Molecular determinants of dynamic protein-protein interactions in the functional cycle of the membrane protein DsbD

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Molecular recognition is of central importance in biology. The molecular determinants shaping recognition of one protein domain by another are incompletely understood, especially in the context of the complex function of molecular machines. Here, we combine NMR experiments and molecular dynamics simulations to elucidate the determinants of recognition of the C-terminal (cDsbD) domain of the transmembrane reductant conductor DsbD by its cognate partner, the N-terminal domain of the protein (nDsbD). As part of the natural cycle of this oxidoreductase, which effectively transfers electrons from the cytoplasm to the periplasm of Gram-negative bacteria, cDsbD and nDsbD toggle between oxidised and reduced states, something that modulates the affinity of the domains for each other and prevents otherwise unproductive reactions. We find that the redox state of cDsbD determines the dissociation rate of cDsbD-nDsbD complexes. Molecular dynamics simulations demonstrate how the redox-state of the active site determines the stability of inter-domain hydrogen bonds and thus the dissociation rate. AlphaFold modelling and atomistic molecular dynamics simulations of full-length DsbD in a realistic bacterial membrane again highlights the close proximity of the periplasmic domains and the importance of tuning the strength of the interactions of the periplasmic domains to enable electron transfer to cognate periplasmic partners such as CcmG. Our AlphaFold models are consistent with in vivo functional assays of DsbD mutants, which together help to reveal for the first-time a putative binding site for thioredoxin on the cytoplasmic side of DsbD.

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