Structural and biochemical analysis of the heterocyclase that acts on a ribosomally synthesized and post-translationally modified peptide identifies the basis for leader peptide activation and facilitates engineering of a constitutively active enzyme. Regioselective modification of amino acids within the context of a peptide is common to a number of biosynthetic pathways, and many of the resulting products have potential as therapeutics. The ATP-dependent enzyme LynD heterocyclizes multiple cysteine residues to thiazolines within a peptide substrate. The enzyme requires the substrate to have a conserved N-terminal leader for full activity. Catalysis is almost insensitive to immediately flanking residues in the substrate, suggesting that recognition occurs distant from the active site. Nucleotide and peptide substrate co-complex structures of LynD reveal that the substrate leader peptide binds to and extends the β-sheet of a conserved domain of LynD, whereas catalysis is accomplished in another conserved domain. The spatial segregation of catalysis from recognition combines seemingly contradictory properties of regioselectivity and promiscuity, and it appears to be a conserved strategy in other peptide-modifying enzymes. A variant of LynD that efficiently processes substrates without a leader peptide has been engineered.

Counting backwards: The cyanobactin class of heterocyclases, exemplified by TruD, possess an almost unique combination of processivity, specificity, chemical versatility, and promiscuity. TruD is shown by biochemical assay to be an adenylase, and processes cysteines in a defined order. The entire substrate leader can be removed and TruD will process a single specific cysteine residue; however the role of leader is to permit processivity through a balance of recognition. ATP/AMP=adenosine tri/monophosphate; PPi=pyrophosphate.

A hierarchical hormonal cascade along the hypothalamic‐pituitary‐adrenal axis orchestrates bodily responses to stress. Although corticotropin‐releasing hormone (CRH), produced by parvocellular neurons of the hypothalamic paraventricular nucleus (PVN) and released into the portal circulation at the median eminence, is known to prime downstream hormone release, the molecular mechanism regulating phasic CRH release remains poorly understood. Here, we find a cohort of parvocellular cells interspersed with magnocellular PVN neurons expressing secretagogin. Single‐cell transcriptome analysis combined with protein interactome profiling identifies secretagogin neurons as a distinct CRH‐releasing neuron population reliant on secretagogin9s Ca²⁺ sensor properties and protein interactions with the vesicular traffic and exocytosis release machineries to liberate this key hypothalamic releasing hormone. Pharmacological tools combined with RNA interference demonstrate that secretagogin9s loss of function occludes adrenocorticotropic hormone release from the pituitary and lowers peripheral corticosterone levels in response to acute stress. Cumulatively, these data define a novel secretagogin neuronal locus and molecular axis underpinning stress responsiveness. Synopsis Bodily responses to acute stress are orchestrated by the hierarchical release of stress hormones along the hypothalamic‐pituitary‐adrenal axis. Corticotropin‐releasing hormone (CRH)‐secreting neurons in the paraventricular nucleus of the hypothalamus are first order neurons in this axis, priming subsequent hormonal cascades upon CRH release into the portal circulation at the median eminence. Secretagogin is identified as the first neuronal Ca²⁺ sensor localized to parvocellular systems. The transcriptome landscape of a novel hypothalamic neuron subtype, which contains secretagogin as Ca²⁺ sensor, is shown. Secretagogin9s Ca²⁺‐dependent protein interactome minimally required for regulated CRH release is defined. Secretagogin is recognized to rate‐limit CRH release, thus limiting hormonal responses to stress.

To cause disease and persist in a host, pathogenic and commensal microbes must adhere to tissues. Colonization and infection depend on specific molecular interactions at the host-microbe interface that involve microbial surface proteins, or adhesins. To date, adhesins are only known to bind to host receptors non-covalently. Here we show that the streptococcal surface protein SfbI mediates covalent interaction with the host protein fibrinogen using an unusual internal thioester bond as a ‘chemical harpoon’. This cross-linking reaction allows bacterial attachment to fibrin and SfbI binding to human cells in a model of inflammation. Thioester-containing domains are unexpectedly prevalent in Gram-positive bacteria, including many clinically relevant pathogens. Our findings support bacterial-encoded covalent binding as a new molecular principle in host-microbe interactions. This represents an as yet unexploited target to treat bacterial infection and may also offer novel opportunities for engineering beneficial interactions.