to determine the ligand-binding affinity of the SBDs and single-molecule Förster resonance energy transfer (smFRET) to relate ligand binding to conformational changes in each of the domains of the tandem. We show that substrate binding and conformational changes are not notably affected by the presence of the adjoining domain in the wild-type protein, and changes only occur when the linker between the domains is shortened. In a proof-of-concept experiment, we combine smFRET with protein-induced fluorescence enhancement (PIFE–FRET) and show that a decrease in SBD linker length is observed as a linear increase in donor-brightness for SBD2 while we can still monitor the conformational states (open/closed) of SBD1. These results demonstrate the feasibility of PIFE–FRET to monitor protein–protein interactions and conformational states simultaneously.

Oxidation of hemoglobin in the lung parenchyma facilitates the differentiation of pneumococci into encapsulated bacteria

“…6A). 50 GlnPQ has been described as a glutamine, and/or glutamic acid transporter in pathogenic bacteria 51 .…”

Exploring the Ligand Binding and Conformational Dynamics of the Substrate-Binding Domain 1 of the ABC Transporter GlnPQ

“…25 Intriguingly, the formation of the SBD1−2 tandem has no significant effect on Asn/Gln binding to SBD1 in tandem (K D of 0.4 ± 0.1 and 180 ± 100 μM, respectively). 26 The solitary SBD2 binds glutamine with a K D of 0.9 ± 0.2 μM, 26 which is also similar to the one in tandem (K D = 0.6 ± 0.2 μM). 26 In addition, it has been shown through uptake experiments that glutamate is transported by both SBD1 and SBD2 domains.…”

“…It has been shown that substrate binding is linked to a conformational change of the corresponding SBD from an apo (ligand unbound) to a closed (ligand bound) holo form. 24,26,27,33,35 In the presence of the ligand, the closed state of the SBDs was observed more frequently, and there was no effect on the lifetime of the closed-liganded state; it was approximately equal to the ligand-free closed state. 26 The binding of substrate leads to the movement of the subdomains relative to each other, as shown in Figure 1B.…”

A new twist on PIFE: photoisomerisation-related fluorescence enhancement

“…More recently, a proof of principle experiment was presented [143], in which smFRET and PIFE were combined to simultaneously probe conformational changes within single protein domains during their interaction with neighbouring protein domains. As an example, the inter-and intra-domain interactions in the tandem substrate-binding domains (SBDs) 1 and 2 of the bacterial ABC import system GlnPQ were visualised (figures 8(D)-(F)).…”