similar sets of cross peaks, indicating their similar modes of binding. Most of the HSQC peaks affected by these compounds were mapped to residues surrounding the pocket (Figure 3B), indicating that the pocket is a preferred binding site for RKIP ligands and RKIP does bind to small organic compounds that are chemically distinct from lipids (Figure 3B). The three compounds include either 2,4-dinitrophynyl amine or 2-bromo- 4-nitrophynyl amine moieties (Figure 3A), suggesting that the 4-nitrophenyl group serves as a common element recognized by RKIP. Among the screened compounds, there were a total of 19 others containing either 2,4-dinitrophynyl amine or 2-bromo-4- nitrophynyl amine moieties that exhibited no sign of RKIP binding, suggesting that the substituted nitrophenyl amine groups are not sufficient for RKIP binding. Newly identified compounds have no effect on RKIP-Raf-1 interaction and RKIP phosphorylation In our previous studies, we have shown that RKIP pocket occupancy by the short-chain lipid, DHPE, causes dissociation of RKIP from Raf-1 [21]. In contrast, we observed that locostatin, which also bound to the RKIP pocket, had no effect on RKIP- Raf-1 interaction [27]; we have attributed locostatin’s inability to perturb RKIP function to its smaller size than DHPE. The size of the three identified compounds is similar to that of locostatin and also had no effect on Raf-1 interaction (Figure 4A). Furthermore, we monitored RKIP phosphorylation by PKC in the presence of the three compounds and used DHPE as a control. In the presence of DHPE, as previously shown, RKIP phosphorylation was reduced in a dose dependent manner (Figure 4B). By contrast, as observed with locostatin, increasing concentrations of these compounds (up to 2.5 mM) had no effect on RKIP phosphory- lation by PKC (Figure 4B). Compound 48 impairs EGF-induced MAPK in HeLa cells We have previously shown that EGF-induced MAPK activity is augmented in HeLa cells depleted of RKIP [1]. If RKIP binding to Raf-1 requires pocket binding, then compounds that bind the RKIP pocket would interfere with downstream signaling to MAPK. However, we showed that locostatin binds to the RKIP pocket with no effect on Raf-1 association nor RKIP phosphor- ylation by PKC and did not change the EGF-induced MAPK activity in HeLa cells in the presence or absence of RKIP [27]. Similarly, two of the compounds (98 and 26) from our screen mimicked the lack of effect by locostatin on EGF-induced MAPK activity (Figure 5). In addition, we observed compound 48 at 20 mM had no effect on wild-type HeLa cells; however compound 48 reduced MAPK activity by 34611% (mean 6 SEM, n = 3) in RKIP depleted cells (Figure 5). To further examine the effect of compound 48, we compared the dose response of compound 26 as a control to that of compound 48 (Figure 6 and 7). As predicted, compound 26 at all concentrations tested had no effect on EGF- induced MAPK activity in HeLa cells in the presence or absence of RKIP (Figure 6). In contrast, the inhibitory effect was dose- dependent for compound 48 with respect to RKIP depleted cells but dose-independent in wild-type RKIP cells (compare Figure 7A with 7B). Compound 48 reduced cell viability in HeLa cells To examine the effect of these compounds on HeLa cells, we assayed cell viability as assessed by metabolic activity in the presence or absence of the compounds from the screen. All compounds examined with the exception of compound 48 had no effect on cell viability. In contrast, compound 48 reduced cell Identification of RKIP Ligands viability by 4366 and 3466% in wild-type and RKIP depleted cells respectively (mean 6 SEM; n = 4; Figure 8). In contrast to the viability assays, MAP kinase assays are assessed after shorter exposure to the drug which may account for the differential response to compound 48 between RKIP-expressing and RKIP- depleted cells observed in the MAP kinase assay. Taken together, these results suggest that compound 48 is toxic at 20 mM and may result in off target effects. Discussion Despite a number of investigations into the ligand binding properties of RKIP, there has remained a lack of detailed quantitative and structural analysis of ligand binding in solution. In this study, we used NMR spectroscopy to address a number of fundamental questions raised by the diversity of ligands reported to bind RKIP and to provide definitive quantitative structural data regarding the ligand specificity of RKIP. Through this effort we provide the first comprehensive structural analysis of ligand binding to the conserved pocket of RKIP in solution. We also found significant pH-dependent changes of HSQC peak intensity for residues surrounding the pocket. Although the loss in peak intensity precluded detailed analysis, these results are indicative of changes in the conformation and/or dynamic properties of the region at lower pH. We found evidence for weak binding of PE at pH 6.0. Likewise, the affinity of PA was higher at pH 6.0 relative to that at pH 7.4. However, lowered pH does not alter the binding of DHPE, DHPG and DHPS, strongly suggesting that the pocket is unaffected by pH change over this range. The difference in the binding affinity of PA between the two pH values can be explained by a change in the protonation state of the phosphate group of PA, suggesting that the microenvironment of the cell will determine the localized effect of ligand binding as well as protein-protein interaction. RKIP binds more strongly to the di-anionic form than the mono-anionic form of the phosphate group. It is, however, possible that a further decrease in pH, particularly in the range near the pKa of carboxylates (4–4.5) may induce substantial changes in the RKIP pocket structure and its ligand-binding properties. Also in this study, we observed that there are conserved His residues that are sensitive to pH which suggests that they may be involved in localized protein-protein interaction and are not involved in ligand binding. While prior structural information regarding binding of ligands to the conserved pocket of RKIP was limited to crystal structures that contained only small anions [17,19], this work significantly expands our knowledge of the chemical space for RKIP ligands and demonstrates a definitive specificity for phospholipids under near physiological conditions. The binding analysis of a drug-like compound, locostatin [30], its precursors and their stereoisomers revealed that the RKIP pocket can also bind to non-lipid compounds with a certain level of stereospecificity [27]. The RKIP ligands identified in this work all bound to the conserved pocket, and no other binding sites were found. These observations strongly suggest that the pocket is the main, if not the only, binding site in RKIP for organic compounds. These initial studies lead to screening for compounds that had a similar structure as the non-lipids described in Table S1. Essentially these were planar hydrophobic compounds that, unlike locostatin, were not capable of chemical cross-linking, which made them easily studied by NMR. These compounds bound to the pocket with an affinity similar to locostatin; however, their side chains allowed for binding to different regions within the RKIP binding pocket. PLoS ONE | www.plosone.org 6 May 2010 | Volume 5 | Issue 5 | e10479