Ligand Binding Study of Human PEBP1/RKIP: Interaction with Nucleotides and Raf-1 Peptides Evidenced by NMR and Mass Spectrometry

Background Human Phosphatidylethanolamine binding protein 1 (hPEBP1) also known as Raf kinase inhibitory protein (RKIP), affects various cellular processes, and is implicated in metastasis formation and Alzheimer's disease. Human PEBP1 has also been shown to inhibit the Raf/MEK/ERK pathway. Numerous reports concern various mammalian PEBP1 binding ligands. However, since PEBP1 proteins from many different species were investigated, drawing general conclusions regarding human PEBP1 binding properties is rather difficult. Moreover, the binding site of Raf-1 on hPEBP1 is still unknown. Methods/Findings In the present study, we investigated human PEBP1 by NMR to determine the binding site of four different ligands: GTP, FMN, and one Raf-1 peptide in tri-phosphorylated and non-phosphorylated forms. The study was carried out by NMR in near physiological conditions, allowing for the identification of the binding site and the determination of the affinity constants KD for different ligands. Native mass spectrometry was used as an alternative method for measuring KD values. Conclusions/Significance Our study demonstrates and/or confirms the binding of hPEBP1 to the four studied ligands. All of them bind to the same region centered on the conserved ligand-binding pocket of hPEBP1. Although the affinities for GTP and FMN decrease as pH, salt concentration and temperature increase from pH 6.5/NaCl 0 mM/20°C to pH 7.5/NaCl 100 mM/30°C, both ligands clearly do bind under conditions similar to what is found in cells regarding pH, salt concentration and temperature. In addition, our work confirms that residues in the vicinity of the pocket rather than those within the pocket seem to be required for interaction with Raf-1.METASU

A minimalistic approach to identify substrate binding features in B1 Metallo-beta-lactamases

The 2-oxoazetidinylacetate sodium salt was synthesized as a model of a minimal P-lactam drug. This compound and the monobactam aztreonam were assayed as substrates of the Metallo-p-lactamase Bell. None of them was hydrolyzed by the enzyme. While the azetidinone was not able to bind Bell, aztreonam was shown to bind in a nonproductive mode. These results provide an explanation for the unability of Metallo-beta-lactamases to inactive monobactams and give some clues for inhibitor design. (c) 2007 Elsevier Ltd. All rights reserved

K_D values of nucleotides derived from NMR and MS spectrometry.

<p>K_D values of nucleotides derived from NMR and MS spectrometry.</p

Binding of GTP to hPEBP1 at pH 6.5/20°C by NMR.

<p>(<b>A</b>) Overlay of ¹H, ¹⁵N HSQC spectra of hPEBP1 270 µM in the absence (black) and presence (red) of GTP 4 mM. (<b>B</b>) Expansion of the selected HSQC region. Overlay of six HSQC spectra of hPEBP1 270 µM with increasing concentration of GTP: 0 mM (black), 0.27 mM (green), 0.54 mM (orange), 1 mM (purple), 2 mM (blue), and 4 mM (red). (<b>C</b>) Plot of CSP versus GTP concentration; data fitted against equation of K_D (see M&M) for the 6 residues indicated on <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036187#pone-0036187-g003" target="_blank">Figure 3A</a>.</p

Binding of the tri-phosphorylated Raf-1 peptide to hPEBP1 by Mass Spectrometry.

<p>(<b>A</b>) ESI mass spectrum of hPEBP1 in complex with the tri-phosphorylated Raf-1 peptide, deconvoluted from 10+, 9+ and 8+ charge states. The complex was formed by incubating 18 µM hPEBP1 with 67.6 µM Raf-1 peptide at 20°C in 20 mM NH₄OAc, pH 6.6. (<b>B</b>) MS-measured hPEBP1 bound fraction as a function of the tri-phosphorylated Raf-1 peptide concentration.</p

Binding site of ligands at hPEBP1 surface at pH 6.5/20°C.

<p>Mapping of amino acid residues whose HSQC peak is significantly affected by (<b>A</b>) GTP, (<b>B</b>) FMN, (<b>C</b>) the tri-phosphorylated Raf-1 peptide, and (<b>D</b>) the non-phosphorylated Raf-1 peptide at the surface of hPEBP1 (X-Ray; PDB 2QYQ). Red = residues in slow exchange; orange = residues in intermediate exchange; yellow = residues in fast exchange. Prolines 74, 111 and 112, which belong to the hPEBP1 pocket but are not detected by HSQC spectrum, are indicated in green. Serine 153 is indicated in cyan as a reference point. (<b>E</b>) hPEBP1 sequence alignment (accession number P30086) indicating the residues defining the binding surface of GTP, FMN, the tri-phosphorylated Raf-1 peptide (3P. Raf-1 peptide), and the non-phosphorylated Raf-1 peptide (Raf-1 peptide). The color code is similar to (D).</p

Multiple sequence alignment of human PEBP1 (hPEBP1, accession number P30086), rat PEBP1 (rPEBP1, accession number P31044) and mouse PEBP2 (mPEBP2, accession number Q8VIN1).

<p>The hPEBP1 residues defining the binding surface of GTP at pH 6.5 and 20°C are colored yellow.</p

Structural basis for the broad-spectrum inhibition of metallo-beta-lactamases by thiols

The development of broad-spectrum metallo-beta-lactamase (MBL) inhibitors is challenging due to structural diversity and differences in metal utilisation by these enzymes. Analysis of structural data, followed by non-denturing mass spectrometric analyses, identified thiols proposed to inhibit representative MBLs from all three sub-classes: B1, B2 and B3. Solution analyses led to the identification of broad spectrum inhibitors, including potent inhibitors of the CphA MBL (Aeromonas hydrophila). Structural studies revealed that, as observed for other B1 and B3 MBLs, inhibition of the L1 MBL thiols involves metal chelation. Evidence is reported that this is not the case for inhibition of the CphA enzyme by some thiols; the crystal structure of the CphA-Zn-inhibitor complex reveals a binding mode in which the thiol does not interact with the zinc. The structural data enabled the design and the production of further more potent inhibitors. Overall the results suggest that the development of reasonably broad-spectrum MBL inhibitors should be possible