Background: Human aminopeptidase N (hAPN) is a dimeric cell surface protease involved in peptide processing, cell adhesion, endocytosis, and signal transduction. Results: Crystal structures of peptide and inhibitor complexes were determined. Conclusion: Unlike other family members, hAPN shows substrate-dependent loop ordering and a novel dimer structure. Significance: A model for catalysis and conformational changes provides mechanistic insights into how hAPN mediates its functional roles.

The coronavirus S-protein mediates receptor binding and fusion of the viral and host cell membranes. In HCoV-229E, its receptor binding domain (RBD) shows extensive sequence variation but how S-protein function is maintained is not understood. Reported are the X-ray crystal structures of Class III-V RBDs in complex with human aminopeptidase N (hAPN), as well as the electron cryomicroscopy structure of the 229E S-protein. The structures show that common core interactions define the specificity for hAPN and that the peripheral RBD sequence variation is accommodated by loop plasticity. The results provide insight into immune evasion and the crossspecies transmission of 229E and related coronaviruses. We also find that the 229E S-protein can expose a portion of its helical core to solvent. This is undoubtedly facilitated by hydrophilic subunit interfaces that we show are conserved among coronaviruses. These interfaces likely play a role in the S-protein conformational changes associated with membrane fusion.Li et al. eLife 2019;8:e51230.

Reported here is a piggyBac transposon-based expression system for the generation of doxycycline-inducible, stably transfected mammalian cell cultures for large-scale protein production. The system works with commonly used adherent and suspension-adapted mammalian cell lines and requires only a single transfection step. Moreover, the high uniform expression levels observed among clones allow for the use of stable bulk cell cultures, thereby eliminating time-consuming cloning steps. Under continuous doxycycline induction, protein expression levels have been shown to be stable for at least 2 mo in the absence of drug selection. The high efficiency of the system also allows for the generation of stable bulk cell cultures in 96-well format, a capability leading to the possibility of generating stable cell cultures for entire families of membrane or secreted proteins. Finally, we demonstrate the utility of the system through the large-scale production (140-750 mg scale) of an endoplasmic reticulum-resident fucosyltransferase and two potential anticancer protein therapeutic agents.glycoprotein | X-ray crystallography

RNA viruses are characterized by a high mutation rate, a buffer against environmental change. Nevertheless, the means by which random mutation improves viral fitness is not well characterized. Here we report the X-ray crystal structure of the receptor-binding domain (RBD) of the human coronavirus, HCoV-229E, in complex with the ectodomain of its receptor, aminopeptidase N (APN). Three extended loops are solely responsible for receptor binding and the evolution of HCoV-229E and its close relatives is accompanied by changing loop–receptor interactions. Phylogenetic analysis shows that the natural HCoV-229E receptor-binding loop variation observed defines six RBD classes whose viruses have successively replaced each other in the human population over the past 50 years. These RBD classes differ in their affinity for APN and their ability to bind an HCoV-229E neutralizing antibody. Together, our results provide a model for alphacoronavirus adaptation and evolution based on the use of extended loops for receptor binding.

Protein O-fucosyltransferase 1 (POFUT1) fucosylates the epidermal growth factor (EGF)-like domains found in cell-surface and secreted glycoproteins including Notch and its ligands. Although Notch fucosylation is critical for development, and POFUT1 deficiency leads to human disease, how this enzyme binds and catalyzes the fucosylation of its diverse EGF-like domain substrates has not been determined. Reported here is the X-ray crystal structure of mouse POFUT1 in complex with several EGF-like domains, including EGF12 and EGF26 of Notch. Overall shape complementarity, interactions with invariant atoms of the fucosylation motif and flexible segments on POFUT1 all define its EGF-like-domain binding properties. Using large-scale structural and sequence analysis, we also show that POFUT1 binds EGF-like domains of the hEGF type and that the highly correlated presence of POFUT1 and fucosylatable hEGFs has accompanied animal evolution.