Auxin is a plant hormone that is central to plant growth and development from embryogenesis to senescence. Auxin signaling is mediated by auxin response transcription factors (ARFs) and Aux/IAA repressors that regulate the expression of a multitude of auxin response genes. ARF and Aux/IAA proteins assemble into homomeric and heteromeric complexes via their conserved PB1 domains. Here we report the first crystal structure of the PB1 complex between ARF5 and IAA17 of Arabidopsis thaliana, which represents the transcriptionally repressed state at low auxin levels. The PB1 domains assemble in a head-to-tail manner with a backbone arrangement similar to that of the ARF5:ARF5 PB1 complex. The ARF5:IAA17 complex, however, reveals distinct points of contact that promote the ARF5:IAA17 interaction over the ARF5:ARF5 interaction. Specifically, surface charges at the interface form salt-bridges that distinguish the homomeric and heteromeric complexes, revealing common and specific interfaces between transcriptionally repressed and derepressed states. Further, the salt-bridges can be reconfigured to switch the affinity between homomeric and heteromeric complexes in an incremental manner. The complex structure combined with quantitative binding analyses would be essential for deciphering the PB1 interaction code underlying the transcriptional regulation of auxin signaling.

The bacterial CRISPR-Cas system provides adaptive immunity against invading phages. Cas9, an RNA-guided endonuclease, specifically cleaves target DNA substrates and constitutes a well-established platform for genome editing. Recently, anti-CRISPR (Acr) proteins that inhibit Cas9 have been discovered, promising a useful off-switch for Cas9 to avoid undesirable off-target effects. Here, we report the solution structure and dynamics of Listeria monocytogenes AcrIIA4 that inhibits Streptococcus pyogenes Cas9 ( Spy Cas9). AcrIIA4 forms a compact monomeric αβββαα fold comprising three antiparallel β strands flanked by three α-helices and a short 3 10 -helix. AcrIIA4 exhibits distinct backbone dynamics in fast and slow timescales at loop regions that form interaction surfaces for Spy Cas9. In particular, the β1–β2 loop that binds to the RuvC domain of Spy Cas9 is highly mobile, and the β1–β2 and α2–α3 loops that bind to the RuvC and C-terminal domains of Spy Cas9, respectively, undergoes conformational exchanges in microsecond-to-millisecond time scales. AcrIIA4 binds to apo- Spy Cas9 with K D ~4.8 μM, which compares to K D ~0.6 nM for AcrIIA4 binding to sgRNA-bound Spy Cas9. Since the binary complex between AcrIIA4 and Spy Cas9 does not compete with the target DNA binding, it can effectively disable the Cas9 nuclease activity by forming a tight ternary complex in the presence of sgRNA.

Modern genetic techniques, enable the use of animal bioreactor systems for the production and functional enhancement of anti-cancer antibodies. Chicken is the most efficient animal bioreactor for the production of anti-cancer antibodies because of its relatively short generation time, plentiful reproductive capacity, and daily deposition in the egg white. Although several studies have focused on the production of anti-cancer antibodies in egg white, in-depth studies of the biological activity and physiological characteristics of transgenic chicken-derived anti-cancer antibodies have not been fully carried out. Here, we report the production of an anti-cancer monoclonal antibody against the CD20 protein from egg whites of transgenic hens, and validated the bio-functional activity of the protein in B-lymphoma and B-lymphoblast cells. Quantitative analysis showed that deposition of the chickenised CD20 monoclonal antibody (cCD20 mAb) from transgenic chickens increased in successive generations and with increasing transgene copy number. Ultra-performance liquid chromatography (UPLC) tandem mass spectrometry (LC/MS/MS) analysis showed that the cCD20 mAb exhibited 14 N-glycan patterns with high-mannose, afucosylation and terminal galactosylation. The cCD20 mAb did not exhibit significantly improved Fab-binding affinity, but showed markedly enhanced Fc-related functions, including complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) compared to commercial rituximab, a chimeric mAb against CD20. Our results suggest that the transgenic chicken bioreactor is an efficient system for producing anti-cancer therapeutic antibodies with enhanced Fc effector functions.

In this study, we elucidated the mechanism underlying atmospheric pressure plasma (APP)-induced green discolouration of myoglobin. Green-coloured pigments are produced upon conversion of myoglobin into sulphmyoglobin, choleglobin, verdoheme, nitrihemin, or nitrimyoglobin. We exposed myoglobin dissolved in phosphate buffer to APP for 20 min and found a decrease in a * value (+redness/−greenness) and increase in b * value (+yellowness/−blueness) ( P < 0.05). In the ultraviolet absorption spectrum, myoglobin treated with APP for 20 min showed absorption peaks at 503 and 630 nm, a spectrum different from that of sulphmyoglobin or choleglobin. The secondary structure and molecular weight of myoglobin were unaffected by APP treatment, excluding the possibility of verdoheme or nitrihemin formation. After APP treatment, nitrite was produced in myoglobin solution that provided a positive environment for nitrimyoglobin formation. However, the addition of 0.5% sodium dithionite, a strong reducing agent, to myoglobin solution resulted in the formation of deoxymyoglobin, which was subsequently converted to nitrosomyoglobin upon APP treatment to yield a desirable red colour. Thus, APP-induced green colouration in myoglobin solution is associated with nitrimyoglobin formation. The addition of the antioxidant resulted in the production of red colour in myoglobin solution after APP treatment owing to nitrosomyoglobin formation.

The plant hormone auxin is involved in all stages of plant development. Aux/IAAs are the transcriptional repressors that bind to the Auxin Response Factors (ARFs) to regulate the gene expression upon auxin release. Aux/IAA have highly conserved C-terminal domains (domains III-IV) that mediate both homotypic and heterotypic interactions between Aux/IAA and ARF family proteins. Recent studies revealed that the conserved domains III-IV share a common beta-grasp fold that oligomerizes in a front-to-back manner. 1,2,3 In particular, Aux/IAA contains a mobile insert helix in the domain III-IV, whereas ARFs do not. Here, we investigated the dynamics of the insert helix using paramagnetic NMR spectroscopy. The insert helix exhibited fast motions in the ps-ns time scale from 15N relaxation data, but the amplitude of the motion is likely limited to the local neighborhood. Our result suggests that the motion of the helix may have functional implications in protein-protein interactions for transcriptional regulations.

Significance Auxin is the central hormone that governs diverse developmental processes in plants. Auxin response is regulated by auxin response transcription factor (ARF) and Aux/IAA transcriptional repressor. ARF and Aux/IAA form homo-oligomers and also hetero-oligomers for transcriptional regulation of auxin-response genes. Mechanistic understanding of how ARF and Aux/IAA change their association states is not well established. This work reports, to our knowledge, the first structure of the oligomerization domain of IAA17, and describes the key determinant that dictates the switch between homo- and hetero-oligomers. While Aux/IAA and ARF use a common scaffold and interface for homotypic and heterotypic associations, the charge composition at the interface determines the affinity and the oligomerization states. Based on the results, we propose a refined model of auxin-induced transcriptional regulation.