Insights into the Role of Side-Chain Team Work in nDsbD_Ox/Red Proteins: Mechanism of Substrate Binding

N-terminal disulfide bond oxidoreductase (nDsbD_Ox/Red) proteins display divergent substrate binding mechanisms depending on the conformational changes to the Phe₇₀ cap, which is also dependent on the disulfide redox state. In nDsbD_Ox, the cap dynamics is complex (shows both open/closed Phe₇₀ cap conformations), resulting in an active site that is highly flexible. So the system's active site is conformationally selective (the active site adapts before substrate binding) toward its substrate. In nDsbD_Red, the cap is generally closed, resulting in induced fit-type binding (adapts after substrate approach). Recent studies predict Tyr₄₀ and Tyr₄₂ residues to act as internal nucleophiles (Tyr_40/42O^-) for disulfide association/dissociation in nDsbD_Ox/Red, supplementing the electron transfer channel. From this perspective, we investigate the cap dynamics and the subsequent substrate binding modes in these proteins. Our molecular dynamics simulations show that the cap opening eliminates Tyr₄₂O^- electrostatic interactions irrespective of the disulfide redox state. The active site becomes highly flexible, and the conformational selection mechanism governs. However, Tyr₄₀O^- formation does not alter the chemical environment; the cap remains mostly closed and plausibly follows the induced fit mechanism. Thus, it is apparent that mostly Tyr₄₂O^- facilitates the internal nucleophile-mediated self-preparation of nDsbD_Ox/Red proteins for binding.