Bacterial Chaperone Domain Insertions Convert Human FKBP12 into an Excellent Protein-Folding Catalyst-A Structural and Functional Analysis.

Many folding enzymes use separate domains for the binding of substrate proteins and for the catalysis of slow folding reactions such as prolyl isomerization. FKBP12 is a small prolyl isomerase without a chaperone domain. Its folding activity is low, but it could be increased by inserting the chaperone domain from the homolog SlyD of E. coli near the prolyl isomerase active site. We inserted two other chaperone domains into human FKBP12: the chaperone domain of SlpA from E. coli, and the chaperone domain of SlyD from Thermococcus sp. Both stabilized FKBP12 and greatly increased its folding activity. The insertion of these chaperone domains had no influence on the FKBP12 and the chaperone domain structure, as revealed by two crystal structures of the chimeric proteins. The relative domain orientations differ in the two crystal structures, presumably representing snapshots of a more open and a more closed conformation. Together with crystal structures from SlyD-like proteins, they suggest a path for how substrate proteins might be transferred from the chaperone domain to the prolyl isomerase domain.

Strengthening an Intramolecular Non-Classical Hydrogen Bond to Get in Shape for Binding.

In this research article, we report on the strengthening of a non-classical hydrogen bond (C-H···O) by introducing electron withdrawing groups at the carbon atom. The approach is demonstrated on the example of derivatives of the physiological E-selectin ligand sialyl Lewisx (1, sLex). Its affinity is mainly due to a beneficial entropy term, which is predominantly caused by the pre-organization of sLex in its binding conformation. We have shown, that among the elements responsible for the pre-organization, the stabilization by a non-classical hydrogen bond between the H-C5 of l-fucose and the ring oxygen O5 of the neighboring d-galactose moiety is essential and yields 7.4 kJ mol-1. This effect could be further strengthened by replacing l-fucose by 6,6,6-trifluoro-l-fucose leading to an improved non-classical H-bond of 14.9 kJ mol-1, i.e., an improved pre-organization in the bioactive conformation. For a series of glycomimetics of sLex (1), this outcome could be confirmed by high field NMR-shifts of the H-C5Fuc, by X-ray diffraction analysis of glycomimetics co-crystallized with E-selectin as well as by isothermal titration calorimetry. Furthermore, the electron-withdrawing character of the CF3-group beneficially influences the pharmacokinetic properties of sLex mimetics. Thus, acid-stability a prerequisite for gastrointestinal stability could be substantially improved.

An artificial nickel chlorinase based on the biotin-streptavidin technology.

Herein, we report on an artificial nickel chlorinase (ANCase) resulting from anchoring a biotinylated nickel-based cofactor within streptavidin (Sav). The resulting ANCase was efficient for the chlorination of diverse C(sp3)-H bonds. Guided by the X-ray analysis of the ANCase, the activity of the artificial chlorinase could be significantly improved. This approach opens interesting perspectives for late-stage functionalization of organic intermediates as it complements biocatalytic chlorination strategies.

Thermodynamics-Guided Design Reveals a Cooperative Hydrogen Bond in DC-SIGN-targeted Glycomimetics.

Due to the shallow and hydrophilic binding sites of carbohydrate-binding proteins, the design of glycomimetics is often complicated by high desolvation costs as well as competition with solvent. Therefore, a careful optimization of interaction vectors and ligand properties is required in the design and optimization of glycomimetics. Here, we employ thermodynamics-guided design to optimize mannose-based glycomimetics targeting the human C-type lectin receptor dendritic cell-specific intercellular adhesion molecule 3 grabbing nonintegrin (DC-SIGN), a pathogenic host factor in viral infections. By exploring ligand rigidification and hydrogen bond engineering, a monovalent glycomimetic with an unprecedented affinity for DC-SIGN in the low µM range was discovered. A matched molecular pair analysis based on microcalorimetric data revealed a stereospecific hydrogen bond interaction with Glu358/Ser360 as the origin of this cooperative and enthalpically dominated interaction. This detailed insight into the binding mechanism paves the way for an improvement of monovalent glycomimetics targeting DC-SIGN.

Analogues of the pan-selectin antagonist rivipansel (GMI-1070).

The selectin family consisting of E-, P- and L-selectin plays dominant roles in atherosclerosis, ischemia-reperfusion injury, inflammatory diseases, and metastatic spreading of some cancers. An early goal in selectin-targeted drug discovery campaigns was to identify ligands binding to all three selectins, so-called pan-selectin antagonists. The physiological epitope, tetrasaccharide sialyl Lewisx (sLex, 1) binds to all selectins, albeit with very different affinities. Whereas P- and L-selectin require additional interactions contributed by sulfate groups for high binding affinity, E-selectin can functionally bind sLex-modified glycolipids and glycoproteins. Rivipansel (3) marked the first pan-selectin antagonist, which simultaneously interacted with both the sLex and the sulfate binding site. The aim of this contribution was to improve the pan-selectin affinity of rivipansel (3) by leveraging a new class of sLex mimetics in combination with an optimized linker length to the sulfate bearing group. As a result, the pan-selectin antagonist 11b exhibits an approximatively 5-fold improved affinity for E-, as well as P-selectin.

An evolved artificial radical cyclase enables the construction of bicyclic terpenoid scaffolds via an H-atom transfer pathway.

While natural terpenoid cyclases generate complex terpenoid structures via cationic mechanisms, alternative radical cyclization pathways are underexplored. The metal-catalysed H-atom transfer reaction (M-HAT) offers an attractive means for hydrofunctionalizing olefins, providing access to terpenoid-like structures. Artificial metalloenzymes offer a promising strategy for introducing M-HAT reactivity into a protein scaffold. Here we report our efforts towards engineering an artificial radical cyclase (ARCase), resulting from anchoring a biotinylated [Co(Schiff-base)] cofactor within an engineered chimeric streptavidin. After two rounds of directed evolution, a double mutant catalyses a radical cyclization to afford bicyclic products with a cis-5-6-fused ring structure and up to 97% enantiomeric excess. The involvement of a histidine ligation to the Co cofactor is confirmed by crystallography. A time course experiment reveals a cascade reaction catalysed by the ARCase, combining a radical cyclization with a conjugate reduction. The ARCase exhibits tolerance towards variations in the dienone substrate, highlighting its potential to access terpenoid scaffolds.