Structural Insights

Here we look at the structural characteristics of QscR, with particular focus in the cross-subunit architecture of the homodimer, protein-ligand and protein-DNA interactions.

AHL-bound QscR as Symmetric Homodimer

The N-terminal Ligand Binding Domain (LBD) forms an α-β-α sandwich which constitutes the binding pocket of AHL, while a canonical helix-turn-helix motif is seen in the DNA Binding Domain (DBD) (see Fig 1). QscR has a cross-subunit conformation whereby extensive dimerization interfaces are formed between LBD of one subunit with both LBD and DBD of the other, facilitating the AHL-bound QscR dimer to bind DNA through the dimerised DBDs. Site-directed mutagenesis confirmed that key residues involved in such interfaces, such as Glu84/Lys121 in LBD and Arg79/Arg42/Asn237 in DBD, were essential for QscR activity (see Fig 2,3).

Fig 1: PyMOL rendered QscR dimer (chains coloured in blue and purple). One molecule of 3OC12-HSL is bound in each LBD. The dimerised DBD forms a DNA binding site. The cross-subunit architecture can be seen from the 'criss-crossing' of one chain with respect to the other.

Fig 2a: LBD-LBD interaction between two chains of QscR. Notice how Gly84 from one chain forms a hydrogen bond (yellow dashed line) with Lys121 from the other chain. Tyr85 from both chains form van der Waals' interaction and is buried in the hydrophobic core. Carbon: grey, Oxygen: red, Nitrogen: dark blue. 

Fig 2b: LBD-DBD interaction between two chains of QscR. LBD-A (green) Arg79 and Arg42 form hydrogen bonds (yellow dashed line) with DBD-B (light blue) Asn237 residue. Numbers on hydrogen bonds represents distance between hydrogen donor and acceptor in Angstrom. Carbon: grey, Oxygen: red, Nitrogen: dark blue.

Fig 3: Activity assays of wild-type (WT) QscR  as positive control and 4 amino acid substitutions by site-directed mutagenesis in E. coli. Each assay was conducted at three different concentrations of AHL (10, 50 and 250 nM for 3OC12-, C12-, and 3OC14-HSLs and 10, 50, and 250 μM for 3OC6-HSL). All four substitutions showed distinct decrease in QscR activity in vivo at all concentrations. Bars show standard deviations. [Source: Lintz et al, 2012]

Acyl Chain of AHL in LBD of QscR

Ser56 residue in LBD interacts primarily with the 3-oxo group in AHL acyl chain through hydrogen bonding via 2 water molecules (see Fig 4). Research shows that S56G mutant has significant lower activity in presence of 3-oxo substituted AHLs than the wild type. In contrast, unsubstituted AHL such as C12-HSL retains about 50% of WT activity, indicating that Ser56 residue plays a role in 3-oxo substituted AHLs recognition selectivity. 

Fig 4: Ser56 hydroxyl side chain interact with 3-oxo group of AHL via hydrogen bonds through 2 water molecules.

Fig 5: Hydrogen bond interaction (yellow dashed line) between 3OC12-HSL (marked with pink dots) and key residues in AHL-binding site. Water molecules are shown as red spheres. Carbon: light blue, Oxygen: red, Nitrogen: dark blue. Numbers on hydrogen bonds represents distance between hydrogen donor and acceptor in Angstrom.

Hydrophobic and Van der Waals' interactions were the major stabilising forces that operate between the acyl chain of AHLs and residues at distal end of AHL-binding pocket which side chains point into the acyl-chain binding pocket. Substituting residues at this region with bulky side chains such as phenylalanine results in significant decreased interactions with C12 AHLs. Interestingly, 3OC6-HSL forms much favourable interactions with QscR G40F mutant at low ligand concentration than WT (see Fig 6). This suggests that through substituting bulky side chains, the reduced binding pocket space results not only in exclusion of larger C12-HSLs, but also creates a better fit for smaller ligand with shorter acyl chain such as 3OC6-HSL. 

Fig 6: G40F mutant interacts much more favourably with 3OC6-HSL (marked *) at low concentration (10 μM) than WT. Concentrations used are the same as Fig 2 above. Bars show standard deviations. [Source: Lintz et al, 2012]

Ligand Recognition

The acyl chain binding region is poorly conserved in the different LuxR family receptors. AHL conformation was found to be similar in QscR and LasR complexes i.e. acyl chain is located in a cavity close to the LBD-DBD dimer interface. However it appears to be completely different in TraR, SdiA and CviR in which the acyl chain of the ligand extends towards the solvent. These differences in binding pockets come down to the length of the acyl chain each receptor specifically binds.

Fig 7: Pymol derived structures of receptors (shown as cartoon) bound to their ligands (shown as spheres). Ligand conformation appears to be similar in QscR and LasR but different in CviR. In CviR the ligand is exposed to the solvent.

Ligand Binding Domain Dimerization Interfaces

LBDs of the different AHL receptors were structurally compared. QscR and LasR LBDs were found to be similar but very different from the LBDs of CviR and TraR. QscR and LasR dimers superimpose well, whereas when we try to superimpose one monomer of TraR or CviR on QscR, the other monomer is displaced and appears to be away from the second monomer of QscR. TraR second monomer undergoes a rotation and CviR monomer undergoes a rotation and a translation.

  

Fig 8: We can see the similarity in structure between QscR (cyan) and LasR (green). Also displacement of the second monomer of CviR (red) which undergoes a rotation and a translation and TraR (pink) which undergoes a rotation of 150°.

DNA Binding

Although TraR and QscR LBDs are very different, their DBDs appear to be very similar (rmsd value of 0.73Å). This similarity allowed the superposition of TraR and QscR DBDs to obtain a model for DNA binding by QscR. The effects of DNA binding were then assessed on the ligand bound receptor complex:

•         Secondary Structure: Studied using Circular Dichroism. In the presence of DNA, more α-helices appeared suggesting an increase in overall stability (Fig.9).

•         Thermal stability: Studied by recording the denaturation temperature, which increased upon DNA binding (Fig. 10).

•         Susceptibility to proteolytic cleavage: Assessed by Trypsin digest followed by SDS-PAGE. Trypsin cleaves the linker region between LBD and DBD. QscR exists primarily in the monomer form at nM concentrations, but achieves an equilibrium of monomer-dimer at 1-3 μM concentrations. Linker exposure is greater when QscR is in the monomer form and less when the receptor exists in a dimer or bound to DNA.

Base sequence in the DNA recognition site was compared between TraR and QscR but found to be mostly different. As a result, no further insight into the molecular basis of specific DNA recognition was achieved.

Fig 9: CD derived graph showing the increase in α-helical content in the DNA-bound form of the receptor (blue) rather than the unbound form (black). [Source: Lintz et al, 2012]

Fig 10: Graph showing the thermal stability of the protein complex in the presence and absence of DNA. We can see that when bound to DNA (blue) the receptor is more resistant to heat and denatures more slowly. [Source: Lintz et al, 2012]

SUMMARY

• QscR adopts a cross-subunit homodimer structure with extensive interaction between LBD of one chain and LBD/DBD of the other
• Ser56 in QscR LBD is involved in 3-oxo selectivitiy of ligand
• Acyl chain of AHL is buried within the LBD in LasR/QscR and exposed in other QscR homologues (TraR/SdiA/CviR)
• LBD-LBD interaction of QscR is very similar to that of LasR
• LBD-DBD interaction of QscR is very similar to that of TraR
• QscR adopts an active DNA-binding state in homodimer form upon ligand binding