Identifying a key spot for electron mediator-interaction to tailor CO dehydrogenase's affinity.

Fe‒S cluster-harboring enzymes, such as carbon monoxide dehydrogenases (CODH), employ sophisticated artificial electron mediators like viologens to serve as potent biocatalysts capable of cleaning-up industrial off-gases at stunning reaction rates. Unraveling the interplay between these enzymes and their associated mediators is essential for improving the efficiency of CODHs. Here we show the electron mediator-interaction site on ChCODHs (Ch, Carboxydothermus hydrogenoformans) using a systematic approach that leverages the viologen-reactive characteristics of superficial aromatic residues. By enhancing mediator-interaction (R57G/N59L) near the D-cluster, the strategically tailored variants exhibit a ten-fold increase in ethyl viologen affinity relative to the wild-type without sacrificing the turn-over rate (kcat). Viologen-complexed structures reveal the pivotal positions of surface phenylalanine residues, serving as external conduits for the D-cluster to/from viologen. One variant (R57G/N59L/A559W) can treat a broad spectrum of waste gases (from steel-process and plastic-gasification) containing O2. Decoding mediator interactions will facilitate the development of industrially high-efficient biocatalysts encompassing gas-utilizing enzymes.

GPCR targeting of E3 ubiquitin ligase MDM2 by inactive ß-arrestin.

E3 ubiquitin ligase Mdm2 facilitates ß-arrestin ubiquitination, leading to the internalization of G protein-coupled receptors (GPCRs). In this process, ß-arrestins bind to Mdm2 and recruit it to the receptor; however, the molecular architecture of the ß-arrestin-Mdm2 complex has not been elucidated yet. Here, we identified the ß-arrestin-binding region (ABR) on Mdm2 and solved the crystal structure of ß-arrestin1 in complex with Mdm2ABR peptide. The acidic residues of Mdm2ABR bind to the positively charged concave side of the ß-arrestin1 N-domain. The C-tail of ß-arrestin1 is still bound to the N-domain, indicating that Mdm2 binds to the inactive state of ß-arrestin1, whereas the phosphorylated C-terminal tail of GPCRs binds to activate ß-arrestins. The overlapped binding site of Mdm2 and GPCR C-tails on ß-arrestin1 suggests that the binding of GPCR C-tails might trigger the release of Mdm2. Moreover, hydrogen/deuterium exchange experiments further show that Mdm2ABR binding to ß-arrestin1 induces the interdomain interface to be more dynamic and uncouples the IP6-induced oligomer of ß-arrestin1. These results show how the E3 ligase, Mdm2, interacts with ß-arrestins to promote the internalization of GPCRs.

Biochemical Characterization of the Num1-Mdm36 Complex at the Mitochondria-Plasma Membrane Contact Site.

Saccharomyces cerevisiae that tethers mitochondria to the plasma membrane and plays a key role in mitochondrial fission. The main components of MECA are Num1 and Mdm36, and it is known that Mdm36 binds to Num1 to enhance mitochondrial tethering. To better understand the biochemical characteristics of the Num1-Mdm36 complex at the molecular level, we purified the coiled-coil domain of Num1, full-length Mdm36, and Num1-Mdm36 complex and identified the oligomeric state and stoichiometric characteristics of the Num1-Mdm36 complex by chemical crosslinking, size-exclusion chromatography coupled with multi-angle light scattering, and isothermal titration calorimetry. Mdm36 exists as a dimer and interacts preferentially with Num1 with a stoichiometry of 2:2, forming a heterotetrameric complex. Furthermore, we narrowed down the specific binding region of Num1, which is essential for interacting with Mdm36, and showed that their binding affinity is strong enough to tether both mitochondrial and plasma membranes. Our biochemical characterizations suggest a stoichiometric model of the Num1-Mdm36 complex at the mitochondria-plasma membrane contact site in budding yeast.

Dissecting the structural features of ß-arrestins as multifunctional proteins.

ß-arrestins bind active G protein-coupled receptors (GPCRs) and play a crucial role in receptor desensitization and internalization. The classical paradigm of arrestin function has been expanded with the identification of many non-receptor-binding partners, which indicated the multifunctional role of ß-arrestins in cellular functions. To elucidate the molecular mechanism of ß-arrestin-mediated signaling, the structural features of ß-arrestins were investigated using X-ray crystallography and cryogenic electron microscopy (cryo-EM). However, the intrinsic conformational flexibility of ß-arrestins hampers the elucidation of structural interactions between ß-arrestins and their binding partners using conventional structure determination tools. Therefore, structural information obtained using complementary structure analysis techniques would be necessary in combination with X-ray crystallography and cryo-EM data. In this review, we describe how ß-arrestins interact with their binding partners from a structural point of view, as elucidated by both traditional methods (X-ray crystallography and cryo-EM) and complementary structure analysis techniques.

Reversible Protein Conjugation on Live Cell Surfaces by Specific Recognition between Coiled-Coil Motifs of Natural Amino Acid Sequences.

In this study, we propose a reversible covalent conjugation method for peptides, proteins, and even live cells based on specific recognition between natural amino acid sequences. Two heptad sequences can specifically recognize each other and induce the formation of a disulfide bond between cysteine residues. We show the covalent bond formation and dissociation between peptides and proteins in cell-free conditions and on the surface of live cells. Because heptad sequences consist of natural amino acids, they are expressed in cells without additional preparation and can be used to selectively conjugate peptides, proteins, and cells.