Atomistic Simulations of the Mechanisms of the Poorly Catalytic Mitochondrial Chaperone Trap1: Insights into the Effects of Structural Asymmetry on Reactivity

The mitochondrial chaperone Trap1 is an ATPase protein that oversees the correct folding of client proteins and exhibits altered activity and/or expression levels in certain cancers. By inducing extensive structural rearrangements, ATP cleavage is essential for Trap1 to fulfill its task; at the same time, Trap1's sluggish ATP turnover represents one of the control switches through which its conformational cycle and inter-residue cross-talk signals mediating it can be subtly tuned. Remarkably, hydrolysis requires Trap1 to adopt a distinctive asymmetric "closed"homodimeric conformation wherein the ATP bound to the "buckled"protomer (Buc) is preferentially cleaved, while hydrolysis in the "straight"protomer (Str) remains unfavored. However, molecular links between asymmetry and Trap1's characteristic reactivity remain elusive, due to intrinsic mechanistic complexity and its protomers' active site similarity. To address this issue, we herein report a detailed computational investigation of Trap1's potential ATPase mechanisms. Through classical molecular dynamics (MD) simulations we monitor how frequently ATP and nucleophilic water WatNuc can attain reactive poses within Buc and Str. Semiempirical hybrid quantum mechanical-molecular mechanical (QM/MM) MD simulations coupled to umbrella sampling and benchmarked with density functional theory calculations are then used to sample reaction free energy barriers within each protomer for two possible hydrolytic pathways. Enzyme-assisted hydrolysis, featuring a metaphosphate-like transition state and a catalytic glutamate deprotonating WatNuc, is found to be favored in both protomers over substrate-assisted hydrolysis. However, we also find that WatNuc's sequestration by another water molecule WatTyr bound to a vicinal tyrosine is far rarer in Buc, proving that such a rare sequestration lowers reaction barriers for the enzyme-assisted pathway. We thus identify the (biologically significant) tyrosine as the main mediator favoring ATP hydrolysis in Buc over Str. Our improved model for Trap1 reactivity is experimentally and structurally consistent and should further aid in the development of selective modulators of the protein's activities.