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Diversity of mechanisms to control bacterial GTP homeostasis by the mutually exclusive binding of adenine and guanine nucleotides to IMP dehydrogenase - PubMed

Diversity of mechanisms to control bacterial GTP homeostasis by the mutually exclusive binding of adenine and guanine nucleotides to IMP dehydrogenase

David Fernández-Justel et al. Protein Sci. 2022 May.

Abstract

IMP dehydrogenase(IMPDH) is an essential enzyme that catalyzes the rate-limiting step in the guanine nucleotide pathway. In eukaryotic cells, GTP binding to the regulatory domain allosterically controls the activity of IMPDH by a mechanism that is fine-tuned by post-translational modifications and enzyme polymerization. Nonetheless, the mechanisms of regulation of IMPDH in bacterial cells remain unclear. Using biochemical, structural, and evolutionary analyses, we demonstrate that, in most bacterial phyla, (p)ppGpp compete with ATP to allosterically modulate IMPDH activity by binding to a, previously unrecognized, conserved high affinity pocket within the regulatory domain. This pocket was lost during the evolution of Proteobacteria, making their IMPDHs insensitive to these alarmones. Instead, most proteobacterial IMPDHs evolved to be directly modulated by the balance between ATP and GTP that compete for the same allosteric binding site. Altogether, we demonstrate that the activity of bacterial IMPDHs is allosterically modulated by a universally conserved nucleotide-controlled conformational switch that has divergently evolved to adapt to the specific particularities of each organism. These results reconcile the reported data on the crosstalk between (p)ppGpp signaling and the guanine nucleotide biosynthetic pathway and reinforce the essential role of IMPDH allosteric regulation on bacterial GTP homeostasis.

Keywords: (p)ppGpp; IMP dehydrogenase; allosteric regulation; bacterial GTP homeostasis; protein structure and function; purine nucleotide biosynthesis.

© 2022 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.

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Figures

FIGURE 1
FIGURE 1

Structure, function, and regulation of eukaryotic IMPDHs. (a) Schematic and simplified scheme of the de novo purine nucleotide biosynthetic pathways. Competitive inhibitors are colored in yellow, while allosteric activators and inhibitors are colored in green and red, respectively. (b) Ribbon representation of an IMPDH tetramer, showing the catalytic domain (light blue) with the substrates NAD (yellow spheres) and IMP (orange spheres) and the regulatory Bateman domain (dark blue) with three GDP molecules (red spheres) bound. (c) Nucleotide binding to the allosteric sites in the Bateman domain promotes tetramer dimerization into octamers with different conformations and catalytic activities. IMPDH is represented as protein surface with the catalytic and regulatory domains light and dark blue colors, respectively. Adenine and guanine nucleotides bound to the Bateman regulatory domain are shown as spheres colored in green and red, respectively. IMPDH, IMP dehydrogenase

FIGURE 2
FIGURE 2

Effects of guanine nucleotides on the catalytic activity of IMPDH in vitro. Graphs showing the normalized initial velocity values (V 0 values in the absence of GTP divided by the respective values in the presence of GTP). The V 0 values used for the normalization of the data are EcIMPDH 16.6 ± 1.4, PaIMPDH 26.0 ± 0.7, BsIMPDH 14.2 ± 0.7, and StcIMPDH 14.0 ± 0.3 nM s−1 (mean ± std. error). Estimated IC50 values for are 455.3 ± 6.3 μM and 147.4 ± 3.9 μM (mean ± std. error) for EcIMPDH and PaIMPDH, respectively. Similar results were obtained for GDP inhibition (Figure S1). BsIMPDH, Bacillus subtilis IMPDH; EcIMPDH, Escherichia coli IMPDH; IMPDH, IMP dehydrogenase; PaIMPDH, Pseudomonas aeruginosa IMPDH; StcIMPDH, Streptomyces coelicolor IMPDH

FIGURE 3
FIGURE 3

Structure of PaIMPDH bound to ATP and GDP. Detailed views of ATP (a) and GDP (b) bound in the Bateman domain to the first and second nucleotide canonical sites, respectively. IMPDH protein is represented in semitransparent blue cartoons with the side chain of key interacting residues shown in sticks. The 2mFo–DFc electron density map, contoured at the 1.6σ level, is shown as a grey mesh. Key protein–nucleotide atomic interactions are represented as orange dashed lines and the coordinated Magnesium atom is shown as an orange sphere. (c) Upper panel: structural superposition of the catalytic domains (white ribbons) of a monomer of PaIMPDH showing the different conformations adopted by the Bateman domain upon ATP (orange ribbons; PDB ID 4DQW) or ATP/GDP (blue ribbons) binding. Lower panel: the conformational switch described in the upper panel, translated to the octameric structures. PaIMPDH octamers are represented as protein surfaces with the same color code as in the upper panel. IMPDH, IMP dehydrogenase; PaIMPDH, Pseudomonas aeruginosa IMPDH

FIGURE 4
FIGURE 4

The ATP/GTP balance modulates the activity of proteobacterial IMPDHs. Heatmap representation of the enzymatic percent activity. V 0 values at different ATP versus GTP concentrations, normalized to the V 0 values in the absence of nucleotide for EcIMPDH (a) and at 1 mM ATP for PaIMPDH (b). The V 0 values used for normalization are EcIMPDH 15.9 and PaIMPDH 16.4 nM s−1 (note that PaIMPDH is inactive in vitro in the absence of ATP 10 ). IMPDH, IMP dehydrogenase; PaIMPDH, Pseudomonas aeruginosa IMPDH

FIGURE 5
FIGURE 5

Effects of ppGpp on the catalytic activity of IMPDH in vitro. Graphs showing the normalized initial velocity values (V 0 values in the absence of ppGpp divided by the respective values in the presence of ppGpp. The V 0 values used for the normalization of the data are EcIMPDH 18.5 ± 1.0, PaIMPDH 26.7 ± 0.9, BsIMPDH 12.9 ± 0.8, and StcIMPDH 12.6 ± 0.4 nM s−1 (mean ± std. error). Estimated IC50 values are 8.9 ± 0.4 μM and 2.0 ± 0.03 μM (mean ± std. error) for BsIMPDH and StcIMPDH, respectively. Similar results were obtained for pppGpp inhibition (Figure S1). BsIMPDH, Bacillus subtilis IMPDH; EcIMPDH, Escherichia coli IMPDH; IMPDH, IMP dehydrogenase; PaIMPDH, Pseudomonas aeruginosa IMPDH; StcIMPDH, Streptomyces coelicolor IMPDH

FIGURE 6
FIGURE 6

Structure of StcIMPDH bound to ATP and ppGpp. (a) Structural superimposition of the Bateman domains of PaIMPDH‐ATP/GDP (blue), StcIMPDH‐ATP‐ppGpp (green), and AgIMPDH‐ATP/GDP (red; PDB ID 5TC3). (b) Detailed view of the ppGpp binding site in the Bateman domain. IMPDH protein is represented in semitransparent green cartoons with the side chain of key interacting residues shown in sticks. The 2mFo–DFc electron density map, contoured at the 1.6σ level, is shown as a grey mesh. Key protein–nucleotide atomic interactions are represented as orange dashed lines and the coordinated Magnesium atoms are shown as orange spheres. (c) The taxonomic distribution of the (p)ppGpp binding site within the Bateman domain is shown. The phylogenetic tree on the left shows the evolutionary relationships among the groups of bacteria (color‐coded according to a) and is extracted from a more detailed analysis shown in Figures S9 and S10. IMPDH, IMP dehydrogenase; PaIMPDH, Pseudomonas aeruginosa IMPDH; StcIMPDH, Streptomyces coelicolor IMPDH

FIGURE 7
FIGURE 7

ppGpp modulates the activity of IMPDH in Actinobacteria and Firmicutes. Heatmap representation of the enzymatic percent activity (V 0 values normalized to the V 0 values in the absence of nucleotide) of BsIMPDH (a) and StcIMPDH (b) at different ATP versus ppGpp concentrations. The V 0 values used for normalization are BsIMPDH 32.9 and StcIMPDH 7.6 nM s−1. BsIMPDH, Bacillus subtilis IMPDH; IMPDH, IMP dehydrogenase; StcIMPDH, Streptomyces coelicolor IMPDH

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