Structural Basis for the Propagation of Homing Endonuclease-Associated Inteins

Inteins catalyze their removal from a host protein through protein splicing. Inteins that contain an additional site-specific endonuclease domain display genetic mobility via a process termed "homing" and thereby act as selfish DNA elements. We elucidated the crystal structures of two archaeal inteins associated with an active or inactive homing endonuclease domain. This analysis illustrated structural diversity in the accessory domains (ACDs) associated with the homing endonuclease domain. To augment homing endonucleases with highly specific DNA cleaving activity using the intein scaffold, we engineered the ACDs and characterized their homing site recognition. Protein engineering of the ACDs in the inteins illuminated a possible strategy for how inteins could avoid their extinction but spread via the acquisition of a diverse accessory domain.Peer reviewe

Mini-Intein Structures from Extremophiles Suggest a Strategy for Finding Novel Robust Inteins

Inteins are prevalent among extremophiles. Mini-inteins with robust splicing properties are of particular interest for biotechnological applications due to their small size. However, biochemical and structural characterization has still been limited to a small number of inteins, and only a few serve as widely used tools in protein engineering. We determined the crystal structure of a naturally occurring Pol-II mini-intein from Pyrococcus horikoshii and compared all three mini-inteins found in the genome of P. horikoshii. Despite their similar sizes, the comparison revealed distinct differences in the insertions and deletions, implying specific evolutionary pathways from distinct ancestral origins. Our studies suggest that sporadically distributed mini-inteins might be more promising for further protein engineering applications than highly conserved mini-inteins. Structural investigations of additional inteins could guide the shortest path to finding novel robust mini-inteins suitable for various protein engineering purposes

Mini-Intein Structures from Extremophiles Suggest a Strategy for Finding Novel Robust Inteins

Inteins are prevalent among extremophiles. Mini-inteins with robust splicing properties are of particular interest for biotechnological applications due to their small size. However, biochemical and structural characterization has still been limited to a small number of inteins, and only a few serve as widely used tools in protein engineering. We determined the crystal structure of a naturally occurring Pol-II mini-intein from Pyrococcus horikoshii and compared all three mini-inteins found in the genome of P. horikoshii. Despite their similar sizes, the comparison revealed distinct differences in the insertions and deletions, implying specific evolutionary pathways from distinct ancestral origins. Our studies suggest that sporadically distributed mini-inteins might be more promising for further protein engineering applications than highly conserved mini-inteins. Structural investigations of additional inteins could guide the shortest path to finding novel robust mini-inteins suitable for various protein engineering purposes

Crystal structures of CDC21-1 inteins from hyperthermophilic archaea reveal the selection mechanism for the highly conserved homing endonuclease insertion site

Self-splicing inteins are mobile genetic elements invading host genes via nested homing endonuclease (HEN) domains. All HEN domains residing within inteins are inserted at a highly conserved insertion site. A purifying selection mechanism directing the location of the HEN insertion site has not yet been identified. In this work, we solved the three-dimensional crystal structures of two inteins inserted in the cell division control protein 21 of the hyperthermophilic archaea Pyrococcus abyssi and Pyrococcus horikoshii. A comparison between the structures provides the structural basis for the thermo-stabilization mechanism of inteins that have lost the HEN domain during evolution. The presence of an entire extein domain in the intein structure from Pyrococcus horikoshii suggests the selection mechanism for the highly conserved HEN insertion point.Peer reviewe

NMR Structure and Dynamics of TonB Investigated by Scar-Less Segmental Isotopic Labeling Using a Salt-Inducible Split Intein

The growing understanding of partially unfolded proteins increasingly points to their biological relevance in allosteric regulation, complex formation, and protein design. However, the structural characterization of disordered proteins remains challenging. NMR methods can access both the dynamics and structures of such proteins, yet suffering from a high degeneracy of NMR signals. Here, we overcame this bottleneck utilizing a salt-inducible split intein to produce segmentally isotope-labeled samples with the native sequence, including the ligation junction. With this technique, we investigated the NMR structure and conformational dynamics of TonB from Helicobacter pylori in the presence of a proline-rich low complexity region. Spin relaxation experiments suggest that the several nano-second time scale dynamics of the C-terminal domain (CTD) is almost independent of the faster pico-to-nanosecond dynamics of the low complexity central region (LCCR). Our results demonstrate the utility of segmental isotopic labeling for proteins with heterogenous dynamics such as TonB and could advance NMR studies of other partially unfolded proteins.Peer reviewe

The Convergence of the Hedgehog/Intein Fold in Different Protein Splicing Mechanisms

Protein splicing catalyzed by inteins utilizes many different combinations of amino-acid types at active sites. Inteins have been classified into three classes based on their characteristic sequences. We investigated the structural basis of the protein splicing mechanism of class 3 inteins by determining crystal structures of variants of a class 3 intein from Mycobacterium chimaera and molecular dynamics simulations, which suggested that the class 3 intein utilizes a different splicing mechanism from that of class 1 and 2 inteins. The class 3 intein uses a bond cleavage strategy reminiscent of proteases but share the same Hedgehog/INTein (HINT) fold of other intein classes. Engineering of class 3 inteins from a class 1 intein indicated that a class 3 intein would unlikely evolve directly from a class 1 or 2 intein. The HINT fold appears as structural and functional solution for trans-peptidyl and trans-esterification reactions commonly exploited by diverse mechanisms using different combinations of amino-acid types for the active-site residues

Substrate specificities of inteins investigated by QuickDrop-cassette mutagenesis

Inteins catalyze self-excision from host precursor proteins while concomitantly ligating the flanking substrates (exteins) with a peptide bond. Noncatalytic extein residues near the splice junctions, such as the residues at the -1 and +2 positions, often strongly influence the protein-splicing efficiency. The substrate specificities of inteins have not been studied for many inteins. We developed a convenient mutagenesis platform termed "QuickDrop"-cassette mutagenesis for investigating the influences of 20 amino acid types at the -1 and +2 positions of different inteins. We elucidated 17 different profiles of the 20 amino acid dependencies across different inteins. The substrate specificities will accelerate our understanding of the structure-function relationship at the splicing junctions for broader applications of inteins in biotechnology and molecular biosciences.Peer reviewe

NERNST: a genetically-encoded ratiometric non-destructive sensing tool to estimate NADP(H) redox status in bacterial, plant and animal systems

NADP(H) is a central metabolic hub providing reducing equivalents to multiple biosynthetic, regulatory and antioxidative pathways in all living organisms. While biosensors are available to determine NADP+ or NADPH levels in vivo, no probe exists to estimate the NADP(H) redox status, a determinant of the cell energy availability. We describe herein the design and characterization of a genetically-encoded ratiometric biosensor, termed NERNST, able to interact with NADP(H) and estimate E NADP(H). NERNST consists of a redox-sensitive green fluorescent protein (roGFP2) fused to an NADPH-thioredoxin reductase C module which selectively monitors NADP(H) redox states via oxido-reduction of the roGFP2 moiety. NERNST is functional in bacterial, plant and animal cells, and organelles such as chloroplasts and mitochondria. Using NERNST, we monitor NADP(H) dynamics during bacterial growth, environmental stresses in plants, metabolic challenges to mammalian cells, and wounding in zebrafish. NERNST estimates the NADP(H) redox poise in living organisms, with various potential applications in biochemical, biotechnological and biomedical research

Illuminating hydrological processes at the soil-vegetation-atmosphere interface with water stable isotopes

Funded by DFG research project “From Catchments as Organised Systems to Models based on Functional Units” (FOR 1Peer reviewedPublisher PDFPublisher PD