PROTAC-induced Protein Functional Dynamics in Targeted Protein Degradation

Reviewer #1 (Public review):

This study by Wu et al. provides valuable computational insights into PROTAC-related protein complexes, focusing on linker roles, protein-protein interaction stability, and lysine residue accessibility. The findings are significant for PROTAC development in cancer treatment, particularly breast and prostate cancers.

The authors' claims about the role of PROTAC linkers and protein-protein interaction stability are generally supported by their computational data. However, the conclusions regarding lysine accessibility could be strengthened with more in-depth analysis. The use of the term "protein functional dynamics" is not fully justified by the presented work, which focuses primarily on structural dynamics rather than functional aspects.

Strengths:

(1) Comprehensive computational analysis of PROTAC-related protein complexes.

(2) Focus on critical aspects: linker role, protein-protein interaction stability, and lysine accessibility.

Weaknesses:

(1) Limited examination of lysine accessibility despite its stated importance.

(2) Use of RMSD as the primary metric for conformational assessment, which may overlook important local structural changes.

Reviewer #2 (Public review):

Summary:

The manuscript reports the computational study of the dynamics of PROTAC-induced degradation complexes. The research investigates how different linkers within PROTACs affect the formation and stability of ternary complexes between the target protein BRD4BD1 and Cereblon E3 ligase, and the degradation machinery. Using computational modeling, docking, and molecular dynamics simulations, the study demonstrates that although all PROTACs form ternary complexes, the linkers significantly influence the dynamics and efficacy of protein degradation. The findings highlight that the flexibility and positioning of Lys residues are crucial for successful ubiquitination. The results also discussed the correlated motions between the PROTAC linker and the complex.

Strengths:

The field of PROTAC discovery and design, characterized by its limited research, distinguishes itself from traditional binary ligand-protein interactions by forming a ternary complex involving two proteins. The current understanding of how the structure of PROTAC influences its degradation efficacy remains insufficient. This study investigated the atomic-level dynamics of the degradation complex, offering potentially valuable insights for future research into PROTAC degradability.

Reviewer #3 (Public review):

The authors offer an interesting computational study on the dynamics of PROTAC-driven protein degradation. They employed a combination of protein-protein docking, structural alignment, atomistic MD simulations, and post-analysis to model a series of CRBN-dBET-BRD4 ternary complexes, as well as the entire degradation machinery complex. These degraders, with different linker properties, were all capable of forming stable ternary complexes but had been shown experimentally to exhibit different degradation capabilities. While in the initial models of the degradation machinery complex, no surface Lys residue(s) of BRD4 were exposed sufficiently for the crucial ubiquitination step, MD simulations illustrated protein functional dynamics of the entire complex and local side-chain arrangements to bring Lys residue(s) to the catalytic pocket of E2/Ub for reactions. Using these simulations, the authors were able to present a hypothesis as to how linker property affects degradation potency. They were able to roughly correlate the distance of Lys residues to the catalytic pocket of E2/Ub with observed DC50/5h values. This is an interesting and timely study that presents interesting tools that could be used to guide future PROTAC design or optimization.

Author response:

Thank you for the reviewers’ thoughtful comments and suggestions! We greatly appreciate the feedback and are committed to address all the points raised by the reviewers to strengthen our manuscript.

We plan to conduct additional local structural analyses to better demonstrate our observations of PROTAC-induced LYS-GLY interactions and lysine associability. Specifically, we will add more in-depth analysis such as computing dihedral entropies and Root Mean Square Fluctuation (RMSF) of nearby side chains and integrating various structural alignments to provide better visualization and understanding of the local structural arrangements. We plan to extend and add simulations when needed. We will review the latest available crystal and cryo-EM structures. If new structures are available, we will incorporate them into our revised analysis and discussion.

In the revision, additional figures will be included to offer a more comprehensive assessment of local conformational changes. We will also ensure that explanations of technical terminology are clear to non-expert readers and will address the grammatical and terminology errors highlighted by the reviewers. We will refine our language to more accurately describe the focus on structural dynamics in our study.