Biology subjects

Dowling, D. P.

Publications and source records attributed to Dowling, D. P..

3 recordsLinked to original sources

bioRxiv2022

Molecular basis of DNA recognition by the HMG-box-C1 module of Capicua

The HMG-box protein Capicua (CIC) is an evolutionarily conserved transcriptional repressor with key functions in development and disease-associated processes. CIC binds DNA using an exclusive mechanism that requires both its HMG-box and a separate domain called C1, but how these domains cooperate to recognize specific DNA sequences is not known. Here we report the crystal structure of the human CIC HMG-box and C1 domains in complex with an 18-base-pair DNA oligomer containing a consensus octameric CIC binding site. We find that both protein domains adopt independent tri-helical structures that pack against opposite sides of the DNA helix. The C1 domain in particular folds into a helix-turn-helix (HTH) structure that resembles the FF phosphoprotein binding domain. It inserts into the major groove of the DNA and plays a direct role in enhancing both the affinity and sequence specificity of CIC DNA binding. Our results reveal a unique bipartite protein module, ensuring highly specific DNA recognition by CIC, and show how this mechanism is disrupted by cancer mutations affecting either the HMG-box or C1 domains.

biochemistry↗

bioRxiv2022

Studies of a siderophore-producing cyclization domain: A refined proposal of substrate binding

Nonribosomal peptide synthetase (NRPS) heterocyclization (Cy) domains generate biologically important ox-/thiazoline modifications in natural products, including in production of compounds targeting disease or siderophores that are important for bacterial pathogenicity. Cy domains share the NRPS condensation domain fold but catalyze consecutive condensation and cyclodehydration reactions via an unknown mechanism. To further understanding of Cy domain catalysis, we report the crystal structure of the second Cy domain (Cy2) of yersiniabactin synthetase from the causative agent of the plague, Yersinia pestis. We find the high-resolution structure of Cy2 adopts a conformation enabling exploration of binding the extended, thiazoline-containing cyclodehydration intermediate for catalysis and the acceptor carrier protein to which it is tethered. We also report complementary electrostatic interfaces between Cy2 and its donor carrier protein that mediate donor binding. Lastly, we explore domain flexibility through the normal mode approximation and identify small-molecule fragment binding sites to inform antibiotic design targeting Cy function. Our results suggest how carrier protein binding may influence global conformation, with consequences for active site catalytic states and inhibitor development.

biochemistry↗

bioRxiv2021

Global Dynamics as Communication Sensors in Peptide Synthetase Cyclization Domains.

Structural biology is the foundation for deriving molecular mechanisms, where snapshots of macromolecules and binding partners inform on mutations that test or modify function. However, frequently, the impact of mutations violates the underpinnings of structural models, and mechanisms become cryptic. This conundrum applies to multidomain enzymatic systems called nonribosomal peptide synthetases (NRPSs), which assemble simple substrates into complex metabolites often with pharmaceutical properties. Engineering NRPSs can generate new pharmaceuticals1-3 but a dynamic domain organization challenges rational design.4-8 Using nuclear magnetic resonance (NMR), we determined the solution structure of a 52 kDa cyclization domain and demonstrate that global intra-domain dynamics enable sensing of substrates tethered to partner domains and draw an allosteric response encompassing the enzymes buried active site and two binding sites 40 [A] apart. We show that a point-site mutation that impedes the domain dynamics globally hampers the allosteric response. We demonstrate this mechanism through NMR experiments that provide atomic-level read-outs of allosteric responses during biochemical transformations in situ. Our results establish global structural dynamics as sensors of molecular events that can remodel domain interactions and illustrate the need for integrating structural dynamics explicitly when deriving molecular mechanisms through mutagenesis and structural biology.

biophysics↗