The synthesis of glycogen in bacteria and starch in plants is allosterically controlled by the production of ADP-glucose by ADP-glucose pyrophosphorylase. Using computational studies, site-directed mutagenesis, and kinetic characterization, we found a critical region for transmitting the allosteric signal in the Escherichia coli ADP-glucose pyrophosphorylase. Molecular dynamics simulations and structural comparisons with other ADP-glucose pyrophosphorylases provided information to hypothesize that a Pro103-Arg115 loop is part of an activation path. It had strongly correlated movements with regions of the enzyme associated with regulation and ATP binding, and a network analysis showed that the optimal network pathways linking ATP and the activator binding Lys39 mainly involved residues of this loop. This hypothesis was biochemically tested by mutagenesis. We found that several alanine mutants of the Pro103-Arg115 loop had altered activation profiles for fructose-1,6-bisphosphate. Mutants P103A, Q106A, R107A, W113A, Y114A, and R115A had the most altered kinetic profiles, primarily characterized by a lack of response to fructose-1,6-bisphosphate. This loop is a distinct insertional element present only in allosterically regulated sugar nucleotide pyrophosphorylases that could have been acquired to build a triggering mechanism to link proto-allosteric and catalytic sites.
MarR (multiple antibiotic resistance repressor) family proteins are bacterial repressors that regulate transcription in response to a wide range of chemical signals. Although specific features of MarR family function have been described, the role of atomic motions in MarRs remains unexplored thus limiting insights into the evolution of allostery in this ubiquitous family of repressors. Here, we provide the first experimental evidence that internal dynamics play a crucial functional role in MarR proteins. Streptococcus pneumoniae AdcR (adhesin-competence repressor) regulates ZnII homeostasis and ZnII functions as an allosteric activator of DNA binding. ZnII coordination triggers a transition from somewhat independent domains to a more compact structure. We identify residues that impact allosteric activation on the basis of ZnII-induced perturbations of atomic motions over a wide range of timescales. These findings appear to reconcile the distinct allosteric mechanisms proposed for other MarRs and highlight the importance of conformational dynamics in biological regulation.
18MarR (multiple antibiotic resistance repressor) family proteins are bacterial repressors that 19 regulate transcription in response to a wide range of chemical signals. 20Although specific features of MarR family function have been described, the role of atomic 21 motions in MarRs remains unexplored thus limiting insights into the evolution of allostery in this 22 ubiquitous family of repressors. Here, we provide the first experimental evidence that internal 23 dynamics play a crucial functional role in MarR proteins. Streptococcus pneumoniae AdcR 24 (adhesin-competence repressor) regulates Zn II homeostasis and Zn II functions as an allosteric 25 activator of DNA binding. Zn II coordination triggers a transition from independent domains to a 26 more compact structure. We identify residues that impact allosteric activation on the basis of 27 Zn II -induced perturbations of atomic motions over a wide range of timescales. These findings 28 reconcile the distinct allosteric mechanisms proposed for other MarRs and highlight the 29 importance of conformational dynamics in biological regulation. 30 31 32 33
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