Bergeron, L. | bioRxiv Science

bioRxiv2023

High germline mutation rates but not extreme population size outbreaks influence genetic diversity in crown-of-thorns sea stars

Lewontins paradox, the observation that levels of genetic diversity ({pi}) among animals do not scale linearly with variation in census population sizes (Nc), is an evolutionary conundrum, where the most extreme mismatches between {pi} and Nc are found for highly abundant marine invertebrates. Yet, whether new mutations influence {pi} relative to extrinsic processes remains unknown for most taxa. Here, we provide the first direct germline mutation rate () estimate for a marine invertebrate, using high-coverage (60x) whole-genome sequencing of wild-caught Acanthaster cf. solaris crown-of-thorns sea stars (Echinodermata). We also provide empirical estimates of adult Nc in Australias Great Barrier Reef to jointly examine the determinants of {pi}. Based on direct observations of 63 de novo mutations across 14 parent-offspring trios, the A. cf. solaris mean was 9.13 x 10-09 mutations per-site per-generation (95% CI: 6.51 x 10-09 to 1.18 x 10-08). This value exceeds estimates for other invertebrates, showing greater concordance with reported vertebrate germline mutation rates. Lower-than-expected Ne ([~]70,000-180,000) and low Ne/Nc values (0.0047-0.048) indicated significant genetic drift and weak influences of contemporary population outbreaks on long-term {pi}. Our findings of elevated and low Ne in A. cf. solaris may help explain high mutational loads and extreme polymorphism levels observed in some marine invertebrate taxa and are consistent with evolving in response to Ne (drift-barrier hypothesis). This study advances our understanding of the processes controlling levels of natural genetic variation and provides new data valuable for further testing hypotheses about mutation rate evolution across animal phyla.

evolutionary biology↗

bioRxiv2021

Atomic-Resolution Prediction of Degrader-mediated Ternary Complex Structures by Combining Molecular Simulations with Hydrogen Deuterium Exchange

Targeted protein degradation (TPD) has emerged as a powerful approach in drug discovery for removing (rather than inhibiting) proteins implicated in diseases. A key step in this approach is the formation of an induced proximity complex, where a degrader molecule recruits an E3 ligase to the protein of interest (POI), facilitating the transfer of ubiquitin to the POI and initiating the proteasomal degradation process. Here, we address three critical aspects of the TPD process: 1) formation of the ternary complex induced by a degrader molecule, 2) conformational heterogeneity of the ternary complex, and 3) assessment of ubiquitination propensity via the full Cullin Ring Ligase (CRL) macromolecular assembly. The novel approach presented here combines experimental biophysical data--in this case hydrogen-deuterium exchange mass spectrometry (HDX-MS, which measures the solvent exposure of protein residues)--with all-atom explicit solvent molecular dynamics (MD) simulations aided by enhanced sampling techniques to predict structural ensembles of ternary complexes at atomic resolution. We present results demonstrating the efficiency, accuracy, and reliability of our approach to predict ternary structure ensembles using the bromodomain of SMARCA2 (SMARCA2BD) with the E3 ligase VHL as the system of interest. The simulations reproduce X-ray crystal structures - including prospective simulations validated on a new structure that we determined in this work (PDB ID: 7S4E) - with root mean square deviations (RMSD) of 1.1 to 1.6 [A]. The simulations also reveal a structural ensemble of low-energy conformations of the ternary complex within a broad energy basin. To further characterize the structural ensemble, we used snapshots from the aforementioned simulations as seeds for Hamiltonian replica exchange molecular dynamics (HREMD) simulations, and then perform 7.1 milliseconds of aggregate simulation time using Folding@home. The resulting free energy surface identifies the crystal structure conformation within a broad low-energy basin and the dynamic ensemble is consistent with solution-phase biophysical experimental data (HDX-MS and small-angle x-ray scattering, SAXS). Finally, we graft structures from the ternary complexes onto the full CRL and perform enhanced sampling simulations, where we find that differences in degradation efficiency can be explained by the proximity distribution of lysine residues on the POI relative to the E2-loaded ubiquitin. Several of the top predicted ubiquitinated lysine residues are validated prospectively through a ubiquitin mapping proteomics experiment.

biophysics↗