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 Phe 70 cap, which is also dependent on the disulfide redox state. In nDsbD Ox , the cap dynamics is complex (shows both open/closed Phe 70 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 Tyr 40 and Tyr 42 residues to act as internal nucleophiles (Tyr 40/42 O - ) 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 Tyr 42 O - electrostatic interactions irrespective of the disulfide redox state. The active site becomes highly flexible, and the conformational selection mechanism governs. However, Tyr 40 O - 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 Tyr 42 O - facilitates the internal nucleophile-mediated self-preparation of nDsbD Ox/Red proteins for binding.