Daly, C. A. | bioRxiv Science

bioRxiv2025

Receptor Allostery Promotes Context-Specific Sonic Hedgehog Signaling During Embryonic Development

Sonic Hedgehog (SHH) signaling functions in temporal- and context-dependent manners to pattern diverse tissues during embryogenesis. The signal transducer Smoothened (SMO) is activated by sterols, oxysterols, and arachidonic acid (AA) through binding pockets in its extracellular cysteine-rich domain (CRD) and 7-transmembrane (7TM) bundle. In vitro analyses suggest SMO signaling is allosterically enhanced by combinatorial ligand binding to these pockets but in vivo evidence of SMO allostery is lacking. Herein, we map an AA binding pocket at the top of the 7TM bundle and show that its disruption attenuates SHH and sterol-stimulated SMO induction. A knockin mouse model of compromised AA binding reveals that homozygous mutant mice are cyanotic, exhibit high perinatal lethality, and show congenital heart disease. Surviving mutants demonstrate pulmonary maldevelopment and fail to thrive. Neurodevelopment is unaltered in these mice, suggesting that context-dependent allosteric regulation of SMO signaling allows for precise tuning of pathway activity during cardiopulmonary development.

developmental biology↗

bioRxiv2021

Engineered chimeras unveil swappable modular features of fatty acid and polyketide synthase acyl carrier proteins

The strategic redesign of microbial biosynthetic pathways is a compelling route to access molecules of diverse structure and function in a potentially environmentally sustainable fashion. The promise of this approach hinges on an improved understanding of acyl carrier proteins (ACPs), which serve as central hubs in biosynthetic pathways. These small, flexible proteins mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural products. Past combinatorial biosynthesis efforts have failed due to incompatible ACP-enzyme pairings. Herein we report the design of chimeric ACPs with features of the actinorhodin polyketide synthase ACP (ACT) and of the E. coli fatty acid synthase (FAS) ACP (AcpP). We evaluate the ability of the chimeric ACPs to interact with the E. coli FAS ketosynthase FabF, which represents an interaction essential to building the carbon backbone of the synthase molecular output. Given that AcpP interacts with FabF but ACT does not, we sought to exchange modular features of ACT with AcpP to confer functionality with FabF. The interactions of chimeric ACPs with FabF were interrogated using sedimentation velocity experiments, surface plasmon resonance analyses, mechanism-based crosslinking assays, and molecular dynamics simulations. Results suggest that the residues guiding AcpP-FabF compatibility and ACT-FabF incompatibility may reside in the loop I, -helix II region. These findings can inform the development of strategic secondary element swaps that expand the enzyme compatibility of ACPs across systems and therefore represent a critical step towards the strategic engineering of unnatural natural products.

biochemistry↗