Insights into the\nRole of Side-Chain Team Work in\nnDsbD<sub>Ox/Red</sub> Proteins: Mechanism of Substrate Binding

N-terminal disulfide bond oxidoreductase (nDsbD<sub>Ox/Red</sub>) proteins display divergent substrate binding mechanisms depending\non the conformational changes to the Phe<sub>70</sub> cap, which is\nalso dependent on the disulfide redox state. In nDsbD<sub>Ox</sub>, the cap dynamics is complex (shows both open/closed Phe<sub>70</sub> cap conformations), resulting in an active site that is highly flexible.\nSo the system’s active site is conformationally selective (the\nactive site adapts before substrate binding) toward its substrate.\nIn nDsbD<sub>Red</sub>, the cap is generally closed, resulting in\ninduced fit-type binding (adapts after substrate approach). Recent\nstudies predict Tyr<sub>40</sub> and Tyr<sub>42</sub> residues to\nact as internal nucleophiles (Tyr<sub>40/42</sub>O<sup>–</sup>) for disulfide association/dissociation in nDsbD<sub>Ox/Red</sub>, supplementing the electron transfer channel. From this perspective,\nwe investigate the cap dynamics and the subsequent substrate binding\nmodes in these proteins. Our molecular dynamics simulations show that\nthe cap opening eliminates Tyr<sub>42</sub>O<sup>–</sup> electrostatic\ninteractions irrespective of the disulfide redox state. The active\nsite becomes highly flexible, and the conformational selection mechanism\ngoverns. However, Tyr<sub>40</sub>O<sup>–</sup> formation does\nnot alter the chemical environment; the cap remains mostly closed\nand plausibly follows the induced fit mechanism. Thus, it is apparent\nthat mostly Tyr<sub>42</sub>O<sup>–</sup> facilitates the internal\nnucleophile-mediated self-preparation of nDsbD<sub>Ox/Red</sub> proteins\nfor binding.