Conserved residues of the Pro103–Arg115 loop are involved in triggering the allosteric response of the Escherichia coli ADP‐glucose pyrophosphorylase

Abstract

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.

The allosteric control mechanism of bacterial glycogen biosynthesis disclosed by cryoEM

“…Kinetic properties of EcAGPase single-point mutants have been previously reported. 32,37,38 The harmonization of these results from different conditions and the interpretation of the kinetics parameters in the context of the structural data provided by X-ray crystallography and cryoEM represent a challenge. To reduce the complexity, we have performed a simplified meta-analysis of…”

“…16 This electron density likely represents the RL2 loop in multiple conformational states, as suggested in our previous crystallographic studies on EcAGPase, as well as in other homologues, where the it could not be modeled except when participating in crystal contacts. 16,[31][32][33] Conversely, the EcAGPase-FBP D2 reconstruction reveals a consensus electron density for the RL2 loop in a relaxed 'free' conformation ( Figures 2 and 5). Moreover, the RL1 (residues Ile72 to His78) and the G-rich loops (residues 26 to 32; a constituent of the SM) appear in different conformations in these reconstructions, revealing the underlying dynamics of these loops.…”

“…The replacement of Trp113 and Gln74 by alanine leads to a remarkable reduction in the activity and abolished the activation by FBP and inhibition by AMP. 37,32 Furthermore, beyond their impact in the enzyme activity, the single point mutation to alanine of RL2 residues 105-111 reduce AMP inhibition. 32 It is worth noting that RL2 residues are mainly conserved among AGPases from different sources, suggesting a common inhibition mechanism ( Figure 6).…”

“…26,27,28,29 The two subunits have different functions; α is the catalytic subunit, whereas β is the regulatory subunit. To date, three crystal structures of AGPases have been reported, those of the bacterial homotetrameric AGPases from Escherichia coli (EcAGPase) 5,16,30 and Agrobacterium tumefaciens (AtAGPase), 31,32 and a recombinant homotetrameric version of the small subunit (α4) of the photosynthetic potato tuber AGPase (StAGPase). 33 The activity of the paradigmatic EcAGPase is enhanced by the high-energy glycolytic intermediate fructose-1,6-bisphosphate (FBP), whereas, the ubiquitous low-energy metabolite AMP inhibits the enzyme.…”

“…However, the organization of the protein molecules inside the crystal packing prevents, in many cases, the visualization of native biologically relevant conformational states associated to the binding of substrates and regulatory molecules, impeding the construction of accurate models to explain allosteric regulation. 16,[31][32][33] In this work, we disclose the cryoEM structures of the homotetrameric EcAGPase (a ca. 200 kDa enzyme, with each protomer of 48.7 kDa), in complex with FBP and AMP, both at 3.0 Å resolution, respectively.…”