Probing the Gate-Opening Transition in the Bacterial ClpP Peptidase Using Molecular Dynamics Simulations and Machine Learning

Preserving proteome integrity is crucial for maintaining cell viability across all kingdoms of life. The bacterial caseinolytic protease (ClpP) plays a critical role in maintaining protein homeostasis by degrading misfolded or damaged proteins within the cellular environment. The N-terminal region of each protomer within the ClpP tetradecamer regulates <i>substrate protein</i> access to the proteolytic chamber, thereby preventing the degradation of essential proteins. Upon binding of an ATPase partner or small molecules, such as acyldepsipeptide (ADEP), ClpP undergoes a spectacular transition from its inactive "closed pore" state to its active "open pore" conformation, widening its central channel to allow substrate entry. In this study, we integrate molecular dynamics simulations and multiclass and binary classification machine learning approaches to probe the structural determinants within the N-terminal region that control the conformational transition underlying the pore opening. SHapley Additive exPlanations (SHAP) analysis reveals that the representative features of the transition between open and closed pore conformations correspond to stabilizing intraloop interactions, including hydrogen bonds, native contacts and intraprotomer salt bridges. Our study also uncovers the effect of perturbation, namely through ADEP removal or charge-reversal/neutralizing point mutations of functionally important residues, on the closed to open pore transition, as well as the direction of change of the structural features.