phosphatidylethanolamine binding protein. Subsequent studies demonstrated that RKIP binds only negatively charged phospha- tidylglycerol in liposomes and lipid monolayers [12]. The macroscopic methods used in these previous studies did not identify the specific binding site(s) for these interactions nor determine site-specific affinity of these ligands to RKIP. Also the effective concentrations of ligands in these methods are very high, and consequently they can detect weak binding events that may not be physiologically relevant. Amino acid sequence analysis of RKIP has identified it as a member of a large protein family named the PEBP family. This family has homologs present from bacteria to mammals [13,14,15,16]. Among the mammalian members of this family, the pocket region between residues 63 and 126 (based on the bovine RKIP numbering scheme [17]) displays a particularly high level of sequence identity [18], suggesting a conserved function for this region. The structures of RKIP and its homologs from a number of species have been determined using x-ray crystallog- raphy [17,19,20,21,22,23]. These crystal structures reveal a globular protein with a beta-sheet core and a prominent solvent exposed pocket. The residues that form the pocket are among the aforementioned highly conserved residues. In three of the five x- ray structures of mammalian RKIP proteins a small ionic ligand is bound in this pocket [17,19], confirming this pocket as a ligand- binding site. While two of these three structures (Protein Data Bank entries 1A44 and 1BEH) contain a small ion from the buffer as the ligand, the third (PDB id 1B7A) contains O-phosphor- ylethanolamine (PE), the soluble polar head group of the membrane phospholipid phosphatidylethanolamine. The RKIP- PE complex is the only crystal-based structural evidence to date for the binding of an organic ion to the pocket. Although PE is a phospholipid head group and the crystal structure may represent the mode of phospholipid–RKIP interaction, this interpretation is still speculative because PE lacks the diacylglycerol moiety that constitutes the majority of the phospholipids. Moreover, the crystals of RKIP with PE bound were formed at pH 4.6 in the presence of 100 mM PE [17], and the other crystal structures of mammalian RKIP with ligands bound to the pocket were also determined at low pH (# pH 5.5). Thus, little is known about the mode(s) of RKIP-ligand interactions under near physiological solution conditions. Although the previous studies have reported a number of RKIP ligands, they collectively lack definitive data regarding binding affinity, the location of the binding site(s) as well as the ligand specificity of RKIP in solution under near physiological condi- tions. The chemical diversity of the previously identified ligands raises fundamental questions about the structural basis for RKIP– ligand interactions: Does the pocket have an extremely broad binding specificity so that it can bind to all of these ligands? If so, what is the chemical space for the pocket ligand? Or alternatively, does RKIP have another binding site distinct from the pocket? To address these questions it is necessary to investigate ligand binding by a method that permits quantitative and structural analysis of protein-ligand interactions under near physiological solution conditions. Solution NMR spectroscopy meets the above criteria for high- resolution and quantitative analysis of protein-ligand interactions. Ligand binding experiments using NMR spectroscopy require relatively low protein concentrations and thus allow for flexibility in the choice of solution conditions. In addition, it has the ability to detect weak interactions and to map specific protein residues affected by ligand binding. These attributes make NMR spectroscopy an ideal tool to perform a comprehensive analysis of the ligand binding properties of RKIP, which was not possible Identification of RKIP Ligands with the previously implemented techniques. Furthermore, it can also determine the site-specific affinity of the interactions [24] providing a significant advantage over the methods previously used to study RKIP-ligand interactions. We have reported the sequence specific resonance assignments of RKIP at pH 7.4 [25], the prerequisite for NMR-based structural analysis [26], providing the foundation for detailed characterization of RKIP-ligand interactions by NMR spectroscopy. Recently, we characterized the binding of locostatin, a putative RKIP inhibitor, to the RKIP pocket [27]. In this study, we used NMR-based screening to assay interactions of RKIP with a number of potential ligands by NMR spectroscopy. Surprisingly, the binding of many com- pounds previously postulated or reported to interact with RKIP was not detectable at near physiological solution conditions even at millimolar concentrations of the compounds. In contrast, we discover three novel ligands for RKIP, and we provide evidence for site-specific binding of these compounds to the pocket and their binding affinity. In addition, we examined the biological effect of these compounds and identified one that had off target effects which reduced MAPK activity. Taken together, this work defines the ligand-binding properties of RKIP and suggest its potential role as a scavenger for hydrophobic compounds within cells. Results NMR assay of ligand binding to RKIP To investigate whether potential non-lipid ligands reported in the literature actually bind to RKIP and, if so, to define their binding site, we tested the interactions between RKIP and these compounds using NMR spectroscopy [21,25]. By comparing the 1H,15N-heteronuclear single-quantum correlation (HSQC) spectra of 15N-labeled rat RKIP (rRKIP) in the presence and absence of a potential ligand, we can detect ligand binding as perturbations to the NMR cross peaks (referred to as ‘‘chemical shift perturbations’’ hereafter) [24]. Note that only the backbone and side chain amide moieties of the 15N-rRKIP give rise to NMR signals while an unlabeled ligand is spectroscopically silent. The recently complet- ed sequence-specific NMR assignments for rRKIP [25] allow us to identify the residues affected by ligand binding and thus to map the binding site. Non-lipid molecules assayed for binding included: O-phosphor- ylethanolamine (PE), the head group of phosphatidylethanol- amine; the nucleotides GDP, GTP and cAMP; the phosphorylated amino acids O-phosphorylserine, O-phosphorylthreonine and O- phosphoryltyrosine; tryptophan, phenylalanine and inorganic phosphate (Pi) (Table S1). Surprisingly, under the near physio- logical solution conditions used (pH 7.4, 100 mM NaCl, 30uC), none of these non-lipid compounds caused significant chemical shift perturbations for the rRKIP residues, even at very high ligand concentrations used (Table S1 and Figure S1). This suggests the importance of examining ligand binding to RKIP in solution under near-physiological conditions. In contrast, a previously identified RKIP ligand, DHPE, caused large perturbations (Figure 1D). Interestingly, although PE had previously been co- crystallized with bovine RKIP at low pH [17], it did not interact with the protein under these more physiological conditions (Figure 1A and C). Together, these results indicate that rRKIP has very weak affinity, if any, to these compounds. Effects of pH on RKIP structure An apparent conundrum in this study is the lack of binding of PE to rat RKIP in solution despite the successful crystallization of PLoS ONE | www.plosone.org 2 May 2010 | Volume 5 | Issue 5 | e10479