Mechanism of ribosome shutdown by RsfS in Staphylococcus aureus revealed by integrative structural biology approach.

For the sake of energy preservation, bacteria, upon transition to stationary phase, tone down their protein synthesis. This process is favored by the reversible binding of small stress-induced proteins to the ribosome to prevent unnecessary translation. One example is the conserved bacterial ribosome silencing factor (RsfS) that binds to uL14 protein onto the large ribosomal subunit and prevents its association with the small subunit. Here we describe the binding mode of Staphylococcus aureus RsfS to the large ribosomal subunit and present a 3.2 Å resolution cryo-EM reconstruction of the 50S-RsfS complex together with the crystal structure of uL14-RsfS complex solved at 2.3 Å resolution. The understanding of the detailed landscape of RsfS-uL14 interactions within the ribosome shed light on the mechanism of ribosome shutdown in the human pathogen S. aureus and might deliver a novel target for pharmacological drug development and treatment of bacterial infections.

Exploring key parameters to detect subtle ligand-induced protein conformational changes using traveling wave ion mobility mass spectrometry.

Evidencing subtle conformational transitions in proteins occurring upon small modulator binding usually requires atomic resolution techniques (X-ray crystallography or NMR). Recently, hyphenation of ion mobility and mass spectrometry (IM-MS) has greatly enlarged the potentials for biomolecular assembly structural characterization. Using the well 3D-characterized Bcl-xL/ABT-737 protein model, we explored in the present report whether IM-MS can be used to differentiate close conformers and monitor collision cross section (CCS) differences correlating with ligand-induced conformational changes. Because comparing CCS derived from IM-MS data with 3D-computed CCS is critical for thorough data interpretation, discussing pitfalls related to protein construct similarity and missing sequence sections in PDB files was of primary importance to avoid misinterpretation. The methodic exploration of instrument parameters showed enhanced IM separation of Bcl-xL conformers by combining high wave heights and velocities with low helium and nitrogen flow rates while keeping a high He/N(2) flow rate ratio (>3). The robustness of CCS measurements was eventually improved with a modified IM calibration method providing constant CCS values regardless of instrument settings. Altogether, optimized IM-MS settings allowed a 0.4 nm(2) increase (i.e., 2%) of Bcl-xL CCS to be evidenced upon ABT-737 binding.

Characterization of novel checkpoint kinase 1 inhibitors by in vitro assays and in human cancer cells treated with topoisomerase inhibitors.

AIMS: We have developed biochemical and cell based assays to characterize small therapeutic molecules that inhibit the DNA damage checkpoint enzyme, Chk1 (Checkpoint kinase 1). MAIN METHODS: To prepare a screen of large chemical libraries, we purified the full-length and the catalytic domain versions of human Chk1. We characterized their properties and then selected full-length Chk1 as the variant most suitable for screening. We then identified and characterized structurally different Chk1 inhibitors in cell based-assays by measuring cytotoxicity and checkpoint bypass activity. KEY FINDINGS: We treated human cells with topoisomerase I inhibitors and demonstrated that at the time of Chk1 inhibitor addition, the cells have damaged DNA and activated Chk1. One Chk1 inhibitor, the indolocarbazole S27888, was active in the checkpoint bypass assay. SIGNIFICANCE: Knowing that the protein kinase inhibitory properties are different for each inhibitor, it seems that only a limited range of inhibitory activity is tolerated by cells. Chk1 has an essential role in determining how cancer cells respond to genotoxic treatments, therefore, inhibitors of this protein kinase are of great medical interest.

All-trans retinoic acid is a ligand for the orphan nuclear receptor ROR beta.

Retinoids regulate gene expression through binding to the nuclear retinoic acid receptors (RARs) and retinoid X receptors (RXRs). In contrast, no ligands for the retinoic acid receptor-related orphan receptors beta and gamma (ROR beta and gamma) have been identified, yet structural data and structure-function analyses indicate that ROR beta is a ligand-regulated nuclear receptor. Using nondenaturing mass spectrometry and scintillation proximity assays we found that all-trans retinoic acid (ATRA) and several retinoids bind to the ROR beta ligand-binding domain (LBD). The crystal structures of the complex with ATRA and with the synthetic analog ALRT 1550 reveal the binding modes of these ligands. ATRA and related retinoids inhibit ROR beta but not ROR alpha transcriptional activity suggesting that high-affinity, subtype-specific ligands could be designed for the identification of ROR beta target genes. Our results identify ROR beta as a retinoid-regulated nuclear receptor, providing a novel pathway for retinoid action.