spans both the catalytic and oligomerization domains, with its adenine moiety bound to the oligomerization domain and its nicotinamide moiety bound to the catalytic domain. (3) The cofactor − substrate complex indicates an additional layer of complexity: while NADPH appears to be bound to all four subunits, only two closed-form subunits show density for the complete thioester, whereas the two open-form subunits represent catalytically incompetent ternary complexes. QM/MM simulations indicate that the substrate is confor-mationally constrained in the closed-form subunits, and highly dynamic in the open-form subunits, suggesting half-site reactivity. (4) Most impressively from a conformational perspective, the all-atom molecular dynamic simulations indicate highly complex allosteric regulation, involving catalytically essential swing and twist motions of peripheral catalytic domains that are coupled both within and across pairs of dimers. Removing the substrate from the closed-form subunits resulted in rapid conformational changes in just a few tens of nanoseconds of simulation time, causing the closed-form subunits to transition to an open-form conformation, coupled with a twist motion of one subunit to the other in each dimer. In the A/C dimer, which opens its closed subunit first, this twisting motion was shown to rotate the catalytic domain of the now-empty open-form active site in subunit A, which would then be expected to push a bound substrate toward the NADPH cofactor in the C-subunit. Further simulations indicated coupling between the dynamics of each dimer pair in the tetrameric complex (Figure that is regulated by the absence or of
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