The molecular circadian clock in mammals is generated from transcriptional activation by the bHLH-PAS transcription factor CLOCK–BMAL1 and subsequent repression by PERIOD and CRYPTOCHROME (CRY). The mechanism by which CRYs repress CLOCK–BMAL1 to close the negative feedback loop and generate 24-hour timing is not known. Here we show that CRY1 competes for binding with coactivators to the intrinsically unstructured C-terminal transactivation domain (TAD) of BMAL1 to establish a functional switch between activation and repression of CLOCK–BMAL1. Mutations within the TAD that alter affinities for coregulators change the balance of repression and activation to consequently change intrinsic circadian period or eliminate cycling altogether. Our results suggest that CRY1 fulfills its role as an essential circadian repressor by sequestering the TAD from coactivators and highlight regulation of the BMAL1 TAD as a critical mechanism for establishing circadian timing.
Biofilm formation is critical for the infection cycle of Vibrio cholerae. Vibrio exopolysaccharides (VPS) and the matrix proteins RbmA, Bap1 and RbmC are required for the development of biofilm architecture. We demonstrate that RbmA binds VPS directly and uses a binary structural switch within its first fibronectin type III (FnIII-1) domain to control RbmA structural dynamics and the formation of VPS-dependent higher-order structures. The structural switch in FnIII-1 regulates interactions in trans with the FnIII-2 domain, leading to open (monomeric) or closed (dimeric) interfaces. The ability of RbmA to switch between open and closed states is important for V. cholerae biofilm formation, as RbmA variants with switches that are locked in either of the two states lead to biofilms with altered architecture and structural integrity.
SUMMARY The C-terminal transactivation domain (TAD) of BMAL1 (Brain and muscle ARNT-like 1) is a regulatory hub for transcriptional coactivators and repressors that compete for binding and consequently contributes to period determination of the mammalian circadian clock. Here, we report the discovery of two distinct conformational states that slowly exchange within the dynamic TAD to control timing. This binary switch results from cis/trans isomerization about a highly conserved Trp-Pro imide bond in a region of the TAD that is required for normal circadian timekeeping. Both cis and trans isomers interact with transcriptional regulators, suggesting that isomerization could serve a role in assembling regulatory complexes in vivo. Towards this end, we show that locking the switch into the trans isomer leads to shortened circadian periods. Furthermore, isomerization is regulated by the cyclophilin family of peptidyl-prolyl isomerases, highlighting the potential for regulation of BMAL1 protein dynamics in period determination.
Widespread resistance to antimicrobial and cancer therapeutics is evolving in every country worldwide and has a direct impact on global health, agriculture and the economy. The specificity and selectivity of bioactive peptide natural products present a possible stopgap measure to address the ongoing deficit of new therapeutic compounds. PepSAVI-MS (Statistically-guided bioActive Peptides prioritized VIa Mass Spectrometry) is an adaptable method for the analysis of natural product libraries to rapidly identify bioactive peptides. This pipeline was validated via screening of the cyclotide-rich botanical species Viola odorata and identification of the known antimicrobial and anticancer cyclotide cycloviolacin O2. Herein we present and validate novel bioactivities of the anthelmintic V. odorata cyclotide, cycloviolacin O8 (cyO8), including micromolar anticancer activity against PC-3 prostate, MDA-MB-231 breast, and OVCAR-3 ovarian cancer cell lines and antifungal activity against the agricultural pathogen Fusarium graminearum. A reduction/alkylation strategy in tandem with PepSAVI-MS analysis also revealed several previously uncharacterized putatively bioactive cyclotides. Downstream implementation of ultraviolet photodissociation (UVPD) tandem mass spectrometry is demonstrated for cyO8 as a method to address traditionally difficult-to-sequence cyclotide species. This work emphasizes the therapeutic and agricultural potential of natural product bioactive peptides and the necessity of developing robust analytical tools to deconvolute nature's complexity.
Traditional medicinal plants are a rich source of antimicrobials; however, the bioactive peptide constituents of most ethnobotanical species remain largely unexplored. Herein, PepSAVI-MS, a mass spectrometry-based peptidomics pipeline, was implemented for antimicrobial peptide (AMP) discovery in the medicinal plant Amaranthus tricolor. This investigation revealed a novel 1.7 kDa AMP with strong activity against Escherichia coli ATCC 25922, deemed Atr-AMP1. Initial efforts to determine the sequence of Atr-AMP1 utilized chemical derivatization and enzymatic digestion to provide information about specific residues and post-translational modifications. EThcD (electron-transfer/higher-energy collision dissociation) produced extensive backbone fragmentation and facilitated de novo sequencing, the results of which were consistent with orthogonal characterization experiments. Additionally, multistage HCD (higher-energy collisional dissociation) facilitated discrimination between isobaric leucine and isoleucine. These results revealed a positively charged proline-rich peptide present in a heterogeneous population of multiple peptidoforms, possessing several post-translational modifications including a disulfide bond, methionine oxidation, and proline hydroxylation. Additional bioactivity screening of a simplified fraction containing Atr-AMP1 revealed activity against Staphylococcus aureus LAC, demonstrating activity against both a Gram-negative and a Gram-positive bacterial species unlike many known short chain proline-rich antimicrobial peptides.
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