Cysteine oxidation and disulfide formation in the ribosomal exit tunnel.

Funder: DFG graduate college: CLiC State of Hesse HMWK: BMRZUnderstanding the conformational sampling of translation-arrested ribosome nascent chain complexes is key to understand co-translational folding. Up to now, coupling of cysteine oxidation, disulfide bond formation and structure formation in nascent chains has remained elusive. Here, we investigate the eye-lens protein γB-crystallin in the ribosomal exit tunnel. Using mass spectrometry, theoretical simulations, dynamic nuclear polarization-enhanced solid-state nuclear magnetic resonance and cryo-electron microscopy, we show that thiol groups of cysteine residues undergo S-glutathionylation and S-nitrosylation and form non-native disulfide bonds. Thus, covalent modification chemistry occurs already prior to nascent chain release as the ribosome exit tunnel provides sufficient space even for disulfide bond formation which can guide protein folding

Essential protein P116 extracts cholesterol and other indispensable lipids for Mycoplasmas

Mycoplasma pneumoniae, responsible for approximately 30% of community-acquired human pneumonia, needs to extract lipids from the host environment for survival and proliferation. Here, we report a comprehensive structural and functional analysis of the previously uncharacterized protein P116 (MPN_213). Single-particle cryo-electron microscopy of P116 reveals a homodimer presenting a previously unseen fold, forming a huge hydrophobic cavity, which is fully accessible to solvent. Lipidomics analysis shows that P116 specifically extracts lipids such as phosphatidylcholine, sphingomyelin and cholesterol. Structures of different conformational states reveal the mechanism by which lipids are extracted. This finding immediately suggests a way to control Mycoplasma infection by interfering with lipid uptake.We thank L. Company and I. Fernández-Vidal for their support during MALS and mass spectroscopy measurements, A. Iborra (Servei de Cultius Cellulars, Anticossos Citometria, UAB) for his assistance with immunizing mice, D. Santos for his assistance in the radioactivity experiment and R. Pérez-Luque and D. Aparicio for their constant support and discussions. J. P. was funded by grants BIO2017-84166-R and PID2021-125632OB-C22 from the ministerio de Ciencia, Innovación y Universidades (MICINN, Spain). I. F. was funded by MICINN-Spain grant PID2021-125632OB-C21. A. S. F. was supported by the Deutsche Forschungsgemeinschaft (FR 1653/14-1 for MS and, FR 1653/6-3 for LS) and the Research Training Group iMOL (GRK 2566/1 for SM)

Isolation of a novel heterodimeric PSII complex via strep-tagged PsbO

The multi-subunit membrane protein complex photosystem II (PSII) catalyzes the light-driven oxidation of water and with this the initial step of photosynthetic electron transport in plants, algae, and cyanobacteria. Its biogenesis is coordinated by a network of auxiliary proteins that facilitate the stepwise assembly of individual subunits and cofactors, forming various intermediate complexes until fully functional mature PSII is present at the end of the process. In the current study, we purified PSII complexes from a mutant line of the thermophilic cyanobacterium Thermosynechococcus vestitus BP-1 in which the extrinsic subunit PsbO, characteristic for active PSII, was fused with an N-terminal Twin-Strep-tag. Three distinct PSII complexes were separated by ion- exchange chromatography after the initial affinity purification. Two complexes differ in their oligomeric state (monomeric and dimeric) but share the typical subunit composition of mature PSII. They are characterized by the very high oxygen evolving activity of approx. 6000 μmol O2⋅(mg Chl⋅h) 1. Analysis of the third (heterodimeric) PSII complex revealed lower oxygen evolving activity of approx. 3000 μmol O2⋅(mg Chl⋅h) 1 and a manganese content of 2.7 (±0.2) per reaction center compared to 3.7 (±0.2) of fully active PSII. Mass spectrometry and time-resolved fluorescence spectroscopy further indicated that PsbO is partially replaced by Psb27 in this PSII fraction, thus implying a role of this complex in PSII repair.</p

Structural insights into photosystem II assembly

International audienceBiogenesis of photosystem II (PSII), nature's water splitting catalyst, is assisted by auxiliary proteins that form transient complexes with PSII components to facilitate stepwise assembly events. Using cryo-electron microscopy, we solved the structure of such a PSII assembly intermediate from Thermosynechococcus elongatus at 2.94 Å resolution. It contains three assembly factors (Psb27, Psb28, Psb34) and provides detailed insights into their molecular function. Binding of Psb28 induces large conformational changes at the PSII acceptor side, which distort the binding pocket of the mobile quinone (QB) and replace the bicarbonate ligand of non-heme iron with glutamate, a structural motif found in reaction centers of non-oxygenic photosynthetic bacteria. These results reveal novel mechanisms that protect PSII from damage during biogenesis until water splitting is activated. Our structure further demonstrates how the PSII active site is prepared for the incorporation of the Mn4CaO5 cluster, which performs the unique water splitting reaction