Highlights d Structures of seven NTD-directed neutralizing antibody complexes with spike or NTD d Structures define distinct recognition classes, one observed in multiple donors d Supersite is glycan free, electropositive, with mobile b-hairpin and flexible loops d Most potent NTD-directed neutralizing antibodies may target this supersite

An artificial heme enzyme was created through self‐assembly from hemin and the lactococcal multidrug resistance regulator (LmrR). The crystal structure shows the heme bound inside the hydrophobic pore of the protein, where it appears inaccessible for substrates. However, good catalytic activity and moderate enantioselectivity was observed in an abiological cyclopropanation reaction. We propose that the dynamic nature of the structure of the LmrR protein is key to the observed activity. This was supported by molecular dynamics simulations, which showed transient formation of opened conformations that allow the binding of substrates and the formation of pre‐catalytic structures.

The impressive rate accelerations that enzymes display in nature often result from boosting the inherent catalytic activities of side chains by their precise positioning inside a protein binding pocket. Such fine‐tuning is also possible for catalytic unnatural amino acids. Specifically, the directed evolution of a recently described designer enzyme, which utilizes an aniline side chain to promote a model hydrazone formation reaction, is reported. Consecutive rounds of directed evolution identified several mutations in the promiscuous binding pocket, in which the unnatural amino acid is embedded in the starting catalyst. When combined, these mutations boost the turnover frequency ( k cat ) of the designer enzyme by almost 100‐fold. This results from strengthening the catalytic contribution of the unnatural amino acid, as the engineered designer enzymes outperform variants, in which the aniline side chain is replaced with a catalytically inactive tyrosine residue, by more than 200‐fold.

The insufficient operational stability of amine transaminases (ATA) constitutes a limiting factor for high productivity in chiral amine synthesis. In this work, we investigated the operational stability of a tetrameric ATA with 92% sequence identity to a Pseudomonas sp. transaminase and compared it to the two commonly used dimeric ATAs from Chromobacterium violaceum and Vibrio fluvialis. In the presence of substrate, all three ATAs featured reduced stability in comparison to their resting stability, but the tetramer showed slower inactivation rates than the dimeric ATAs. Kinetic and thermodynamic analysis revealed an amine donor induced inactivation mechanism involving accumulation of the less stable aminated enzyme−cofactor intermediate. Dissociation of the enzyme−PMP complex forms the unstable apoenzyme, which can rapidly unfold. Crystal structure analysis shed light on the structure−function relationship suggesting that the cofactor−ring binding element is stabilized in the quaternary structure conferring higher operational stability by minimizing PMP leakage and apoenzyme formation. In contrast to the common practice, increasing the amine acceptor content improved the stability and substrate turnover of dimeric ATAs. An extra supply of the pyridoxal cofactor (PLP) enhanced the stability of dimeric and tetrameric ATAs but reduced the transamination activity. The ATA inactivation mechanism described here provides valuable aspects for both process development and protein engineering.

SummaryNumerous antibodies that neutralize SARS-CoV-2 have been identified, and these generally target either the receptor-binding domain (RBD) or the N-terminal domain (NTD) of the viral spike. While RBD-directed antibodies have been extensively studied, far less is known about NTD-directed antibodies. Here we report cryo-EM and crystal structures for seven potent NTD-directed neutralizing antibodies in complex with spike or isolated NTD. These structures defined several antibody classes, with at least one observed in multiple convalescent donors. The structures revealed all seven antibodies to target a common surface, bordered by glycans N17, N74, N122, and N149. This site – formed primarily by a mobile β-hairpin and several flexible loops – was highly electropositive, located at the periphery of the spike, and the largest glycan-free surface of NTD facing away from the viral membrane. Thus, in contrast to neutralizing RBD-directed antibodies that recognize multiple non-overlapping epitopes, potent NTD-directed neutralizing antibodies target a single supersite.

Antibodies with heavy chains that derive from the VH1-2 gene constitute some of the most potent SARS-CoV-2-neutralizing antibodies yet identified. To provide insight into whether these genetic similarities inform common modes of recognition, we determined structures of the SARS-CoV-2 spike in complex with three VH1-2-derived antibodies: 2-15, 2-43, and H4. All three utilize VH1-2-encoded motifs to recognize the receptor-binding domain (RBD), with heavy chain N53I enhancing binding and light chain tyrosines recognizing F486 RBD . Despite these similarities, class members bind both RBD-up and -down conformations of the spike, with a subset of antibodies utilizing elongated CDRH3s to recognize glycan N 343 on a neighboring RBD – a quaternary interaction accommodated by an increase in RBD separation of up to 12 Å. The VH1-2-antibody class thus utilizes modular recognition encoded by modular genetic elements to effect potent neutralization, with VH-gene component specifying recognition of RBD and CDRH3 component specifying quaternary interactions.

Antibodies that potently neutralize SARS-CoV-2 target mainly the receptor-binding domain or the N-terminal domain (NTD). Over a dozen potently neutralizing NTD-directed antibodies have been studied structurally, and all target a single antigenic supersite in NTD (site 1). Here we report the cryo-EM structure of a potent NTD-directed neutralizing antibody 5-7, which recognizes a site distinct from other potently neutralizing antibodies, inserting a binding loop into an exposed hydrophobic pocket between the two sheets of the NTD β-sandwich. Interestingly, this pocket has been previously identified as the binding site for hydrophobic molecules including heme metabolites, but we observe their presence to not substantially impede 5-7 recognition. Mirroring its distinctive binding, antibody 5-7 retains a distinctive neutralization potency with variants of concern (VOC). Overall, we reveal a hydrophobic pocket in NTD proposed for immune evasion can actually be used by the immune system for recognition.

We present an artificial metalloenzyme based on the transcriptional regulator LmrR that exhibits dynamics involving the positioning of its abiological metal cofactor. The position of the cofactor, in turn, was found to be related to the preferred catalytic reactivity, which is either the enantioselective Friedel–Crafts alkylation of indoles with β-substituted enones or the tandem Friedel–Crafts alkylation/enantioselective protonation of indoles with α-substituted enones. The artificial metalloenzyme could be specialized for one of these catalytic reactions introducing a single mutation in the protein. The relation between cofactor dynamics and activity and selectivity in catalysis has not been described for natural enzymes and, to date, appears to be particular for artificial metalloenzymes.