Encounter-state over-anchoring governs productive PETase binding on PET surfaces

Abstract

Polyethylene terephthalate (PET) hydrolysis by Ideonella sakaiensis PETase (IsPETase) begins at a heterogeneous solid-liquid interface, yet the molecular basis of productive surface recognition remains poorly resolved. Here, we combined a Martini 3 coarse-grained PET model with G[o]Martini protein dynamics to investigate IsPETase binding to an extended PET surface. A four-state kinetic model, comprising unbound, encounter, docked, and pre-catalytic states, shows that productive binding is not limited by adsorption itself, but by a post-adsorption re-registration step that converts surface-bound encounter complexes into productively aligned configurations. The simulations reveal a stage-dependent role of conformational flexibility: flexible surface loops facilitate early capture, whereas excessive flexibility promotes misregistered hydrophobic contacts, over-stabilizes non-productive encounter states, and lowers the overall probability of productive commitment. Analysis of productive trajectories further identifies three microscopic reorientation modes by which the enzyme reaches the pre-catalytic state after adsorption. Comparative simulations of engineered PETase variants uncover a flexibility-driven speed-yield trade-off, in which increased flexibility accelerates successful binding events but reduces productive yield through encounter-state over-anchoring. Guided by this mechanism, we formulated a landscape-based design strategy that either weakens encounter-specific anchors or reinforces product-like contacts, leading to mutations that improve productive-binding yield. These results identify post-adsorption alignment as the key kinetic bottleneck in PETase surface recognition and provide a mechanistic framework for designing enzymes that operate at heterogeneous polymer interfaces.