Binding studies on DcuS-PD

DcuS senses a number of C4-dicarboxylates like fumarate (Kd 5 mM), succinate etc [122].

The isolated DcuS-PD was used for the binding studies of the effectors to the domain by NMR spectroscopy. During the addition of fumarate, sharpening of peaks were observed for some of the amino acids of the periplasmic domain in ¹⁵N-¹H HSQC spectra (Figure 4.10).

The affected residues cluster in a well defined region in the structure of the periplasmic domain of DcuS involving amino acid residues 107-168 (Figure 4.12 Left panel). This region in the structure correspond to the binding pocket of citrate in the periplasmic domain of CitA. There were no chemical shift changes observed during the titration. Sharpening of peaks were not observed when the protein sample contained a detergent (CHAPS) used for the titration with fumarate. This suggest that the detergent might have shielded the binding pocket of DcuS-PD.

Figure 4.10:Ratio of peak heights in ¹⁵N-¹H HSQC spectrum after the addition of 30-fold excess of fumarate to DcuS-PD. Titrations were carried out with addition of properly buffered sodium fumarate to a solution of protein at 1.2 mM. Residue that shows the maximum peak intensity is Q144

Figure 4.11: Plot of chemical shift changes in ¹⁵N-¹H HSQC when D-tartrate (50-fold excess) is added to periplasmic domain of DcuS (1.2 mM). The chemical shift plotted here is the sum of¹⁵N and¹H^N chemical shift, properly scaled using the method given in chapter 2, section 2.2.9. Residue that shows the maximum chemical shift change is G140.

Figure 4.12: (Right) Structure of DcuS periplasmic domain showing the residues most affected by fumarate titration. The residues, represented as sticks in the structure are R107, A113, I116, K121, A128, N134, A136, A143, Q144, A145, R147, F149 and T150. (Left) Structure of DcuS periplasmic domain showing the residues most affected by the tartrate titration. The residues shown as sticks on the structure are V89, K91, L96, F97, V100, H110, A113, Q114, Q118, K121, D124, N134, G140, A145, and T150. Common residues that are affected both by fumarate and tartrate titration are represented in green colour and the residues individually affected by the ligand titration are shown in blue sticks.

Figure 4.13: Electrostatic surface potential of DcuS-PD with positive and negative potentials coloured blue and red respectively . This reveals a positive charged surface region in the putative binding site of DcuS-PD. Residues of this region of the protein are mainly affected by the ligand titration.

D-tartrate is regarded as a non-physiological stimuli of DcuS [132], but is structurally closely related to other C4 di-carboxylates and binds to DcuS with a higher affinity than fumarate (apparent Kd is 0.5 mM [132]). Chemical shift changes were observed for a number of residues when DcuS-PD was titrated with D-tartrate (Figure 4.11). Most of these residues belong to the same region that was affected by fumarate binding. Such chemical shift changes were observed only for D-tartrate and not for L-tartrate. There was a set of common residues that were affected both by the fumarate and tartrate titration (Ala113, Lys121, Asn134, Ala145 and Thr150-refer Figure 4.12 : Residues coloured in green). Other residues were specifically affected by fumarate (Arg107, Ile116, Ala128, Ala136, Ala143, Gln144, Arg147 and Phe149-refer Figure 4.12: Residues coloured in red in the right panel) or D-tartrate addition (Val89, Lys91, Leu96, Phe97, Val100, His110, Gln114, Gln118, Asp124 and Gly140-refer Figure 4.12: Residues coloured in blue in the left panel). A comparison of the affected residues reveal that fumarate and tatrate bind to the same positively charged binding pocket in the DcuS-PD domain (Figure 4.13). In addition, some residues located outside this defined binding pocket are specifically affected by D-tartrate and more non-polar residues are affected by fumarate than D-tartrate, the latter containing two additional hydroxyl groups. Titration experiments were also performed with nitro-propionate (known to be sensed by DcuS at higher affinity than fumarate, Kd 0.4 mM [132]) as ligand, but unfortunately no visible effects were observed in the spectrum during the titration.

Alignment of the amino acid sequence of the periplasmic domain of DcuS from E.Coli with those from the citrate sensor CitA fromKlebsiella pneumoniae[127, 133] and other DcuS and CitA proteins revealed conserved residues in the members of the CitA/DcuS family (Figure 4.14). Most of the conserved residues are hydrophobic and might be structurally relevant. Charged residues are conserved in both types of proteins (D102, R107, H110 and R147).

Mutation of the charged residue like R147, H110 to a non polar residue like Alanine com-pletely repress dcuB’-’lacZ reporter gene fusion, indicating loss of effector binding. Another

set of mutation for amino acid residues that are specifically conserved in DcuS-type proteins (M103, F120, F149 and Q159) also impair DcuB’-’lacZ expression [132].

Figure 4.14: Comparison of the amino acid sequences of the periplasmic sensor domains of C4-dicarboxylate or citrate sensory histidine kinases. Amino acid residues conserved in both types of proteins are boxed; amino acids conserved in only one of the types of are boxed and shaded.

Amino acid residues of E. coli DcuS that were changed by site-directed mutagenesis are indi-cated by arrows. BS,Bacillus subtilis; BH,Bacillus halodurans, CG,Corynebacterium glutamicum;

CP,Clostridium perfringens; EC,Escherichia coli; KP,Klebsiella pneumoniae; OI,Oceanobacillus ihejensis; SC, Streptomyces coelicolor; SF, Shigella flexneri; SP, Streptococcus pyogenes; ST, Salmonella typhimurium; VC, Vibrio cholerae. This figure has been reproduced from [132].

The wild type periplasmic domain used in the study was well folded (See Figure 4.15 peaks in blue) and the residues R107, H110, and R147, found from the mutational studies to be essential for fumarate binding appear to be in close proximity in the structure, suggesting that the binding motif is retained in the periplasmic domain. In order to observe the influence of mutation on the effector binding on DcuS-PD, ¹⁵N-¹H HSQC spectra of the mutants were measured. ¹⁵N-¹H HSQC spectra of the mutants H110A and F120M showed very small

chemical shift dispersion in proton dimension which are characteristic of unfolded proteins ( Figure 4.15 panel B and C). For R147A, severe line broadening of resonances were observed denoting a high oligomerization state of the protein( Figure 4.15 panel D). Moreover R147A-PD was highly unstable and precipitated in the NMR tube very quickly. Unfolding and aggregation of these mutants indicate a loss of structure and stability in these mutants.

This study illustrates that these residues are very important for the fold of DcuS-PD. The decrease in the binding affinity of ligands in the biological assay could partly be due to these point mutations which are related to the loss of the three dimensional structure of mutated sensory domain.

4.2.5 Conclusion

Solution structure of the ligand free periplasmic domain of DcuS was determined. Structure reveals a PAS fold with the putative binding site of the ligand formed by the beta sheet and the inter strand loops. Titration experiments were carried out with dicarboxylates namely, fumarate and tartrate. The affected residues cluster around the putative binding pocket of DcuS-PD. Upon fumarate titration only sharpening of peaks were observed indicating a stabilization of the structure with the binding of fumarate. Upon titration with the non-physiological stimuli tartrate, small chemical shift changes were observed for a subset of peaks in the HSQC spectrum. The presence of two hydroxyl group in tartrate makes this C4 di-carboxylate to bind to DcuS-PD with much higher affinity. Because of the presence of two more charges, more polar residues are affected upon tartrate titration. However, even with tartrate, no major conformational changes could be observed in the C-terminus of the protein to indicate signal transduction.

Figure 4.15: (A) DcuS periplasmic domain with the residues that are mutated are indicated by sticks. The residues shown are H110, F120 and R147. (B) ¹⁵N-¹H HSQC of DcuS-PD mutant H110A. The protein was unfolded with small dispersion in chemical shift in the proton dimen-sion.(C) ¹⁵N-¹H HSQC of DcuS-PD mutant F120A. This protein is also unfolded with small dis-persion in chemical shift in the proton dimension. (D) Overlay of ¹⁵N-¹H HSQC of wild type DcuS periplasmic domain (blue) with R147A mutant of DcuS-PD (red). Severe line broadening is observed for the peaks of the mutant in the HSQC. Large number of peaks were missing when compared to peaks in the HSQC of the wild type DcuS-PD.

4.3 Periplasmic domain of the sensory domain of the