Electrophilic biocompatible warheads, particularly cysteine-reactive acrylamides, have enabled the development of covalent inhibitor drugs and chemical biology probes, but cysteine is rarely present in protein binding sites. Therefore, expansion of...

Thalidomide and its derivatives are molecular glues that bind cereblon (CRBN), a component of an E3 ubiquitin ligase complex, and mediate protein interactions with neosubstrates resulting in their polyubiquitination and...

The ability to rapidly and selectively modulate cellular protein levels using small molecules is essential for studying complex biological systems. Degradation tags, such as dTAG, allow for selective protein removal with a specific degrader molecule, but their utility is limited by the large tag size (>12 kDa) and the low efficiency of fusion product gene knock-in. Here, we describe the development of a short 24 amino acid peptide tag that enables cellbased quantification and covalent functionalization of proteins to which it is fused. The minimalistic peptide, termed HiBiT-SpyTag, incorporates the HiBiT peptide for protein level quantification and SpyTag, which forms a spontaneous isopeptide bond in the presence of the SpyCatcher protein. Transient expression of dTAG-SpyCatcher efficiently labels HiBiT-SpyTag-modified BRD4 or IRE1α in cells, and subsequent treatment with the dTAG13 degrader results in efficient protein removal without the need for full dTAG knock-in. We also demonstrate the utility of HiBiT-SpyTag for validating the degradation of the endoplasmic reticulum (ER) stress sensor IRE1α, which led to the development of the first PROTAC degrader of the protein. Our modular HiBiT-SpyTag system represents a valuable tool for the efficient development of degraders and for studying other proximity-induced pharmacology.

Cytochrome P460s are heme enzymes that oxidize hydroxylamine to nitrous oxide. They bear specialized “heme P460” cofactors that are cross-linked to their host polypeptides by a post-translationally modified lysine residue. Wild-type N. europaea cytochrome P460 may be isolated as a cross-link-deficient proenzyme following anaerobic overexpression in E. coli. When treated with peroxide, this proenzyme undergoes maturation to active enzyme with spectroscopic and catalytic properties that match wild-type cyt P460. This maturation reactivity requires no chaperonesit is intrinsic to the protein. This behavior extends to the broader cytochrome c′β superfamily. Accumulated data reveal key contributions from the secondary coordination sphere that enable selective, complete maturation. Spectroscopic data support the intermediacy of a ferryl species along the maturation pathway.

Cytochrome P460s are heme enzymes that oxidize hydroxylamine to nitrous oxide as part of the biogeochemical nitrogen cycle. They bear unique “heme P460” cofactors that are cross-linked to their host polypeptides by a post-translationally modified lysine residue. Wild-type N. europaea cytochrome P460 may be isolated as a cross-link deficient proenzyme following anaerobic overexpression in E. coli. When treated with peroxide, This proenzyme undergoes complete maturation to active enzyme with spectroscopic properties that match wild-type cyt P460. Together, these data indicate that the cofactor is primed to undergo this covalent modification by virtue of the protein fold. A putative mechanism analogous to that used by heme oxygenases to degrade hemes is proposed.