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Cas13a are single-molecule effectors of the Class II, Type VI family of CRISPR-Cas systems that are part of the bacterial and archaeal defense systems. These RNA-guided and RNA-activated RNA endonucleases are characterized by their ability to cleave target RNAs complementary to the crRNA-spacer sequence, as well as bystander RNAs in a sequence-unspecific manner. Due to cleavage of cellular transcripts they induce dormancy in the host cell and thus protect the bacterial population by aborting the infectious cycle of RNA-phages. Here we report the structural and functional characterization of a Cas13a enzyme from the photo-auxotrophic purple bacteria Rhodobacter capsulatus. The X-ray crystal structure of the RcCas13a-crRNA complex reveals its distinct crRNA recognition mode as well as the enzyme in its contracted, pre-activation conformation. Using site-directed mutagenesis in combination with mass spectrometry, we identified key residues responsible for pre-crRNA processing by RcCas13a in its distinct catalytic site, and elucidated the acid-base mediated cleavage reaction mechanism. In addition, RcCas13a cleaves target-RNA as well as bystander-RNAs in Escherichia coli which requires its catalytic active HEPN (higher eukaryotes and prokaryotes nucleotide binding) domain nuclease activity. Our data provide further insights into the molecular mechanisms and function of this intriguing family of RNA-dependent RNA endonucleases that are already employed as efficient tools for RNA detection and regulation of gene expression., (© 2022. The Author(s).)

Genetically encoded and ribosomally synthesised peptides and small proteins act as important regulators in fundamental cellular processes, including gene expression, development, signalling and metabolism. Moreover, they also play a crucial role in eukaryotic and prokaryotic defence against microorganisms. Extremely diverse in size and structure, they are often subject to extensive post-translational modification. Recent technological advances are now allowing the analysis of the whole cellular transcriptome and proteome, revealing the presence of hundreds of long-overlooked alternative and short open reading frames (short ORFs, or sORFs) in mRNA and supposedly noncoding RNAs. However, in many instances the biological roles of their translational products remain to be elucidated. Here we provide an overview on the intriguing structural and functional diversity of ribosomally synthesised peptides and newly discovered peptides and small proteins., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Caseinolytic protease P (ClpP) is the proteolytic component of the ClpXP protein degradation complex. Eukaryotic ClpP was recently found to act within the mitochondria-specific unfolded protein response (UPR mt ). However, its detailed function and dedicated regulation remain largely unexplored. A small molecule (D9) acts as a potent and species-selective activator of human ClpP (hClpP) by mimicking the natural chaperone ClpX. Structure-activity relationship studies highlight the importance of a halogenated benzyl motif within D9 that interacts with a unique aromatic amino acid network in hClpP. Mutational and structural studies suggest that this YYW motif tightly controls hClpP activity and regulates substrate turnover by interaction with cognate ligands. This signature motif is unique to ClpP from higher organisms and does not exist in tested bacterial homologues, allowing a species-selective analysis. Thus, D9 is a versatile tool to analyze mechanistic features of hClpP., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Inteins carry out protein-splicing reactions, which are used in protein chemistry, protein engineering and biotechnological applications. Rearrangement of the order of the domains in split-inteins results in head-to-tail cyclisation of the target sequence, which can be used for genetic encoding and expression of libraries of cyclic peptides (CPs). The efficiency of the splicing reaction depends on the target sequence. Here we used mass spectrometry to assess in vivo cyclic peptide formation from different hexameric target sequences by the DnaE split-inteins from Synechocystis sp. and Nostoc punctiforme, revealing a strong impact of the target sequence and of the intein on the intracellular peptide concentration. Furthermore, we determined the crystal structures of their pre-splicing complexes, which allowed us to identify F-block Asp17 as crucial for the DnaE-mediated splicing reaction., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Rhodopsin is a membrane protein from the G protein-coupled receptor family. Together with its ligand retinal, it forms the visual pigment responsible for night vision. In order to perform ultrafast dynamics studies, a time-resolved serial femtosecond crystallography method is required owing to the nonreversible activation of rhodopsin. In such an approach, microcrystals in suspension are delivered into the X-ray pulses of an X-ray free-electron laser (XFEL) after a precise photoactivation delay. Here, a millilitre batch production of high-density microcrystals was developed by four methodical conversion steps starting from known vapour-diffusion crystallization protocols: (i) screening the low-salt crystallization conditions preferred for serial crystallography by vapour diffusion, (ii) optimization of batch crystallization, (iii) testing the crystal size and quality using second-harmonic generation (SHG) imaging and X-ray powder diffraction and (iv) production of millilitres of rhodopsin crystal suspension in batches for serial crystallography tests; these crystals diffracted at an XFEL at the Linac Coherent Light Source using a liquid-jet setup.

Structural information of the different conformational states of the two prototypical light-sensitive membrane proteins, bacteriorhodopsin and rhodopsin, has been obtained in the past by X-ray cryo-crystallography and cryo-electron microscopy. However, these methods do not allow for the structure determination of most intermediate conformations. Recently, the potential of X-Ray Free Electron Lasers (X-FELs) for tracking the dynamics of light-triggered processes by pump-probe serial femtosecond crystallography has been demonstrated using 3D-micron-sized crystals. In addition, X-FELs provide new opportunities for protein 2D-crystal diffraction, which would allow to observe the course of conformational changes of membrane proteins in a close-to-physiological lipid bilayer environment. Here, we describe the strategies towards structural dynamic studies of retinal proteins at room temperature, using injector or fixed-target based serial femtosecond crystallography at X-FELs. Thanks to recent progress especially in sample delivery methods, serial crystallography is now also feasible at synchrotron X-ray sources, thus expanding the possibilities for time-resolved structure determination.