Solution structures of the Bacillus cereus metallo-β-lactamase BcII and its complex with the broad spectrum inhibitor R-thiomandelic acid

Metallo-β-lactamases, enzymes which inactivate β-lactam antibiotics, are of increasing biological and clinical significance as a source of antibiotic resistance in pathogenic bacteria. In the present study we describe the high-resolution solution NMR structures of the Bacillus cereus metallo-β-lactamase BcII and of its complex with R-thiomandelic acid, a broad-spectrum inhibitor of metallo-β-lactamases. This is the first reported solution structure of any metallo-β-lactamase. There are differences between the solution structure of the free enzyme and previously reported crystal structures in the loops flanking the active site, which are important for substrate and inhibitor binding and catalysis. The binding of R-thiomandelic acid and the roles of active-site residues are defined in detail. Changes in the enzyme structure upon inhibitor binding clarify the role of the mobile β3–β4 loop. Comparisons with other metallo-β-lactamases highlight the roles of individual amino-acid residues in the active site and the β3–β4 loop in inhibitor binding and provide information on the basis of structure–activity relationships among metallo-β-lactamase inhibitors

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

Solution structures of the Bacillus cereus metallo-β-lactamase BcII and its complex with the broad spectrum inhibitor R-thiomandelic acid

Metallo-β-lactamases, enzymes which inactivate β-lactam antibiotics, are of increasing biological and clinical significance as a source of antibiotic resistance in pathogenic bacteria. We describe the high resolution solution NMR structures of the Bacillus cereus metallo-β-lactamase, BcII, and of its complex with R-thiomandelic acid, a broad spectrum inhibitor of metallo-β-lactamases. This is the first reported solution structure of any metallo-β-lactamase. There are differences between the solution structure of the free enzyme and previously reported crystal structures in the loops flanking the active site, which are important for substrate and inhibitor binding and catalysis. The binding of R-thiomandelic acid and the roles of active site residues are defined in detail. Changes in the enzyme structure upon inhibitor binding clarify the role of the mobile β3-β4 loop. Comparisons with other metallo-β-lactamases highlight the roles of individual amino-acid residues in the active site and the β3-β4 loop in inhibitor binding and provide information on the basis of structure-activity relationships among metallo-β-lactamase inhibitors

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

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

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

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

An alternative flexible conformation of the E. coli HUbeta(2) protein: structural, dynamics, and functional aspects.

The histone-like HU protein is the major nucleoid-associated protein involved in the dynamics and structure of the bacterial chromosome. Under physiological conditions, the three possible dimeric forms of the E. coli HU protein (EcHUalpha(2), EcHUbeta(2), and EcHUalphabeta) are in thermal equilibrium between two dimeric conformations (N(2) I(2)) varying in their secondary structure content. High-temperature molecular dynamics simulations combined with NMR experiments provide information about structural and dynamics features at the atomic level for the N(2) to I(2) thermal transition of the EcHUbeta(2) homodimer. On the basis of these data, a realistic 3D model is proposed for the major I(2) conformation of EcHUbeta(2). This model is in agreement with previous experimental data