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

Abstract

Targeted protein degradation (TPD) has recently emerged as a powerful approach for removing (rather than inhibiting) proteins implicated in diseases. A key step in TPD 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 using atomistic simulations: 1) formation of the ternary complex induced by a degrader molecule, 2) conformational heterogeneity of the ternary complex, and 3) degradation efficiency via the full Cullin Ring Ligase (CRL) macromolecular assembly. The novel approach described here combines experimental biophysical data with molecular dynamics (MD) simulations to accurately predict ternary complex structures at atomic resolution. We integrate hydrogen-deuterium exchange mass spectrometry (HDX-MS, which measures the solvent exposure of protein residues) directly into the MD simulation algorithm to improve the efficiency and accuracy of the ternary structure predictions of the bromodomain of the cancer target SMARCA2 with the E3 ligase VHL, as mediated by three different degrader molecules. The simulations accurately reproduce X-ray crystal structures--including a new structure that we determined in this work (PDB ID: 7S4E)--with root mean square deviations (RMSD) of 1.1 to 1.6 Å. The simulations also reveal a structural ensemble of low-energy conformations of the ternary complex. Snapshots from these simulations are used as seeds for additional simulations, where we perform 5.7 milliseconds of aggregate simulation time using Folding@home, the world's largest distributed supercomputer. The detailed free energy surface captures the crystal structure conformation within the low-energy basin and is consistent with solution-phase experimental data (HDX-MS and SAXS). Finally, we graft a structural ensemble of the ternary complexes onto the full CRL and perform enhanced sampling simulations. Our results suggest that differences in degradation efficiency may be related to the proximity distribution of lysine residues on the POI relative to the E2-loaded ubiquitin. We make source code and the simulation and experimental datasets from this work publicly available for researchers to further advance the field of induced proximity modulation.

A Different Kind of Restraint Suitable for Molecular Dynamics Simulations

“…We recently published our work on atomic-resolution prediction of degrader-mediated ternary complex structures [15] where we also studied the dynamic nature of the full CRL-VHL-degrader-SMARCA2 complex via meta-eABF and generated a diverse set of putative closed con- formations that place the E2-loaded ubiquitin close to lysine residues on SMARCA2. The results from the meta-eABF simulation are used to seed additional simulations for unbiased ensemble-scale sampling [15]. It was this meta-eABF path CV simulation where we first had encountered a serious problem with penalty restraints and found a solution using the barrier restraint described in this work.…”

Modeling the effect of cooperativity in ternary complex formation and targeted protein degradation mediated by heterobifunctional degraders

“…The target in the ternary complexes of different HBF molecules may have different ubiquitination rates because of the difference in their structural ensembles, 27,28 which affects the exposure of lysines on the target surface to ubiquitination. 29 If ubiquitination is fast, a transient ternary complex is sufficient for ubiquitination to take place, and fast dissociation of the ternary complex-corresponding to a low cooperativity-leads to both high turnover of the HBF molecules and rapid production of free, polyubiquitinated target ready for degradation; in contrast, high cooperativity-and thus a very stable ternary complex-may hinder the catalytic turnover and slow down the degradation.…”

Charting functional E3 ligase hotspots and resistance mechanisms to small-molecule degraders

“…Among others, structures (i) present a static snapshot of an otherwise dynamic system that may sample multiple states, (ii) might lack resolution especially at dynamic interfaces, (iii) don’t consider stoichiometry found in a cellular environment and (iv) often depend on truncated protein constituents that might further lack posttranslational modifications of critical importance. Complementary in solution technologies, such as Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) and small-angle X-ray scattering, can provide a more dynamic perspective towards the dissection of critical determinants of drug-induced ternary complex formation 24,25 . However, many of the aforementioned aspects and limitations similarly apply.…”

“…Complementary in solution technologies, such as Hydrogen Deuterium Exchange Mass Spectrometry (HDX-MS) and small-angle X-ray scattering, can provide a more dynamic perspective towards the dissection of critical determinants of drug-induced ternary complex formation 24,25 . However, many of the aforementioned aspects and limitations similarly apply.…”

“…25.1, Broad Institute GitHub Repository). Reads were sorted and duplicate reads filtered using the SortSam and MarkDuplicates Picard tools.…”