Using Single Molecule FRET to Understand Substrate Binding Domains

By Bryony Ackroyd

Twitter: @BryonyAckroyd

ABC transporters can be either import or export systems for cells. They consist of two transmembrane domains (TMDs) and two cytoplasmic nucleotide binding domains (NBDs). ABC importers also use substrate binding domains (SBDs) or substrate binding proteins (SBPs). SBPs are separate proteins present in the periplasm, however SBDs are fused to the TMDs. Some ABC transporters even have two or three SBDs fused together in tandem. Although this is a known phenomenon, very little is understood about the system and how ABC transporters are able to interact with multiple and structurally distinct SBDs. In the work carried out in this particular paper the group focusses on GlnPQ from L. lactis, a Gram-positive bacterium, that imports asapargine, glutamine and glutamate via two different SBDs.

Although there are crystal structures and NMR data available for SBDs, not much is known about the mechanism of ligand binding e.g. induced fit or conformational selection. Bearing this is mind Poolman et al., used a unique combination of techniques to probe the conformational dynamics of the SBDs, single-molecule Forster resonance energy transfer (smFRET) coupled with isothermal titration calorimetry (ITC). Using this strategy the group was able to provide mechanistic insight into the transport mechanisms of ABC importers, showing that the SBDs of GlnPQ bind ligands via an induced-fit mechanism.

The SBDs can be in one of four states, closed-ligand bound (CL), open (O), partially closed (PC) or closed (C). The induced-fit mechanism of binding triggers the CL state from the O state, however in the conformation-selection model the SBD can be in the PC or C states without a ligand bound. Ligand binding stabilises the PC state and therefore pushes the SBD to the CL conformation. These differing conformational states were examined via smFRET and the changes between states was observed via FRET efficiency. The experiment was designed so that the O conformation of the SBD gave a low FRET efficiency and the closed conformations gave higher FRET efficiency. Fluorophores were designed on the SBDs to be between 3-6 nm apart in both the closed and open states.

The SBD1 of GlnPQ binds asparagine with high affinity and glutamine with low affinity whereas SBD2 solely binds glutamine with a high affinity.

slide1
Single molecule dynamics of SBDs probed with smFRET. (a) Schematic showing immobilisation of histidine tagged SBDs to a PEG-biotin coated surface in a flow cell. The surface scan on the right is shown in flase colour, orange indicates double-labelled SBDs, green is SBDs with only donor fluorophore and red is SBDs with only acceptor fluorophores. (b-d) Representative fluorescence time traces, blue is donor signal, red acceptor signal, grey FRET signal and orange is the fit. These graphs show that the FRET efficiency of SBD1 and SBD2 increased as the concentration of substrate increased. This indicates that closing of SBD1 and SBD2 increased as substrate concentration increased, in keeping with the induced-fit model.

By measuring the fluorescence emitted from an SBD immobilised on a surface when varying concentrations of substrate were added, it was possible to determine the conformational state of the SBD and therefore whether the induced-fit or conformational-selection model was being employed by the SBD. Poolman et al., showed that in the absence of ligand the SBDs of GlnPQ were continuously in the O conformation and not in the PC or C conformations, therefore demonstrating the induced-fit model is used by the SBDs of GlnPQ.

This clever and unique technique was able to beautifully show the different conformations of the SBDs and conformational changes that occur within SBDs during ligand binding. Hopefully this technique will be employed more widely in the future to elucidate ligands, binding mechanisms and conformations of other SBDs and SBPs.

 

Source: Poolman et al., (2015). Conformational dynamics in substrate-binding domains influences transport in the ABC importer GlnPQ. Nature Structural and Molecular Biology 22, 57–64.

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