Comparison of structures of DcuS-PD and CitAP

DcuS of E.Coli and CitA of Klebsilla Pneumonia are homologous proteins. DcuS senses a wide range of C4 dicarboxylates while CitA is highly specific for citrate. Their periplasmic domains have 25% sequence identity and 70% sequence homology. The structures of DcuS-PD and CitAP are similar and reveal a PAS fold. The backbone r.m.s.d for DcuS-DcuS-PD struc-ture with citrate bound and citrate free monomer A and B are 3.5 ˚A, 4.09 ˚A and 4.38

˚A respectively (see Figure 4.38 ). Except for the small beta strand (S2) in citrate bound CitAP, secondary structural elements of citrate bound CitAP and DcuS-PD are conserved.

In DcuS-PD, the N terminal helix is slightly bent inward with the beta sheets. Binding site for ligands in DcuS-PD and CitAP are conserved. As seen from the Figure 4.38, the binding site is more open in DcuS-PD than citrate bound structure of CitAP, but is less so from the citrate free CitAP. Consequently the β sheets are less bent in the DcuS-PD structure compared to citrate bound CitAP. The residues of DcuS-PD affected by fumarate binding are located in the 4 mainβ strands and in the major and minor loops. In CitAP the residues having direct hydrogen bonds with citrate are also located in the four β strands and the major and minor loops. The residue showing major peak intensity increase on binding of fumarate is Q144 which is in the minor loop region of DcuS-PD. The largest chemical shift

Figure 4.38: Comparison of the DcuS-PD structure with citrate-free monomer and citrate-bound monomer structures. A) Superposition of the DcuS-PD structure (green) with the citrate free monomer A structure of CitAP (cyan), r.m.s.d : 4.09 ˚A . B) Superposition of the DcuS-PD structure (green) with the citrate free monomer B structure of CitAP (pale cyan), r.m.s.d : 4.38 ˚A. C) Superposition of the DcuS-PD structure (green) with the citrate bound structure of CitAP (blue), r.m.s.d : 3.5 ˚A. Notable difference are in the minor loop and the N and C terminal regions. The N terminal helix of DcuS-PD is curved in with the beta sheets and helix at the C terminal region is well formed in the case of DcuS-PD. The structure of DcuS-PD resembles the citrate bound structure of CitAP, with the binding pocket slightly more open than the citrate bound structure of CitAP.

Figure 4.39: Citrate free CitAP is characterized by major line broadening due to conformational exchange. The unassigned residues are plotted on the structure of citrate free monomer A of CitAP(left). DcuS-PD also show line broadening. Residues of DcuS-PD showing very low peak intensity in the HSQC are plotted on the NMR structure of DcuS-PD (right).

change upon tartrate addition was observed for residue G140 which again is in the minor loop region in the structure of DcuS-PD. This is in accordance with the large conformational differences in the minor loop region between the citrate-free and citrate-bound CitAP X-ray structure.

Characteristic of citrate free CitAP in solution is the severe line broadening (missing peaks) in the ¹⁵N-¹H HSQC spectrum due to conformational exchange. These missing peaks are mostly from residues in the first three β strands and the part of the major loop region and helix H3 (residues from 64-111). Dcus-PD also showed chemical exchange broadening.

15N-¹H HSQC spectrum of DcuS-PD showed very small peak intensities for a number of residues. Het-NOE values of these residue were in the 0.7 to 0.8 range, very similar to rest of the residues in the sequence, suggesting a rigid structure in these parts of the protein.

Residues corresponding to the peaks showing small intensities are mapped onto the structure of DcuS-PD. This region in the structure of DcuS-PD is similar to the unassigned regions in citrate free CitAP (Figure 4.39) .

4.3.13 Conclusion

In this section, the NMR solution studies on the periplasmic domain of CitA (CitAP) with and without the ligand are presented. In the course of this study the X-ray crystallographic structures of CitAP with and without the ligand were determined. The description of these structures is also presented.

The X-ray structures reveal a PAS fold similar to DcuS-PD and other PAS domains. The citrate bound structure was determined without the non physiological molybdate which was present in the previously determined X-ray structure.

There were two conformers of CitAP (monomer A and monomer B) in the asymmetric unit of the citrate free CitAP crystal structure. A part of the major loop could not be traced in monomer A and the C terminal residues were missing in monomer B. This suggests that the citrate-free CitAP is flexible in the C terminal and also in some parts the major loop region.

But fortunately specific conformational changes were observed between the structures of the citrate-free and -bound form. Based on this, a possible mechanism for signal transduction is discussed.

By NMR titration it was shown that in solution molybdate might induce a different struc-tural response than with only citrate bound to CitAP. Molybdate also induces aggregation of the protein. The newly determined bound structure did indeed differ in the dimer inter-face and slight change in the binding of citrate to CitAP from previously published X-ray structure. Previous studies suggest that CitAP might also sense sodium ion. A sodium ion was tentatively localized in the citrate bound structure of CitAP. With NMR titrations, this is found to be true in solution as well.

NMR backbone assignments for 90% of the peaks seen in the ¹⁵N-¹H HSQC were obtained for both the bound and free form of CitA. The NMR solution structures of CitAP could not be determined because of the large number of missing peaks in NMR spectra due to chem-ical/conformational exchange broadening. Nevertheless, with the available assignments, the chemical shift difference and the secondary structural propensity difference between the free and the bound forms of the CitAP protein were determined. These values indicate significant difference in chemical shifts and secondary structural propensities in the C-terminal regions

of the protein. The model proposed here for signal transduction is based on the structures of the citrate free and bound CitAP. This is also consistent with the present NMR data and the previous knowledge of signal transduction mechanisms.

Correlation co-efficients from residual dipolar coupling analysis of citrate free CitAP suggest that monomer A might be relatively more closer to the solution structure than monomer B.

The correlation coefficient of RDCs of citrate free CitAP fits even better with the citrate bound structure of CitAP. RDCs of citrate-free CitAP therefore have a major contribution from the citrate-bound CitAP structure (RDC analysis indicate that the population of cit-rate bound structure would be 80% and that of the monomer A of citcit-rate free form to be 20%). This suggest that the binding pocket of citrate is already formed in the citrate free solution of CitAP. CitAP might require the two transmembrane helices to keep the N- and C-terminal parts of the protein together, and without the transmembrane part the protein is not confined to one conformational space. Smaller Het-NOE values in the N- and C-terminal regions indicate greater flexibility in these regions of the protein, hence providing greater freedom for the protein to sample different conformational space.

.