Physical basis of the inducer-dependent cooperativity of the Central glycolytic genes Repressor/DNA complex

The Central glycolytic genes Repressor (CggR) from Bacillus subtilis belongs to the SorC family of transcription factors that control major carbohydrate metabolic pathways. Recent studies have shown that CggR binds as a tetramer to its tandem operator DNA sequences and that the inducer metabolite, fructose 1,6-bisphosphate (FBP), reduces the binding cooperativity of the CggR/DNA complex. Here, we have determined the effect of FBP on the size, shape and stoichiometry of CggR complexes with full-length and half-site operator sequence by small-angle X-ray scattering, size-exclusion chromatography, fluorescence cross-correlation spectroscopy and noncovalent mass spectrometry (MS). Our results show that CggR forms a compact tetrameric assembly upon binding to either the full-length operator or two half-site DNAs and that FBP triggers a tetramer–dimer transition that leaves a single dimer on the half-site or two physically independent dimers on the full-length target. Although the binding of other phospho-sugars was evidenced by MS, only FBP was found to completely disrupt dimer–dimer contacts. We conclude that inducer-dependent dimer–dimer bridging interactions constitute the physical basis for CggR cooperative binding to DNA and the underlying repression mechanism. This work provides experimental evidences for a cooperativity-based regulation model that should apply to other SorC family members

Studying protein–protein affinity and immobilized ligand–protein affinity interactions using MS-based methods

This review discusses the most important current methods employing mass spectrometry (MS) analysis for the study of protein affinity interactions. The methods are discussed in depth with particular reference to MS-based approaches for analyzing protein–protein and protein–immobilized ligand interactions, analyzed either directly or indirectly. First, we introduce MS methods for the study of intact protein complexes in the gas phase. Next, pull-down methods for affinity-based analysis of protein–protein and protein–immobilized ligand interactions are discussed. Presently, this field of research is often called interactomics or interaction proteomics. A slightly different approach that will be discussed, chemical proteomics, allows one to analyze selectivity profiles of ligands for multiple drug targets and off-targets. Additionally, of particular interest is the use of surface plasmon resonance technologies coupled with MS for the study of protein interactions. The review addresses the principle of each of the methods with a focus on recent developments and the applicability to lead compound generation in drug discovery as well as the elucidation of protein interactions involved in cellular processes. The review focuses on the analysis of bioaffinity interactions of proteins with other proteins and with ligands, where the proteins are considered as the bioactives analyzed by MS

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

Structural studies of metal ligand complexes by ion mobility-mass spectrometry

The final publication is available at Springer via http://dx.doi.org/10.1007/s12127-013-0122-8Collision cross sections (CCS) have been measured for three salen ligands, and their complexes with copper and zinc using travelling-wave ion mobility-mass spectrometry (TWIMS) and drift tube ion mobility-mass spectrometry (DTIMS), allowing a comparative size evaluation of the ligands and complexes. CCS measurements using TWIMS were determined using peptide and TAAH calibration standards. TWIMS measurements gave significantly larger CCS than DTIMS in helium, by 9 % for TAAH standards and 3 % for peptide standards, indicating that the choice of calibration standards is important in ensuring the accuracy of TWIMS-derived CCS measurements. Repeatability data for TWIMS was obtained for inter- and intra-day studies with mean RSDs of 1. 1 % and 0. 7 %, respectively. The CCS data obtained from IM-MS measurements are compared to CCS values obtained via the projection approximation, the exact hard spheres method and the trajectory method from X-ray coordinates and modelled structures using density functional theory (DFT) based methods. © 2013 Springer-Verlag Berlin Heidelberg

Physical basis of the inducer-dependent cooperativity of the Central glycolytic genes Repressor/DNA complex.

The Central glycolytic genes Repressor (CggR) from Bacillus subtilis belongs to the SorC family of transcription factors that control major carbohydrate metabolic pathways. Recent studies have shown that CggR binds as a tetramer to its tandem operator DNA sequences and that the inducer metabolite, fructose 1,6-bisphosphate (FBP), reduces the binding cooperativity of the CggR/DNA complex. Here, we have determined the effect of FBP on the size, shape and stoichiometry of CggR complexes with full-length and half-site operator sequence by small-angle X-ray scattering, size-exclusion chromatography, fluorescence cross-correlation spectroscopy and noncovalent mass spectrometry (MS). Our results show that CggR forms a compact tetrameric assembly upon binding to either the full-length operator or two half-site DNAs and that FBP triggers a tetramer-dimer transition that leaves a single dimer on the half-site or two physically independent dimers on the full-length target. Although the binding of other phospho-sugars was evidenced by MS, only FBP was found to completely disrupt dimer-dimer contacts. We conclude that inducer-dependent dimer-dimer bridging interactions constitute the physical basis for CggR cooperative binding to DNA and the underlying repression mechanism. This work provides experimental evidences for a cooperativity-based regulation model that should apply to other SorC family members

Analysis of ADCs by Native Mass Spectrometry

International audienceMass spectrometry performed in nondenaturing conditions (native MS) has proven its utility for the quantitative and qualitative analysis of antibody-drug conjugates (ADCs), especially when ADCs’ subunits involve noncovalent interactions (i.e., cysteine-conjugated ADCs). Its hyphenation to ion mobility spectrometry (IM-MS) allows differentiation of gas-phase ions based on their rotationally averaged collision cross section providing an additional dimension of conformational characterization of ADCs. More recently, size exclusion chromatography (SEC) appeared as an interesting technique to perform online buffer exchange in an automated way prior to native MS/IM-MS analysis. Online SEC-native MS/IM-MS allows the global structural characterization of ADCs and the assessment of some critical quality attributes (CQAs) required for ADC release on the market, such as drug load distribution (DLD), drug-to-antibody ratio (DAR), the average DAR (DARav), and the relative amount of unconjugated mAb