As pH was titrated below pH 6.0, a large number of peaks exhibited a dramatic loss in peak intensity (Figure 1B), characteristic of an increase in motion on the ms-ms timescale [26]. These peaks were mapped to residues in the loops that form the binding pocket. Because of the peak intensity decrease, we could not accurately determine the pKa values for these residues, which prevented us from identifying the origin of the pH-induced structural perturbations in the pocket region. Nevertheless, these results clearly indicate that the pocket conformation and/or dynamics are sensitive to pH change and that these loop regions are able to sample multiple conformations while rRKIP retains the overall, folded conformation. There were other HSQC peaks that exhibited significant pH- dependent migration but no significant decrease in intensity. We were able to determine the pKa value for 46 residues of this class. We did not find a clear clustering pattern in the distribution of residues with similar pKa values, except for two discrete sites distal to the conserved pocket corresponding to regions adjacent to solvent exposed histidines at positions 23 and 159 (data not shown). Moreover, the residues near His-23 and -159 do not exhibit ligand-induced chemical shift perturbations at pH 7.4, suggesting that these sites and the pocket are structurally independent and pH-dependent structural changes at these sites do not affect ligand binding to the conserved pocket. Effects of pH on ligand binding by RKIP The above results suggest that an increased flexibility at lower pH might alter the ligand-binding properties of RKIP. To investigate the effect of lower pH on ligand binding, a number of ligands tested at pH 7.4 were reexamined for binding to RKIP at pH 6.0. This pH value was chosen because below this pH value we observed a significant loss of spectral quality. In the presence of PE and O-phosphoryltyrosine at pH 6.0, a number of residues in the loops surrounding the ligand pocket of RKIP displayed significant broadening of amide proton peaks in the HSQC spectra. However, chemical shift perturbations for pocket residues were very small, only slightly above background in these spectra (Figure 1E), much smaller than those observed for phospholipid compounds (Figure 1D). These results suggest that PE and O- phosphoryltyrosine have very low affinity for RKIP at this pH. The phosphate group of PE has a reported pKa of ,5.8, Identification of RKIP Ligands suggesting that the affinity of PE may be much higher at lower pH than at 6.0. However, due to the limitations discussed above, we were not able to investigate this possibility. Our data suggests that the presence of PE in the crystal structure of bovine RKIP (PDB id: 1b7a) may be a product of the weak affinity of PE at low pH coupled with its high concentrations used during crystallization. Two of the four compounds exhibiting binding at pH 7.4, DHPG and DHPS, demonstrated similar patterns of chemical shift perturbations at pH 6.0 as previously described [21]. Titration of the phospholipids at pH 6.0 confirmed interactions with similar affinity for DHPG and DHPS to that observed at pH 7.4 (Table S1). In contrast, while PA binding at pH 6.0 perturbed a similar subset of residues as it did at pH 7.4, the trajectory of the peak shifts was different. As PA was titrated in, the HSQC peaks did not change their positions but their intensities decreased, and a new set of peaks appeared (Figure 2A). This behavior is characteristic of slow interconversion between the free and ligand-bound states [26]. Furthermore, the affinity of PA for RKIP was at least 3-fold higher at pH 6.0 than at pH 7.4 (Figure 2B). The mono-substituted phosphate moiety of PA has a pKa of 8.0 [29] indicating that its protonation state is sensitive to changes in pH between pH 7.4 and pH 6.0. In comparison, the pKa values of the other phospholipids examined in this study are expected to be much lower, as the pKa of the phosphate group of dimyristoyl-phosphatidylethanolamine has been calculated to be ,1.7 [29]. The remarkable similarities between the peak shift patterns observed for DHPE, DHPG and DHPS at pH 6.0 and pH 7.4 are in stark contrast with the differences seen for PA binding at the two pH values, suggesting that the higher affinity for PA at pH 6.0 results from the protonation of the ligand and not from a pH-dependent effect on the RKIP binding pocket itself. Together, these results suggest that the compounds that showed no significant binding in our NMR assays (Table S1) have very weak affinity at best to RKIP in near physiological pH range. NMR-based chemical library screen reveals three novel RKIP ligands The failure of previously reported compounds, except for the short-chain lipid, to bind RKIP at physiological pH has revised our understanding of RKIP’s ligand binding properties. RKIP is Figure 2. A portion of RKIP HSQC spectra illustrating the difference in the DHPA-induced chemical shift perturbations of residue 182 at pH 7.4 and 6.0. (A) DHPA titration at pH 7.4 causes the peak to continuously shift, characteristic of fast exchange kinetics. (B) Titration at pH 6.0 causes the peak intensity to decrease with a concomitant appearance of a new peak, characteristic of slow exchange kinetics. The arrows indicate the trajectories of cross peak movements. Panel (A) shows data with DHPA concentrations of 0, 0.05 0.25, 0.5, 1.0, 2.5 and 7.5 mM, and (B) data with 0, 0.05, 0.25, 0.5, 1.0, 2.5 and 4.5 mM DHPA. doi:10.1371/journal.pone.0010479.g002 PLoS ONE | www.plosone.org 4 May 2010 | Volume 5 | Issue 5 | e10479